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Intra- and intermolecular interactions in proteins Studies of marginally hydrophobic transmembrane α-helices
and protein-protein interactions.
Linnea E. Hedin
© Linnea E. Hedin, Stockholm 2010
ISBN 978-91-7447-111-3
Printed in Sweden by US-AB, Stockholm 2010
Distributor: Department of Biochemistry and Biophysics, Stockholm University
To my parents
Abstract
Most of the processes in a living cell are carried out by proteins.
Depending on the needs of the cell, different proteins will interact
and form the molecular machines demanded for the moment. A
subset of proteins called integral membrane proteins are responsible
for the interchange of matter and information across the biological
membrane, the lipid bilayer enveloping and defining the cell. Most
of these proteins are co-translationally integrated into the membrane
by the Sec translocation machinery.
This thesis addresses two questions that have emerged during the
last decade. The first concerns membrane proteins: a number of α-
helices have been observed to span the membrane in the obtained
three-dimensional structures even though these helices are predicted
not to be hydrophobic enough to be recognized by the translocon for
integration. We show for a number of these marginally hydrophobic
protein segments that they indeed do not insert well outside of their
native context, but that their local sequence context can improve the
level of integration mediated by the translocon. We also find that
many of these helices are overlapped by more hydrophobic
segments. We propose, supported by experimental results, that the
latter are initially integrated into the membrane, followed by post-
translational structural rearrangements. Finally, we investigate
whether the integration of the marginally hydrophobic TMHs of the
lactose permease of Escherichia coli is facilitated by the formation
of hairpin structures. However our combined efforts of
computational simulations and experimental investigations find no
evidence for this.
The second question addressed in this thesis is that of the
interpretation of the large datasets on which proteins that interact
with each other in a cell. We have analyzed the results from several
large-scale investigations concerning protein interactions in yeast
and draw conclusions regarding the biases, strengths and weaknesses
of these datasets and the methods used to obtain them.
Populärvetenskaplig sammanfattning
Av alla de molekyler som finns i och bygger upp en cell är det
proteinerna som är de verkliga arbetshästarna och som står för de
flesta processer som äger rum i cellen. Cellens behov varierar
kontinuerligt, och olika proteiner interagerar med varandra för att
bilda de molekylära verktyg som behövs för stunden.
En undergrupp av proteiner som kallas membranproteiner
ansvarar för utbyte av materia (näring, etc) och information över det
biologiska membranet, det dubbla lager av lipider som omsluter och
definierar cellen. Många sjukdomar är sammankopplade med
membranproteiner som inte fungerar som de ska och studier av
membranproteiner är av högsta vikt för att kunna förstå och bota
dessa sjukdomar.
De flesta membranproteiner sätts in i lipidbilagret samtidigt som
de syntetiseras, via ett maskineri som kallas Sec-translokonet.
Proteinernas byggstenar, aminosyrorna, har olika kemiska
egenskaper. För att kännas igen av translokonet måste ett
proteinsegment bestå av aminosyror som är hydrofoba, som löser sig
bättre i fett än i vatten.
Den här avhandlingen tar upp två frågor som uppstått det senaste
decenniet: Den första frågan rör de proteinsegment som är trädda
genom membranet. Enligt vad som är känt idag är vissa av dessa
segment inte tillräckligt hydrofoba för att kännas igen av
translokonet och stoppas in i membranet. Trots detta syns det tydligt
i tredimensionella bilder av membranproteiner att dessa marginellt
hydrofoba segment faktiskt är inbäddade i membranet. Vi har
undersökt hur detta kan förklaras.
Vi visar för ett antal av dessa segment att de i sig själva inte är
tillräckliga för att effektivt sättas in i membranet, men att egenskaper
hos de angränsande proteinsegmenten kan vara tillräckliga för att få
dem att sättas in. Vi finner också att många av dessa mindre
hydrofoba segment överlappas av mer hydrofoba dito. Vi presenterar
resultat som föreslår att translokonet initialt känner igen de mer
hydrofoba, överlappande segmenten och att den slutliga strukturen
fås genom att proteinet skiftar sin position i membranet efter att
proteinsyntesen avslutats.
Slutligen undersöker vi proteinet laktospermeas och huruvida
dess många marginellt hydrofoba segment sätts in i membranet på
grund av täta interaktioner med det närmaste föregående eller
efterföljande membraninbäddade segmentet. Vi använder både
datorsimuleringar och experimentella undersökningar, men hittar
inget som talar för det ovanstående.
Den andra frågan rör tolkningen av den stora mängden
information om vilka proteiner som interagerar med varandra. Vi har
analyserat resultaten från flera storskaliga undersökningar av
interaktioner i jästceller och drar slutsatser angående styrkorna och
svagheterna i dessa dataset samt de metoder som använts för att ta
fram dem.
List of papers
This thesis is based on the following publications:
Paper I
Paper II
Paper III
Paper IV
Hedin LE*, Öjemalm K*, Bernsel A, Hennerdal A,
Illergård K, Enquist K, Kauko A, Cristobal S, von Heijne
G, Lerch-Bader M, Nilsson I, Elofsson A. “Membrane
insertion of marginally hydrophobic transmembrane
helices depends on sequence context.” (2010) Journal of
Molecular Biology 396(1):221-9
Kauko A*, Hedin LE*, Thebaud E, Cristobal S, Elofsson
A, von Heijne G. “Repositioning of transmembrane
alpha-helices during membrane protein folding.” (2010)
Journal of Molecular Biology 397(1):190-201
Vereshchaga Y*, Hedin L*, Cristobal S, Elofsson A,
Lindahl E. “Insertion Properties of Marginally
Hydrophobic Helices in the LacY Lactose Permease
Transporter” (Manuscript.)
Björklund AK, Light S, Hedin L, Elofsson A.
“Quantitative assessment of the structural bias in protein-
protein interaction assays.” (2008) Proteomics
8(22):4657-67
* Equal contribution
Reprints were made with permissions from the publishers.
Contents
Abbreviations
Chapter 1 Introduction ....................................................................... 13
Chapter 2 Biological membranes ....................................................... 15
2.1 Biological membranes define the cell ........................................ 15
2.2 Biological membranes form due to the hydrophobic effect ....... 16
2.3 The hydrophobic effect is caused by water ................................ 16
2.4 Basic structure of phospholipid membranes .............................. 17
2.5 Biological membranes consist of more than lipids .................... 18
Chapter 3 Proteins in general ............................................................ 19
3.1 Protein structure and folding in general ..................................... 19
3.2 More on the quartenary structure of proteins, protein-protein
interactions ................................................................................ 21
3.3 Evolutionary evolved functional substructures, protein domains
................................................................................................... 22
Chapter 4 Membrane proteins, what do they look like? .................. 23
4.1 Structure and topology of α-helical membrane proteins ............ 24
4.1.1 The primary structure of a transmembrane helix is adapted to
its surroundings .................................................................... 24
4.1.2 Secondary structure in transmembrane segments can be
irregular ................................................................................ 25
4.1.3 Other structural elements of helical membrane proteins ...... 26
4.2 Forces and motifs implied in the folding of helical membrane
proteins ...................................................................................... 26
4.2.1 Intramolecular forces stabilizing the native fold .................. 27
4.2.2 Lipid-protein interactions affecting the folded protein 28
Chapter 5 Biogenesis and bilayer integration of α-helical membrane
proteins .............................................................................. 31
5.1 The Sec translocon channel ....................................................... 32
5.2 The versatile translocon ............................................................. 33
5.3 Other systems for insertion of membrane proteins .................... 34
Chapter 6 How is topology decided during biogenesis and folding?
........................................................................................... 37
6.1 Charged residues in loops, the positive inside rule .................... 38
6.2 Hydrophobicity as recognized by the translocon ....................... 39
6.3 Marginally hydrophobic transmembrane helices do exist.......... 39
6.4 Length of the hydrophobic segment .......................................... 40
6.5 Reentrant loops .......................................................................... 41
6.6 Other factors .............................................................................. 41
Chapter 7 Structure analysis ............................................................. 43
7.1 Obtaining three-dimensional structures ..................................... 43
7.2 In silico investigations ............................................................... 44
7.3 Determining membrane protein topology experimentally ......... 45
7.3.1 N-linked glycosylation as topology reporter and molecular
ruler ...................................................................................... 46
7.4 Determining the propensity to integrate into the membrane ...... 46
7.4.1 Model proteins, LepI and LepII ........................................... 47
7.4.2 In vitro protein expression ................................................... 48
7.5 Determining quartenary protein structure, the composition of
protein complexes ...................................................................... 48
Chapter 8 Summary of the papers .................................................... 51
8.1 Paper I. ....................................................................................... 51
8.2 Paper II. ..................................................................................... 52
8.2 Paper III. .................................................................................... 52
8.4 Paper IV. .................................................................................... 53
8.5 Final thoughts ............................................................................ 55
Acknowledgements ............................................................................. 57
Bibliography ........................................................................................ 59
Abbreviations
DOPC
ER
LC
MALDI-TOF
MP
MS/MS
mTMH
RM
SDS-PAGE
TM
TMH
Å
1,2-dioleoyl-sn-glycero-3-phosphocholine
Endoplasmic Reticulum
Liquid Chromatography
Matrix Assisted Laser Desorption/Ionization – Time
of Flight
Membrane Protein
Tandem Mass Spectrometry Marginally hydrophobic Transmembrane Helix
Rough Microsomes
Sodium dodecyl sulphate polyacrylamide gel
electrophoresis
Transmembrane
Transmembrane Helix
Ångström, 1 Å = 0.1 nm
Amino acids, one and three letter codes:
Any amino acid - Xaa - X Leucine - Leu - L
Alanine - Ala - A Lysine - Lys - K
Arginine - Arg - R Methionine - Met - M
Asparagine - Asn - N Phenylalanine - Phe - F
Aspartic Acid - Asp - D Proline - Pro - P
Cysteine - Cys - C Serine - Ser - S
Glutamine - Gln - Q Threonine - Thr - T
Glutamic acid - Glu - E Tryptophan - Trp - W
Glycine - Gly - G Tyrosine - Tyr - Y
Histidine - His - H Valine - Val - V
Isoleucine - Iso - I
13
Chapter 1
Introduction
The basic building blocks of life are water, nucleotides (DNA, RNA), lipids
and amino acids. Throw in some ions and sugars and you are pretty much set.
DNA carries the information, RNA helps putting the information to use, lipids
make up the biological membranes that separate a cell from the rest of the
world and the cellular organelles from the rest of the cell. Amino acids are put
together into proteins according to the information encoded in the DNA.
These proteins can in turn form larger complexes, varying in composition
depending on the function. Proteins are what make a cell work; they are the
workhorses and small factories of the cell. They give structure and mobility
(cytoskeleton) and take care of molecular synthesis and degradation. They are
involved in energy metabolism and allow exchange of information and
material both within the cell and with the outside (receptors, transporters,
channels), etc. Especially the latter tasks involve a certain category of
proteins, integral membrane proteins. It is easy to imagine that malfunctioning
membrane proteins can result in severe problems for a cell and, in a
multicellular being, the whole organism. In fact, more than half of the targets
in the pharmaceutical industry are membrane proteins1; 2
. To understand how a
chain of amino acids folds properly and gets integrated into the lipid bilayer is
thus an important problem to solve, and not only out of pure curiosity.
This thesis addresses the question of how less hydrophobic protein
segments get incorporated into the membrane. It also deals with large-scale
investigations of which proteins form complexes together and the importance
of knowing about method biases when interpreting results from these
investigations.
I will start by presenting a general picture of a biological membranes and the
proteins within them, what they look like and why. I will then give a short
overview of the major machinery needed to insert proteins into biological
membranes and the factors affecting the insertion process. I will also briefly
review other machineries for insertion and translocation. Thereafter I discuss
ways to study protein structure, including how to study quaternary structure by
deciphering the composition of protein complexes. In the end the articles and
the work included in this thesis are summed up and finally I share some final
thoughts on the subject.
15
Chapter 2
Biological membranes
This chapter gives a brief introduction to biological membranes, the tiny
barriers that distinguish cells from the rest of the world.
2.1 Biological membranes define the cell
Cells are the basic unit of life, defined and demarcated by their cell
membranes. Most unicellular organisms manage all the processes needed to
live and propagate within one cell, while the vast majority of multicellular
organisms are composed by differentiated cells specializing in different
functions. From an evolutionary point of view, all living organisms can be
divided into three major domains, or kingdoms: Bacteria, Archaea and
Eukarya. This division is often useful when discussing different molecular
aspects of life. Bacteria and archaeans are generally simpler than eukaryotes.
For instance, bacteria and archaea are most often unicellular while
eukaryotes usually are multicellular. There are however exceptions like
facultative multicellular prokaryotes3; 4
, colonial cyanobacteria5 and the
eukaryotic, unicellular yeasts and protists. Not only are most eukaryotes
comprised by cells specialized in different ways, but all eukaryotic cells have
organelles, internal compartments which are enclosed by membranes and
specialize in different cellular functions, see figure 1. Organelles in Archaea
and Bacteria are much rarer, but some examples do exist6; 7
.
Figure 1. A schematic
picture of an animal cell.
ER, Endoplasmic reti-
culum. Adapted from
http://en.wikipedia.org/wik
i/File:Biological_cell.svg,
accession date July 2010.
16 Chapter 2 • Biological membranes
The exact processes taking place in each organelle can vary between
different cell types, but these are commonly the major tasks: Peroxisomes
and lysosomes specialize in synthesis and breakdown of macromolecules,
the nucleus in replication and transcription of DNA, the smooth endoplasmic
reticulum in a wide range of metabolic reactions, the rough endoplasmic
reticulum in protein synthesis, and the Golgi in protein modifications.
Mitochondria have two membranes, one inner and one outer, and specialize
in energy conversion. Chloroplasts, the photosynthetic plant organelles (not
shown here) also have double membranes and even have an organelle of
their own, the thylakoid. The thylakoids is responsible for converting
electromagnetic energy (light) into chemical energy.
2.2 Biological membranes form due to the hydrophobic
effect
Membranes exist, and thus cells and thus you yourself, due to the same
principle that makes oil and water mix poorly. Fatty, or hydrophobic, “water-
fearing” surfaces in an aqueous environment will end up interacting with
each other. This is called the hydrophobic effect, and it drives the formation
of biological membranes and the folding of a chain of amino acids into a
functional protein8. Phospholipids, the basic building blocks of a biological
membrane, have one hydrophobic and one hydrophilic, “water-loving” end
and will therefore form bilayers.
2.3 The hydrophobic effect is caused by water
The hydrophobic effect is in truth more of a hydrophilic effect. Due to the
high electronegativity of oxygen and the small size of the hydrogen atom,
water molecules are electric dipoles, with a partial negative charge on the
oxygen atom and a partial positive charge on each hydrogen atom. Water
molecules will therefore engage in hydrogen bonds, strong electrostatic
interactions (see paragraph 4.2), with each other and other polar molecules.
When a non-polar, hydrophobic entity is placed in water, it either falls out of
solution as it cannot interact electrostatically with the solvent, or the water
molecules form a cage-like structure around it, keeping it solubilized, see
figure 2. This structure is more ordered than the structure adopted by free
water molecules and is energetically less favorable. In an aqueous solution,
the total hydrophobic surface area will be minimized if the hydrophobic
entities present in the solution come together. Minimizing the hydrophobic
17
area minimizes the number of water molecules needed to encapsulate it,
which in turn stabilizes the system energetically9.
2.4 Basic structure of phospholipid membranes
Phospholipids in biological membranes are amphiphatic. They consist of a
hydrophobic hydrocarbon tail and a hydrophilic phosphate-containing
headgroup. As mentioned, the hydrophobic effect will cause them to form
bilayers, consisting of two sheets of lipids with their hydrophobic tails facing
each other, see figure 3. The hydrophilic headgroups shield the tails, forming
an interface between the hydrophobic core of the membrane and the aqueous
surroundings10
. There is no sharp border between the hydrophobic core and
the surrounding water as this interface region provides a zone of gradually
changing hydrophobicity, see figure 3. In 1992, Wiener and White
determined the structure of a bilayer of pure DOPC lipids11
. The
hydrophobic core was about 30 Å thick, while the interface region extended
about 15 Å on either side, see figure 3. The tails of the lipids can be of
different lengths and stiffnesses and the size and electrical charge of the
headgroup can differ. Steroidic lipids like cholesterol are very rigid and
confer stability9. The features and properties of a biological membrane,
thickness, fluidity, curvature, pressure, etc, will vary with the lipid
composition12
. It varies from organism to organism and between organelles
Figure 2. (A) A water molecule is dipolar with partial positive charges on the hydrogen atoms
(δ+) and a partial negative charge on the oxygen atom (δ-). The dashed lines represent the
nonbonding electron orbitals. (B) Each water molecule can form a maximum of four
hydrogen bonds. (C) A lipid in water. When confronted with a non-polar entity, like the
hydrophobic tail of a phospholipid, water molecules form a cage-like structure around it.
Water molecules not forming the “cage” have been omitted.
18 Chapter 2 • Biological membranes
in the same cell, even between leaflets of the same bilayer. It is worth to
mention that Archaea, often found in extreme habitats, employ lipids that
differ substantially from the bacterial and eukaryotic ones, even though the
hydrophilic part of the head groups can be the same13
.
Figure 3. (A) A ribbon representation of a phosphatidylcholine lipid with a C17- and a C14-
hydrocarbon tail. Cyan, carbon; red, oxygen; blue, nitrogen; yellow, phosphate. Hydrogens
are omitted for clarity. (B) A simulated phospholipid bilayer. Molecular groups colored as in
A. Without (left) and with (right) surrounding water molecules. (C) The structure of a DOPC-
bilayer along the membrane normal, shown as the probability of finding different chemical
groups at a given distance from the center of the hydrocarbon core. Adapted from White and
Wimley (1999)14.
2.5 Biological membranes consist of more than lipids
One of the earliest models of a cell membrane depicted it as an ocean of
lipids with proteins floating around in it15
. The truth has been shown to be
somewhat different16; 17
. In some membranes, the lipids act more as a
cohesive between proteins with up to 30 000 molecules of integrated
membrane proteins per µm2 membrane
18; 19
19
Chapter 3
Proteins in general
Out of 20 basic amino acids, a plethora of different structures can be built.
The human genome is at the moment estimated to contain 20,000 to 25,000
genes encoding proteins20
. Considering alternative splicing events and post-
translational modifications, the number of proteins with different chemical
composition is estimated to be more than fifty times higher21
.
3.1 Protein structure and folding in general
Proteins are heteropolymers of amino acids, with the amino acids linked in a
chain by peptide bonds. The amino acid side chains, or residues, protrude
from the chain and vary in structure, size and electric charge. For a protein to
be functional, its polypeptide chain needs to be properly folded into its
native conformation. Most of the information on how to reach the native fold
is present in the amino acid sequence itself, in the nature of the side chains
and the order in which they are linked22
. To quote Anfinsen from his Nobel
laureate lecture in 1972 on the folding of proteins: "The native conformation
is determined by the totality of interatomic interactions and hence by the
amino acid sequence, in a given environment." In an aqueous environment
like the cytoplasm, the hydrophobic effect will cause hydrophilic residues
and the polar peptide backbone to be hydrated by water molecules while
hydrophobic parts are hidden in the protein interior. In the hydrophobic core
of the membrane the situation is the opposite and the presence of hydrophilic
residues and the backbone will be energetically unfavorable.
Protein structure is often discussed in terms of different levels of order:
1. Primary structure, the sequence of amino acids.
2. Secondary structure, local folding of the chain. The most widely
occuring secondary structures of regular patterns are α–helices and
β-sheets, see figure 4.
3. Tertiary structure, the final folding of the chain, its spatial
arrangements of and interactions between elements of secondary
structure, see figure 4.
20 Chapter 3 • Proteins in general
4. Quaternary structure, interactions between and arrangement of two
or more folded chains, or subunits, into a larger functional protein
complex, see figure 5.
Local interactions form first and a polypeptide can follow a large number of
different folding paths to reach the most energetically favored structure.
There exist no more than one or possibly a few native conformations for any
given naturally occurring protein23
.
Figure 4. (A) A polypeptide of five residues, side chains denoted by R1-5. The polar nature is
shown for one of the peptide bonds (highlighted in grey). (B) An α –helix is stabilized by
hydrogen bonds between residues i, i+4. Each helical turn rises 5.4 Å. (C) A protein with a
mainly helical tertiary structure, its two β–sheets indicated by an arrow. Cartoon
representation. (D) The same protein from the same viewing angle, surface representation.
21
3.2 More on the quartenary structure of proteins,
protein-protein interactions
“A functional protein within the cell” most often refers to a complex of
interacting individual protein chains. The composition of protein complexes
can vary depending on what is needed for the moment in terms of cellular
architecture, regulation, metabolism, and signaling. Many proteins are part
of larger networks of protein-protein interactions in the cell. Some of the
proteins have high connectivity, interacting with a large number of different
partners. These “hub proteins” are often important for cell survival24
. Some
examples of protein complexes that are relevant for this thesis: (1) The Sec
translocon, which will be discussed to some length in chapter 5. The core of
this protein is a heterotrimer that can combine with different interaction
partners to translocate varying substrates across the membrane or inserting
them into it, see figure 5A. (2) The ribosome, the molecular machine
synthezising all proteins, consists of two subunits. In Archaea, the subunits
comprise in total about 50 relatively small protein chains and three RNA
chains, see figure 5B. (3) Chaperones are proteins involved in more transient
interactions, preventing misfolding events by binding to unfolded or
misfolded proteins until these can fold properly. However, the interaction
conveys neither information nor functionality to the interactants as a group
and the chaperone and its substrate are not considered forming a relevant
quarternary structure.
Figure 5. Examples of
protein complexes. Cartoon
representation, each subunit
is colored differently. (A)
The SecYEβ complex of the
Sec translocon. PDB file
1RH5. (B) The largest (50S)
of the two subunits of a
prokaryotic ribosome. The
orange bands represent
ribosomal RNA. PDB file
2X9S.
22 Chapter 3 • Proteins in general
3.3 Evolutionary evolved functional substructures, protein
domains
Structure gives function. A protein, even a monomeric one, that performs
several tasks like binding a ligand, hydrolyzing ATP, etc, usually uses a
distinct substructure for each task. More often than not, these substructures
are autonomously folding units, called domains, indicating modularity
among protein folds. Indeed, it has been shown that during evolution,
proteins often have evolved by means of deletion, duplication or addition of
domains on the genetic level25; 26
. It has been estimated that out of a total
number of at least 10 000 observed folds, 80% of all protein sequences will
fold into one of 400 common folds, emphasizing how protein domains have
been reused during evolution27
. In paper IV, protein interactions are partly
discussed in terms of interacting domains.
23
Chapter 4
Membrane proteins, what do they look like?
There are peripheral and integral membrane proteins. The peripheral ones
are loosely attached to lipids or proteins in the membrane via electrostatic or
van der Waals interactions, or covalently linked to a lipid anchor28
. The
integral ones traverse the membrane and experience the varying milieus of
the hydrophobic core, the interface region and the aqueous surroundings
outside of the bilayer. Integral
membrane proteins minimize
the cost of harboring the polar
polypeptide backbone within
the membrane by engaging its
polar groups in hydrogen
bonds. The two major
structural classes of membrane
proteins present two different
ways of doing this. (1) β-barrel
membrane proteins form
cylinders of amphiphatic β-
strands and (2) α-helical
membrane proteins span the
membrane with one or more α-
helices. See figure 6. The β-
barrels occur in the outer membrane of some bacteria and in the eukaryotic
organelles of bacterial origin, mitochondria and chloroplasts28
. Two to three
percent of the genes in Gram-positive bacteria have been estimated to
encode β-barrels29
. In comparison, about 25 % of the open reading frames in
most fully sequenced genomes have been estimated to encode α –helical
membrane proteins30
. From here on “membrane proteins” (MPs) refer to
integral α-helical membrane proteins.
Figure 6. The two major structural types of
membrane proteins, cartoon representation. (A)
Helical bundle. Bacteriorhodopsin, PDB code
1NTU. (B) β-barrel. Phosphoporin, PDB code
1PHO.
24 Chapter 4 • Membrane proteins, what do they look like?
4.1 Structure and topology of α-helical membrane proteins
In addition to the four levels of structure, membrane proteins can be
described by their topology, what segments that are transmembrane and
extramembrane, cytosolic or extracytosolic (lumenal, periplasmic, etc.)
Transmembrane helices are described according to their orientation, whether
their N-terminal is cytosolic (Nin) or extracytosolic (Nout). Extramembrane
segments, loops, are simply described as cytosolic or extracytosolic.
4.1.1 The primary structure of a transmembrane helix is
adapted to its surroundings
With respect to the different
milieus a transmembrane helix
traverses, it is not surprising that
there is a statistical difference in
distribution of the amino acids in
different parts of the helix, see
figure 710; 31; 32
. The majority of side
chains found within the core of the
membrane are hydrophobic.
However, as more 3D-structures
are obtained, many examples of
charged residues and sequences
causing irregular secondary
structure within the hydrophobic
core have been observed 33
.
Charged and polar residues are better tolerated at the helix termini due to
snorkeling
Charged or polar amino acids are energetically better tolerated towards the
termini 31; 34
. Their side chains will “snorkel”, orienting themselves so that
they approach the interfacial, aqueous regions 35; 36
. This allows them to pull
hydrating waters into the hydrocarbon part of the bilayer and create polar
microenvironments for themselves, as seen in simulations 37
. Obviously a
long side chain is advantageous for snorkeling 37; 38
, see figure 8. However,
not all polar or charged residues in transmembrane segments are found in
positions allowing snorkeling.
Figure 7. Statistical distribution of amino
acids in different positions in a TMH.
25
Figure 8. Snorkeling of side chains. Simulated model systems with the denoted amino acid
placed symmetrically with an offset of 5 positions from the center of the hydrocarbon core
(indicated by arrows). Note the bend induced by the shorter Asp side chains. Adapted from
Johansson and Lindahl (2006)37.
Tryptophans and tyrosines anchor themselves in the interface region
Tryptophan and tyrosine are especially favored in the interface region10; 39; 40;
41 and can virtually anchor a segment within the membrane
42. This rather
tight anchoring, employed to a higher degree by β-barrel proteins than by α–
helical MPs43
, seems to be due to both steric and electrochemical factors.
The large, flat and rigid ring structures of the Trp and Tyr side chains limit
access to the hydrocarbon core while the electric structure of the aromatic
rings favors interaction with the groups in the interface region. In addition,
both Trp and Tyr have one polar group each. Phenylalanine is also aromatic,
but entirely hydrophobic, and is not biased towards the interface region10; 40
.
4.1.2 Secondary structure in transmembrane segments can be
irregular
A coil is a segment where the peptide forms no regular secondary structure,
neither a helix nor a β-sheet, hence exposing its polar backbone, see figure 9.
Especially in channels and transporters, this feature occurs frequently and is
conserved and important for function44
. Coils are enriched in proline and
glycine residues. In TM helices, they especially confer a higher degree of
structural flexibility, creating swivels and hinges and also positioning side
chains properly for interactions45; 46
. Coils can be introduced into the lipid
bilayer as short kinks or longer breaks in the secondary structure of a
transmembrane helix, or as part of a reentrant loop or region (see the
following paragraph)10; 44
.
26 Chapter 4 • Membrane proteins, what do they look like?
4.1.3 Other structural elements of helical membrane proteins
Reentrant regions are segments
that penetrate the lipid bilayer
without traversing it, entering
and exiting on the same side, see
figure 9. They have been
estimated to occur in at least
10% of all multispanning
membrane proteins, mostly in
ion and water channels47; 48
. As
defined by Viklund et al in 2006,
they can be (1) helix-turn-helix
and are, if long enough,
sometimes hard to distinguish
from two transmembrane
helices, (2) shorter segments of coil-helix or helix-coil and (3) even shorter
segments of purely irregular coil structure47; 48
. Reentrant loops are enriched
in prolines and small residues, especially glycines and alanines48
.
Polar backbone groups can be made available for hydrogen bonding by
other means than forming a coil. The π-helix bulge, or α-aneurysm, occurs
when a hydrogen bond is formed between residues in positions n, n+5
instead of the regular pattern of n→ n+4. This frees one carbonyl group
(C=O) for polar interactions. A tighter bond pattern of n→ n+3 can also
occur and the segment is then called 310-helix49; 50
. Interface helices are
found in the interface region, running roughly parallel to the membrane
surface. They are amphiphatic and on average nine residues long as reported
in 2005 by Granseth et al 10
.
4.2 Forces and motifs implied in the folding of helical
membrane proteins
The native fold of a membrane protein is mostly stabilized by interactions
between its TM helices. The current understanding of the factors stabilizing
a native fold is that van der Waals forces and hydrogen bonds dominate,
aided by the occasional salt bridge and aromatic interaction. Also, the lateral
pressure in the lipid bilayer, as well as the thickness of the bilayer, affects
protein stability.
Figure 9. Example of a reentrant region and
coiled segments within the membrane. The
membrane borders are shown as red and blue
lines.
27
4.2.1 Intramolecular forces stabilizing the native fold
Van der Waals forces
It is still debated which the major factor that stabilizes membrane protein
folds is, but the total sum of the van der Waals interactions is a good
candidate 51
. Van der Waals forces occur between all atoms and molecules:
Non-polar molecules can become temporary dipoles if their total electron
density gets unequally distributed as the electrons move about. If close
enough, this dipole can induce a temporary dipole in the electron density of a
neighboring non-polar molecule, allowing electrostatic interaction. The
interaction gets stronger the closer the molecules are. The information on
how tight TM helices pack in the membrane is ambiguous 52; 53; 54; 55
, but the
residues in transmembrane regions do tend to be buried more often than
residues in soluble proteins or extramembrane regions56
, allowing a higher
number of van der Waals interactions if not necessarily stronger ones.
Hydrogen bonds
A hydrogen bond is the interaction between an atom with a non-bonded pair
of electrons (O, N, etc) and a hydrogen atom covalently bonded to a strongly
electronegative atom (N, F, O). The electronegative atom attracts the
electron density of the small hydrogen atom, decentralizing it and leaving
the hydrogen with a substantial positive partial charge57
. Carbon can also
take the place of the electronegative atom, resulting in a weaker hydrogen
bond. In membrane proteins, the TMH backbones can be involved in such
weak hydrogen bonds, CαH∙∙∙O 58; 59
. Even though the stabilizing effect of the
weaker bonds has been disputed 60; 61; 62
, it is likely to be substantial for
helices at close distances 60; 63
64
. Strong hydrogen bonds involving Asn, Asp,
Gln and Glu have been shown to drive helix oligomerization 65; 66; 67; 68; 69
.
The strength of any type of hydrogen bond depends on the distance, the
chemistry and relative arrangement of the involved atoms, and the nature of
the surrounding milieu 70; 71
. Even small structural changes can rather easily
break a hydrogen bond 62
.
Motifs allowing tighter packing of helices, thereby increasing stabilization
A number of motifs involving small side chains allow tight packing of TM
helices. The small residues are spaced in such a way that their side chains
end up on the same side of the helix, either i, i+4 or i, i+7, creating a surface
that allows close proximity. Statistics show that Glycine zippers like GxxxG,
(G, A, S)xxxGxxxG and GxxxGxxx(G, S, T) occur in more than 10 percent
of all known MP structures72. These motifs usually form right-handed
crossings of the helices. The motif GxxxG has been extensively studied 72; 73;
74; 75 and can readily, depending on sequence context, induce dimerization of
28 Chapter 4 • Membrane proteins, what do they look like?
TM helices76; 77
. However, the majority of helix-helix interaction interfaces
seem to be formed by small residues in a i, i+7 pattern 76
, resulting in left-
handed crossing at small angles and knobs-into-holes packing, where side
chains i and i+3 are interdigitated between four side chains of the other TMH 78
. Tightly packed helices are stabilized by stronger van der Waals
interactions due to closer proximity of the interacting helices. Many helix-
helix interfaces are also stabilized by hydrogen bonds, both “classical” bonds
and the weaker bonds involving the peptide backbone, Cα-H∙∙∙O 64; 79; 80
.
Hildebrand et al have shown that the residues participating in hydrogen
bonds are more conserved than other buried residues 64; 79; 80
, and that the
packing preferences differ between channels and transporters and other
membrane proteins, mostly because these classes of protein are enriched in
different amino acids. In channels and transporters, the helix-helix interfaces
are not as tightly packed. Also, hydrogen bonds occur more frequently and
are often found in proximity to water-filled cavities providing alternative
bonding partners, allowing weaker bonds suitable for the structural
rearrangements needed for functionality 64; 79; 80
.
Aromatic residues can drive helix dimerization
A recent study by Sal-Man et al showed that the motif aromatic-xx-aromatic
is overrepresented in bacteria and that the motifs (Q, W, Y)-xx-(Q, W, Y)
could drive self-oligomerization of TMHs in a model system. The
oligomerization effect was stronger if the aromatic residues were close to the
interface region (see paragraph 4.1.)81
. Another study showed that
tryptophans in various positions promote homo-oligomerization and ruled
out that it was due to anchoring effects 82
.
Ion pairing/salt bridges
Salt bridges are electrostatic interactions between ions of opposite charge.
Within the hydrophobic milieu of the lipid bilayer, the di-electric constant is
very low and the interaction of a salt bridge is therefore strong. As briefly
mentioned in paragraph 4.1.1, charged residues (protonated Lys, Arg, His
and deprotonated Asp, Glu) are not common in transmembrane protein
segments. However, intramembrane salt bridges have been observed and are
of both structural and functional importance for their proteins 83; 84; 85
.
4.2.2 Lipid-protein interactions affecting the folded protein
The lipid composition of the bilayer affects the conformation of membrane
proteins in more ways than one.
29
Lateral pressure in the membrane
Lipid molecules have different geometries, depending on the size of the head
group and how saturated their hydrocarbon tails are. Simply put, lipids
shaped as cylinders are bilayer-forming and lipids shaped like cones
introduce curvature 86
. If non-bilayer lipids are forced into the bilayer a stress
is introduced in the membrane. The stress is also affected by the charge
states of the lipid headgroup87
. The lateral pressure profile through the
membrane, or membrane curvature, differs with lipid composition, and
affects the folding and insertion of some proteins 88
.
Hydrophobic mismatch between membrane and protein
The lengths of the lipid hydrocarbon tails and the TM segments of
membrane proteins are not always matched. To compensate a mismatch,
long TM segments either tilt or distort via coils, or the surrounding lipid tails
extend. The tails of lipids surrounding a short TM segment compensate by
compression 89; 90; 91
. Mismatch has been shown to affect the functionality of
several proteins 92; 93; 94; 95; 96; 97; 98
, partly probably due to effects on helix
dimerization99
. Eukaryotic cells have also been suggested to utilize
hydrophobic mismatch to sort membrane proteins in the exocytic pathway 100; 101; 102; 103
.
31
Chapter 5
Biogenesis and bilayer integration of α-
helical membrane proteins
I have discussed what membrane proteins look like and how their final
structure is stabilized. But how does it all begin, how are they integrated into
the membrane? The vast majority of proteins are synthesized in the cytosol,
where the ribosomes translate mRNA codons into amino acids and add them
to the growing polypeptide chain. Soluble cytosolic proteins simply fold as
they emerge from the ribosome. For proteins meant for secretion or
integration into a lipid bilayer, the process is more complicated and there are
both co-translational and post-translational pathways for translocation across
or insertion into the membrane.
The most primary and well-studied system is the SRP-dependant Sec
pathway. Its major components are the signal recognition particle (SRP), the
signal recognition particle receptor (SR) and the core of the secretase (Sec)
translocon, which forms a pore through the membrane (reviewed in 14; 104
).
This system is present in all organisms studied so far105
.
After translation is initiated, there are two major steps in membrane
protein biogenesis, see figure 10:
1. Targeting to the Sec translocon. The SRP will recognize and bind a highly
hydrophobic stretch of the nascent polypeptide chain, the signal peptide,
when it emerges from the ribosome. In eukaryotes, but not in
prokaryotes, this arrests the chain elongation until the ribosome contacts
the translocon. SRP then binds to its receptor in the ER membrane
(eukaryotes) or in the plasma membrane (prokaryotes), which catalyses
the transfer of the ribosome-nascent chain-complex to the translocon106;
107.
2. Translocation/integration. The exit tunnel in the ribosome is aligned with
the translocon pore108
. When the signal peptide reaches the translocon it
either reorients, placing the N-terminal in the cytosol, giving the protein
an Nin-orientation, or it does not, resulting in an Nout-orientation. As
translation and chain elongation proceeds, the nascent chain passes
through the translocon channel. Segments that are of sufficient length and
hydrophobicity will enter into the lipid bilayer109
.
32 Chapter 5 • Biogenesis and bilayer integration of membrane proteins
Figure 10. SRP-dependant co-translational insertion of an ER membrane protein. Adapted
from Rapoport (2007)14.
5.1 The Sec translocon channel
The Sec core translocon is a heterotrimer in all organisms studied, two of its
subunits are highly conserved and essential while the third is not105
. It is
denoted Sec61αβγ in eukaryotes, SecYEG in eubacteria and SecYEβ in
Archaea. The major component is the pore protein, subunit α/Y. In 2004, a
leap in the study of membrane protein integration was made when van den
Berg et al presented the high-resolution three-dimensional structure of a
closed SecYEβ translocon channel110
, see figure 11. Some of the most
interesting features of the pore is (1) a proposed lateral gate, where the pore
opens like a clamshell towards the bilayer, exposing the translocating peptide
to the lipids and allowing it to partition between the aqueous channel and the
bilayer110; 111; 112; 113
. (2) A plug that closes the non-cytosolic end and prevents
translocation110; 114
. (3) A ring of hydrophobic residues in the middle of the
pore, shaping it into an hourglass and further sealing it so there can be no ion
leakage across the membrane. In the open state the ring most likely prevents
unspecific interactions between the channel and the translocating protein 112;
115; 116; 117. The active translocon has been observed to be a multimer of Sec
heterotrimers and other proteins, but the actual number of subunits and their
arrangement are controversial118; 119; 120
. During translocation/insertion, the
pore dimensions seem to adjust to the nascent protein chain121
. An elongating
hydrophobic segment is thought to bind to a specific site, wedging into the
33
gate and inducing structural changes that eventually will displace the plug
and open the gate 110; 122; 123; 124
.
Figure 11. The SecYEβ translocon pore .(A) Top view from the cytosolic side. (B) Side view.
SecE and Secβ are shown in black, SecY in grey. The lateral gate in SecY is shown in orange
and the pore plug and ring in red. PDB code 1RH5.
5.2 The versatile translocon
So, the Sec heterotrimer, or a multimer thereof, forms the primary translocon
channel. By combining with different proteins responsible for transfer of
peptides to the channel, chaperone function and movement through the pore,
the translocon translocates/integrates substrates of varying characteristics.
Other proteins implied in the SRP-dependant pathway
In bacteria, the YidC protein is needed for the co-translational integration of
many membrane proteins125; 126; 127; 128
. In eukaryotes, the monomeric TRAM
(translocation-associated membrane protein) appears to fulfill a similar
function129; 130; 131; 132
. Also, the tetrameric translocon-associated protein
complex, TRAPαβγδ 133
, has been shown to be essential for the binding of
some signal sequences to the eukaryotic translocon134
. (Note that although
the names are confusingly similar, TRAM and TRAP are two completely
separate proteins.) Cross-linking experiments indicate that these proteins
contact the nascent peptide as it emerges from the translocon, facilitating
integration into the bilayer128; 129; 135; 136
. In 2005, Ménétret et al captured the
structure of a mammalian translocon-ribosome complex at low resolution.
Their data suggests a complex of one ribosome, four Sec heterotrimers and
two TRAP molecules118
. Recently, the cytosolic protein SYD (Suppressor of
34 Chapter 5 • Biogenesis and bilayer integration of membrane proteins
SecY Dominance) was suggested to recognize misassembled SecY channels
in gram negative bacteria, acting as a quality control system137
.
Post-translational translocation
The Sec translocon is also involved in post-translational translocation of
proteins. After synthesis, aggregation is prevented by cytosolic chaperones
binding to the protein before it is transferred to the translocation machinery.
In eubacteria like E. coli, the translocating protein is pushed through the pore
in an ATP-demanding process by the cytosolic SecA protein instead of the
ribosome. A complex of SecD, SecF and YajC also participates in the
process138; 139
. SecA is also needed for the translocation of the extracellular
domains of several membrane proteins utilizing the co-translational
pathway140. In yeast, and most likely all eukaryotes, the translocating protein
instead moves through the pore by Brownian motion after the signal
sequence binds to the translocon. The luminal chaperone and ATPase BiP
binds the protein and prevents movement back into the cytosol. This process
also requires the additional transmembrane Sec62/63 complex.
Modifying proteins
Signal peptidase141
will cut off the signal peptide and release proteins for
secretion into the exoplasm. For MPs, the signal sequence is either cleaved
off or integrated into the membrane as a TMH. In eukaryotes, a modifying
enzyme attaching N-linked glycans to the protein is also found in the close
vicinity of the translocon (see section 7.3.1).
5.3 Other systems for insertion of membrane proteins
Even though most membrane proteins seem to be inserted via the SRP-
dependant Sec pathway, there are other insertion mechanisms. These are
found in the bacterial membranes and in the membranes of the eukaryotic
mitochondria, chloroplasts and peroxisomes. A very brief review of these
systems will be given here.
Mitochondria lack the Sec-translocon completely. Instead this dual-
membrane organelle is equipped with an elaborate system for import and
insertion of membrane proteins, involving six major protein complexes and a
number of chaperones142; 143
. Of these proteins, the Oxa1 protein has
homologues in bacteria, some Archaea and chloroplasts125; 144
. As mentioned,
the homologue in E.coli, YidC, assists the SecYEG translocon in insertion
of some proteins into the cell membrane125; 126; 128; 144
and has recently been
shown to act as a translocon on its own125; 145; 146
. An interesting case has
been reported where the N-terminal of a E.coli protein is inserted via the
35
YidC only pathway and the C-terminal subsequently is inserted via
SecYEG/YidC147
.
In peroxisomes, some membrane proteins are imported co-translationally
in a not yet completely elucidated process while others come in vesicles
budded off from the ER148; 149
. Chloroplasts have translocons of the inner and
outer membranes (TOC and TIC) as well as the twin arginine translocon
(Tat) that translocates folded proteins across the thylakoid membrane and
seems to be involved in the insertion of some integral membrane proteins150;
151; 152; 153. Tat is also found in Bacteria, Archaea and plant mitochondria
154.
From here on, “translocon” will refer to the Sec-translocon as engaged in the
SRP-dependant pathway.
37
Chapter 6
How is topology decided during biogenesis
and folding?
Deciphering the topology signals and folding of a membrane protein are
often described as separate processes in a simplified two-step model155; 156
. In
this model, topology is established first, as the helices are thought to insert
individually and to be stable on their own in the membrane. Thereafter the
helices are envisioned to interact with each other within the membrane and
form the final tertiary structure. Although this model may be true for some
proteins156
, it is too simplified for others. There are several examples on
helices of low hydrophobicity, marginally hydrophobic helices, which are
not inserting well on their own, but are transmembrane in the folded
protein44; 157; 158; 159; 160; 161
. During the folding of aquaporin1, the prospective
TMH2 and TMH4 are even extramembrane after passing the translocon and
are pulled into the membrane as TMH3 later flips 180 degrees, assisted by
the nascent C-terminal helix bundle120
. In addition, it is known that some TM
helices interact transiently with proteins associated with the pore upon
entering into the lipid bilayer (see section 5.2). Emerging evidence also show
that helices can stay in close proximity to the translocon until stable
interactions can be formed with subsequent helices162
and that some even can
be retained within the channel due to specific interactions163
.
Still, the three major factors affecting how a peptide chain is threaded
through the membrane seem to be hydrophobicity (will a segment partition
into the bilayer?), length of the hydrophobic segment (can it span the
membrane?) and the presence of charged residues in the loops (will a
segment be retained in the cytosol?). For some segments, the signals to adopt
a certain topology can be so strong that they force an intermediate segment
to become transmembrane, despite low hydrophobicity160
. Timing of the
elongation and insertion can also affect topology, as the proper segments
need to be available at the correct moment, and so on164
.
38 Chapter 6 • How is topology decided?
6.1 Charged residues in loops, the positive inside rule
Positively charged, basic residues are strong topologenic signals with a
preference for the cytosol. A simple way to predict the topology of a
membrane protein is to localize the most hydrophobic parts (putative TMHs)
and then summarize the number of positively charged residues in the loops
on either side of the membrane. The side sporting the highest number of
positively charged residues is likely to face the cytosol165
. This effect is
called the positive inside rule and has been shown to hold for most
organisms41; 165; 166; 167
. Acidic residues do not effect topology as strongly and
show no statistical preference for loops on either side of the membrane 41; 168
.
However, they seem to have a greater influence on topology in Sec-
independent translocation169
.
Effect of cytosolic basic residues on the insertion of TM segments
If placed downstream of and close to a transmembrane segment of Cin-
orientation, a single Arg or Lys can lower the apparent free energy of
insertion of that segment with ~0.5 kcal/mol 170
.The effect is additive, the
more residues of positive charge, the lower the free energy. Also, a stretch of
six lysines has been shown to affect insertion from a distance of up to ~25
residues away for a single-spanning protein and up to ~12 residues away for
a TMH in a multispanning protein, when expressed in an in vitro system170;
171. Histidines are not charged at physiological pH, but have a similar effect
if the pH is lowered168
.
Possible mechanisms underlying the positive inside rule
The exact mechanisms behind the positive inside rule are not yet fully
understood, and they may differ slightly between Archaea, Bacteria and
Eukarya. For example, the retaining effect of positive charges is more
pronounced in E.coli than in microsomes (miniature eukaryotic ER
membrane)172
, most likely due to the much stronger electrochemical
potential across the bacterial inner cell membrane. The ER membrane
potential seems to be non-existing. In E. coli, the dominance of positive
charges over negative charges as determinants of topology has been
proposed to be due to the lipid composition of the cell membrane. Several
E. coli proteins have been shown to be dependent on the presence of lipids
with a net zero charge to achieve the proper topology173; 174; 175
. It is thought
that these lipids dilute the otherwise strong negative charge of the lipid head
groups so that the acidic residues (aspartic and glutamic acid) stay
protonized and thus uncharged176
. Specific interactions with the translocon
have been reported to contribute to the orientation of the first TMH, the
signal sequence177; 178
. Also, electrostatic interactions between anionic
lipids and positively charged residues have been proposed to help retaining
positive charges in the cytosol179; 180; 181; 182
.
39
6.2 Hydrophobicity as recognized by the translocon
Traditionally, the hydro-
phobicity of an amino
acid has been
determined by its ability
to partition between water
and an organic solvent
(See 183
for benchmarking
of experimental and
statistical hydrophobicity
scales). A more recent
method includes the
energetically important
effect of the protein
backbone on insertion39;
184; 185. There is also a
biological hydrophobicity
scale available, the Hessa scale186
. Rather than looking at the hydrophobicity
of the individual amino acids or side chains, the Hessa scale gives
information on how each side chain will affect the recognition of a helix as
transmembrane by the translocon. See figure 12. ΔGapp ≤ 0 kcal/mol means
that the amino acid favors recognition by the translocon. Positional effects,
as discussed in paragraph 4.1.1, are also taken into consideration. For the
work presented in this thesis, the Hessa scale and tools for predicting the
change in free energy upon insertion based on this scale were employed.
6.3 Marginally hydrophobic transmembrane helices do
exist
A number of TM segments have been observed in experiments where they
did not insert well into the membrane without the presence of other TM
segments120; 159; 187; 188
. Also, as discussed in paper I, the primary sequences of
many TM helices seen in three-dimensional structures do not indicate them
to be transmembrane. For instance, (LacY) of E. coli189
and the glutamate
transporter homologue of P. horikoshii (GltPh)44; 190; 191
both contain several
helices of low hydrophobicity, see figure 13. We take a closer look at GltPh in
paper II, and LacY is examined in paper III
As with coils, many marginally hydrophobic helices contain functionally
important residues, some of them responsible for interactions with other
subunits. However, such interactions are not likely to form at the stage of
integration into the membrane.
Lundin et al showed recently that two Asn- or Asp-mediated hydrogen
Figure 12. The Hessa scale. Apparent change in free
energy (ΔGapp upon translocon-mediated insertion
into the ER membrane. Adapted from Hessa et al
(2007) [1].
40 Chapter 6 • How is topology decided?
bonds to a well-integrating TMH can improve the insertion of a marginally
hydrophobic one192
. In paper I, we investigate the effect of the sequence
context on the insertion of marginally hydrophobic helices. Ota et al
(1998)160
reported an example of well inserting segments with strong
topological signals forcing a less hydrophobic segment to insert into the
membrane, which sustains the findings we present in paper I. In paper II, we
present evidence indicating that some marginally hydrophobic TMHs are not
initially recognized as transmembrane and could be repositioned as a post-
translational event.
Figure 13. Marginally hydrophobic helices. (A) LacY, the lactose permease from Escherichia
coli. PDB code 2cfq. (B) GltPh, the glutamate transporter homologue from Pyrococcus
horikoshii. PDB code 2nwl. The TMHs are colored according to their predicted insertion
propensity, given as the apparent change in free energy upon integration into the membrane
(ΔG). A segment of ΔG≤0 has a reasonable insertion propensity, while a segment of ΔG≥0
does not. Prediction of hydrophobicity was carried out with the ΔG predictor, see section 7.2.
Positively charged residues (Lys, Arg) are marked with circles in light pink and negatively
charged residues (Asp, Glu) are marked with circles in cyan. Borders of the membrane are
indicated by red and blue lines.
6.4 Length of the hydrophobic segment
A transmembrane segment has to span the bilayer. A perfect α-helix raises
5.4 Å per turn, each turn employing 3.6 amino acids. To span the
hydrophobic part of the bilayer, which is about 30 Å wide, a segment of
41
about 20 residues is needed. However, it has been reported that helices of 12-
40 residues can be accommodated in biological membranes10; 38; 193; 194; 195
.
The short helices need to be highly hydrophobic31
and the membrane bilayer
seems to distort to harbor these segments The longer helices can either be
slightly distorted, tilted relative to the normal of the membrane196
or force
the lipids to stretch, introducing another kind of lipid mismatch than the
short segments90
. (See also section 4.2.2.)
6.5 Reentrant loops
The topological signals resulting in reentrant loops are not yet well
understood and may very well vary depending on the nature and length of
the segment. As we propose in paper II for the ClcA H+/Cl
− exchange
transporter of E. coli197
, long reentrant loops à helix-turn-helix are likely to
insert as hairpins and then reposition themselves relative to the membrane
during later folding events. Detailed biochemical studies from the laboratory
of Sakaguchi have presented examples of both co- and post-translational
integration of reentrant loops198; 199
.
6.6 Other factors
Helices interacting with each other before entering the lipid bilayer can
“hide” charged or polar residues in interhelical hydrogen bonds69
,
increasing their apparent hydrophobicity. Also intrahelical ionic bonds,
between residues on the same side of a helix, have been shown to improve
insertion 200
. Regardless of charges, just the plain presence of other
polypeptide entities can effect insertion in a favorable way158; 162; 201
. This has
been explained by the greater capacity of polypeptides to retain hydration
water201
. Another factor affecting insertion may be when the α–helical
structure is formed. It has been shown that α –helices can form already in the
ribosomal exit tunnel202
. However, segments of low helix-forming propensity
may not become helical until a very late state during the integration into the
lipid bilayer 203
.
43
Chapter 7
Structure analysis
The function of a protein depends on its structure and folding. The three-
dimensional structures of proteins are thus of uttermost interest. For
example, once transmembrane protein segments were thought to be perfect
α-helices, long enough to span the lipid bilayer. As more 3D-structures of
MPs have been obtained, it has become clear that TM elements of higher
complexity are commonly occurring, including the previously described
coils and marginally hydrophobic TMHs. As discussed earlier, these
elements are generally important for the function of the protein. This
knowledge allows us to better understand the mechanisms behind protein
functionality and the effect of the primary sequence on folding. For the final
structure of membrane proteins, topology is a vital part. There are three ways
to attain information on topology: (1) Solve the three-dimensional structure.
This is especially challenging for membrane proteins, but gives detailed
information on all aspects of the structure, not only the topology. (2)
Investigate the topology by biochemical means (see section 7.3). (3) Predict
the topology of a protein of interest based on previous knowledge. A number
of in silico topology predictors have been developed as the collective dataset
has grown during the last decades30; 204; 205; 206; 207; 208
. However, so far there
are no tools integrating predictions of topology, coils, helix packing8; 209
,
surface accessibility, and so on, into a detailed prediction of membrane
protein structure. Many more analyses of membrane protein structures are
needed.
7.1 Obtaining three-dimensional structures
The most commonly used techniques for solving three-dimensional
structures are X-ray crystallography210; 211; 212
and nuclear magnetic
resonance (NMR) 213; 214; 215
, both allowing resolution down to the atomic
level (about 1.5 Å or lower). Electron microscopy (EM) gives images of
lower resolution, revealing the general shape of a protein. Due to their poor
solubility and dependence on the lipid bilayer for proper structure,
membrane proteins are not easy targets. However, the techniques for
purification and preparation of the targets, as well as the visualization
techniques themselves, are constantly under development, and the number of
solved structures of membrane proteins has increased steadily since the first
44 Chapter 7 • Structure analysis
high-resolution structure was solved in 1985216
. Nonetheless, the number of
solved structures for soluble proteins is by far higher. In July 2010, there
were 61,860 structures of proteins in the protein data bank (PDB)217
, 4920 of
these turned up in a search on the word “membrane” and 2205 in a search on
the word “transmembrane”. The orientation of membrane proteins (OPM)
database218
includes “all unique experimental structures of transmembrane
proteins and some peripheral proteins and membrane-active peptides” found
in PDB. In July 2010, OPM included 925 structures.
7.2 In silico investigations
A number of predictors and simulation methods are available. I will only
address the in silico tools that I’ve used in my research.
Predicting insertion propensity
To predict insertion propensity of a segment, either computational
simulations can be performed or knowledge gathered experimentally can be
applied.
As mentioned earlier, the study by Hessa et al31
gathered information on
how hydrophobicity, segment length and position of each amino acid in the
segment affect the insertion propensity. The authors incorporated their data
into equation 1 and later on in the “ΔG predictor” available at
http://dgpred.cbr.su.se/, a tool for calculating the ΔG for a specific segment
or for finding potential TM segments within a longer sequence.
Equation 1. The first term sums the individual, position-dependant (i) contributions for each
amino acid (aa) in the sequence. The second term takes the helical dipole moment into
consideration. The last term was fitted and b1, b2, and b3 were optimized to account for effects
due to length (l), based on the data from the study of Hessa et al. See 31 for details.
45
A Monte Carlo-based simulation can give information on both insertion and
peptide interactions
Monte Carlo methods are especially useful in studying systems with a large
number of coupled degrees of freedom, such as a system containing peptides
and a membrane. In a Monte Carlo-based simulation, a number of
computational steps are carried out, each step performing random changes of
the parameters of interest (here the conformation and location of the
polypeptide(s)). The change resulting in the most stable system, the system
of the lowest all-atom potential energy, is selected and the process is iterated
a set number of times. To appreciate the propensity of a polypeptide to insert
into the membrane, several runs are performed, starting with the peptide(s)
in different (random) positions. The fraction of obtained low-energy states
where the peptide(s) end up traversing the membrane is then regarded as the
insertion propensity. If more than one peptide is included, possible
interactions between the peptides can also be evaluated219; 220
.
Prediction of protein topology
The earliest predictors of protein topology were based on (non-biological)
hydrophobicity plots221
, but performed poorly. This is perhaps not so
surprising considering the issues discussed in chapter six and section 5.2.
Later methods were mostly based on statistics, using experimentally derived
datasets to train computational models to recognize TMHs, interface regions,
etc30; 48; 222; 223
. When needed in our work, we used Scampi, a method
evaluating a primary protein sequence without previous training, employing
the biological Hessa hydrophobicity scale and the positive inside rule in a
simple model204
.
7.3 Determining membrane protein topology
experimentally
How can the topology of a membrane protein be determined? One way is to
utilize a modification that can occur in only one of the compartments
separated by the membrane, like glycosylation224
or cysteine labeling173; 225
.
By gradually introducing acceptor sites for this modification in different
positions and analyzing if the modification comes about or not, the topology
can be deciphered. Reporter proteins that will be active only on one side of
the membrane is a variation on the same theme, but can in general only give
information on the location of the protein termini. Another approach is
“membrane shaving”, where all extramembrane protein loops are cut into
pieces by a peptidase226; 227
. The individual peptides in the resulting mix are
then identified using mass spectrometry, which might require specific
modification of the peptides228
, depending on the protease used.
46 Chapter 7 • Structure analysis
7.3.1 N-linked glycosylation as topology reporter and
molecular ruler
The enzyme Oligosaccharyl Transferase is found on the lumenal side of the
ER membrane. It recognizes the peptide sequence X1NX2(S/T)X3 and will
attach one sugar moiety to the asparagine-residue (N). The process is co-
translational229
. The attached oligosaccharide increases the molecular mass
of the protein with approximately 2 kDa230
, and glycosylated and
unglycosylated forms of the protein can easily be separated by SDS-PAGE.
In the recognition sequence, X1-3 cannot be Pro. NXT is preferred over NXS,
as the recognition of the latter sequon is more affected by the residues in
positions X1-3231; 232
.
The active site of the transferase is located at a fixed distance from the
membrane. Half-maximal glycosylation will be achieved if the acceptor site
is (a) 14 residues N-terminal of a transmembrane segment or (b) 10 residues
C-terminal of the most C-terminal TM segment. The position of a TM
segment can thus be determined by finding the “minimal glycosylation
distance” (MGD), the position where the acceptor-asparagine is glycosylated
at half-maximal efficiency233; 234; 235
.
7.4 Determining the propensity to integrate into the
membrane
The method used in this thesis to determine insertion propensity of a protein
segment employs N-linked glycosylation. A model protein carrying the
segment of interest and two glycosylation recognition sites is expressed in an
in vitro system. Unglycosylated, singly and doubly glycosylated protein
species are separated by SDS-PAGE and the signal intensities from the
bands on the gel are quantified. The insertion propensity is reported as the
fraction of inserted protein species and is determined by the glycosylation
status of the protein construct. The apparent change in Gibbs free energy
upon insertion (ΔGapp) can be calculated for this system, using a simplified
model where the translocon-mediated integration, the partioning of the
protein segment between the aqueous pore of the translocon and the
hydrophobic bilayer, is regarded purely as an equilibrium process31
. See
equations 2 and 3.
Where Keq is the equilibrium constant, finserted is the fraction of protein
species where the segment was inserted into the membrane, R is the gas
47
constant and T is the absolute temperature.
7.4.1 Model proteins, LepI and LepII
As mentioned earlier, a highly hydrophobic signal peptide is required for a
nascent polypeptide to enter the translocon-mediated insertion pathway. To
be able to determine how well a less hydrophobic segment insert into the
membrane, either the entire native protein is used or the segment of interest
can be isolated by fusing it to a model protein. In this work, the model
proteins were two modified versions of the well-studied E. coli membrane
protein Leader peptidase B (Uniprot P00803), called LepI and LepII236
.
Leader peptidase B is a protein of 324 amino acid residues and two TMHs.
Both its short N-terminus and the large C-terminal domain face the lumen
when expressed in an in vitro system of rabbit reticulocyte lysate172; 234; 237
. In
LepI, the fused segment replaces a part of the C-terminal loop. In LepII it
replaces the second TMH. The N-terminal of the segment can thus be
positioned either in the lumen (Nout, in LepI) or in the cytosol (Nin, in LepII)
depending on which model protein that is used, see figure 14. Both model
proteins carry two glycosylation recognition sites, placed on appropriate
distances from the lipid bilayer in a manner such that the location of the
fused segment (inserted/not inserted) is mirrored by the glycosylation status
(see figure 14). An apparently systematic, yet unexplained, difference of
about 1 kcal/mol in free energy upon insertion between the two models has
been reported. That is, any segment is likely to insert more efficiently when
introduced into LepII compared to LepI236
.
To study segments containing several transmembrane parts, extra
glycosylation sites can be introduced by altering the segment sequence.
Figure 14. Lep model proteins. Grey rods (TMH1, TMH2), native transmembrane helices of
LepB. Black rod, segment to be investigated. G1, G2, G3, positions for glycosylation
recognition sites. The sites recieve a glycan (Y) if translocated across the membrane to the
lumenal side. A) Lep I. One glycosylation indicates insertion of the segment of interest. (B)
LepII. Two glycosylations indicate integration of the segment of interest into the membrane.
Note that the N-terminal af LepII is longer than for LepI, in order to ensure glycosylation of
48 Chapter 7 • Structure analysis
site G3.( (C) Representative picture of a LepI-construct expressed in the presence (+) or
absence (-) of rough microsomes (RM). Unglycosylated (○), singly (●) and doubly (●●)
glycosylated forms are indicated. Mr, molecular ruler, protein sizes in kDa.
7.4.2 In vitro protein expression
Intact cells are not needed for protein expression. Cell lysates containing all
the necessary molecules are enough and facilitates labeling and purification.
During the last four decades a number of in vitro systems have been made
commercially available, prokaryotic as well as eukaryotic238
. They all
contain a transcription/translation system as well as the signal peptide
receptor system. When supplied with a polymerase of choice, some of these
systems can conveniently transcribe and translate a protein directly from a
DNA plasmid or even a PCR fragment under the control of the appropriate
promoter sequence. To facilitate a eukaryotic ribosome binding to the
mRNA and ensuring a high rate of translation, it is important to include the
Kozak consensus sequence239
upstream of the gene to be expressed.
In a lysate in vitro system, many proteins are already present. To facilitate
visualization of the newly synthesized proteins only, expression can be
carried out using radioactive amino acids, followed by proper visualization
methods. Radioactivity also facilitates quantification of the expressed
proteins.
To study the biogenesis of membrane proteins, the system is
supplemented with rough microsomes, small vesicular fragments of rough
ER.
7.5 Determining quartenary protein structure, the
composition of protein complexes
To fully understand the biological system that is a cell, a map of its protein-
protein interactions is needed. The last decade or so, a number of techniques
for deciphering how and when the proteins in a cell interact have been
developed. The major categories are complementation assays, affinity
purification in combination with mass spectrometry, and single molecule
techniques, as extensively reviewed by24; 240; 241; 242; 243
. Also, blue native
PAGE and clear native PAGE are two most useful techniques for
determining the composition of protein complexes244
245
. The latter three
methods are not suited for high-throughput work. During the latest decade,
several large scale high-throughput efforts employing complementation
assays and affinity purification have been carried out to map the protein
interactome, the set of all protein-protein interactions in a cell246; 247; 248; 249;
250; 251. Most were carried out in the yeast Saccharomyces cerevisiae, a
unicellular eukaryotic organism of about 6000 gene-encoded proteins252
. In
49
paper IV, we present a comparison between datasets obtained with common
high throughput techniques and a collection of individual biochemical
studies.
Methods for elucidating protein interactions
In complementation assays, the direct pairwise interaction between two
proteins is investigated. The proteins are recombinantly fused to two halves
of a reporter protein and if they interact, the two halves will reconstitute the
functional reporter protein. In yeast, due to the possibility of mating, this
allows high-throughput screening. The original method is called yeast two
hybrid (Y2H)253
, where the reconstituted protein is a transcription factor that
initiates the synthesis of the actual reporter protein. This method does not
work well for membrane proteins, which cannot enter the nucleus. However,
there are modified versions where the reporter protein is fused to protein
fragments that upon complementation will cause the reporter to be released
from the protein complex243; 254
. There is also a fluorescent complementation
method, BiFC255
and fluorescent methods based on fluorescent/biolumi-
nescent resonance energy transfer240
.
In affinity purification, a protein of interest, the bait, is recombinantly
tagged with a protein segment with high affinity for a ligand. After in vivo
expression, the tag allows purification of the bait and, if the proper
purification conditions are chosen, its interaction partner(s). This method
allows detection of protein complexes, even subunits that do not interact
directly with the bait. However, the interactions have to be strong to survive
the purification procedure. In yeast, the most commonly used tag consists of
an IgG-binding peptide separated from a calmodulin binding peptide by a
recognition site for a protease. This double tag allows purification in two
steps, minimizing the number of false interaction partners. The procedure is
often referred to as TAP, tandem affinity purification246; 247; 248; 256; 257
.
51
Chapter 8
Summary of the papers
8.1 Paper I - Membrane insertion of marginally
hydrophobic transmembrane helices depends on
sequence context
In this paper we studied helices that are not predicted to be sufficiently
hydrophobic to be recognized by the translocon, but still are transmembrane
in the obtained three-dimensional structure of the protein. We searched for
transmembrane segments that were marginally hydrophobic, with a predicted
change in free energy upon insertion of less than or equal to 1.4 kcal/mol,
corresponding to an insertion efficiency of 8% in our experimental model
system. To allow for some flexibility regarding correct placement with
regard to the membrane, five upstream and five downstream residues were
added to the segment reported to be transmembrane. The subsegment
predicted to be of the highest hydrophobicity within this expanded sequence,
but still marginally hydrophobic, was then considered in the experimental
investigations. Out of 819 transmembrane helices from known 3D-structures
of integral membrane proteins, 136 TMHs (or 17 %) were predicted to be of
such low hydrophobicity.
We screened 16 of the segments predicted to be marginally hydrophobic
and found that most of them were indeed not inserting efficiently when
isolated from their native protein. When we included the flanking segments
up to the neighboring helices or at maximum 20 residues, we found several
examples where the insertion improved, of which most could be explained
by the “positive inside”-rule. For five of the marginally hydrophobic helices
we also included well-inserting flanking segments consisting of either the N-
terminal neighboring helix, the C-terminal neighboring helix, or both these
helices. This improved the insertion of three of the five helices. Of the other
two, one is known not to be inserted by the translocon, but rather via a post-
translational structural rearrangement, and the other one has not been
reported before. Statistical analysis of a homology-reduced version of the
dataset, containing 388 TMHs from 102 proteins, showed that the number of
marginally hydrophobic helices increases with the total number of TMHs in
the protein, that they are less common as the first, N-terminal TMH, and that
they constitute a higher fraction of the helices in channels and transporters
than in other proteins. We could also see that marginally hydrophobic TMHs
52 Chapter 8 • Summary of the papers
are more buried (less exposed to the lipid bilayer) than other TM helices.
However, buried helices were mostly observed in structures of protein
complexes, and were usually buried by other protein chains rather than via
intramolecular contacts, thus not making it likely to affect the initial
integration of the TMH into the membrane.
8.2 Paper II - Repositioning of transmembrane alpha-
helices during membrane protein folding
The glutamate transporter homologue from P. horikoshii, GltPh, has a
complicated three-dimensional structure with several marginally
hydrophobic transmembrane helices, some disrupted by coils within the lipid
core of the membrane. In this paper we showed that some of these segments
have overlapping, more hydrophobic regions that are more efficiently
recognized by the translocon. We could also establish that at least one of the
overlapping segments is initially integrated into the membrane rather than
the segment seen in the structure. In a non-redundant dataset of membrane
proteins of known three-dimensional structures, a substantial number of
transmembrane helices were predicted to be poorly recognized by the
translocon, but to have overlapping, better recognized segments. We
concluded that post-translational repositioning of TM helices in relation to
the membrane may not be uncommon during the folding of multispanning
membrane proteins.
8.3 Paper III - Insertion Properties of Marginally
Hydrophobic Helices in the LacY Lactose Permease
Transporter
The E. coli lactose permease LacY contains several marginally hydrophobic
TM helices. We investigated whether these helices, especially those in the C-
terminal end of the protein, initially integrate into the lipid bilayer as helical
hairpins. We combined molecular simulations based on the Monte Carlo-
method and the same in vitro, glycosylation based experimental setup as in
papers I and II. Surprisingly enough, our results indicate that the marginally
hydrophobic TM helices of the C-terminal half of LacY do not integrate into
the membrane as helical hairpins. This could imply that interactions need to
be established between several of the TMHs before they are collectively
released from the translocon machinery into the lipid bilayer.
.
53
8.4 Paper IV - Quantitative assessment of the structural
bias in protein-protein interaction assays
The interchange of information and matter in a cell depends on interactions
between proteins. The last decade, a number of large-scale investigations of
the yeast interactome have been carried out, employing tandem affinity
purifications (TAP) coupled to mass-spectrometry, or yeast-two-hybrid
(Y2H) techniques. However, the overlap between the resulting datasets is
surprisingly small, indicating systematic differences in detection between the
experimental setups. In order to characterize the biases for each system, we
have analyzed two available TAP-datasets (Krogan248
, Gavin246
) and Y2H
data as collected from the IntAct database and compared them to manually
curated data collected from small-scale experiments.
Note that Y2H identifies direct pairwise interactions and is known to give
a high degree of false positives, while TAP, due to the purification steps,
identifies protein complexes with relatively strong interactions. For TAP, the
results to a higher degree depend on variations in handling, purification
protocol and choice of mass spectrometry (MS) method for identifying the
interactants. Neither method is suited for membrane proteins.
Previous studies have suggested disordered regions and repeating
domains to be important for protein-protein interactions. Certain non-
repeating protein domains have also been proposed to be especially
interactive. In addition, protein size and abundance are, even though not
completely orthogonal to the other properties, also of interest when
analyzing protein-protein interactions.
Krogan reported more interactants per bait than did Gavin, which could be
due to differences in handling of samples or that in addition to the MALDI-
TOF mass spectrometry method used by Gavin, Krogan also used the more
sensitive LC-MS/MS approach. By comparing with the manually curated
datasets and data on co-localization and biological process annotation, we
conclude that the quality of the Gavin dataset is slightly higher. To facilitate
comparison, we reduced the Krogan dataset to a size similar to Gavin,
whereby a similar quality was achieved.
We found that (1) abundance was the most important factor in the TAP
experiments, not surprising as abundant proteins are easier to detect with
MS. (2) Repeating domains were overrepresented among interacting proteins
in all the datasets. (3) Disordered regions were overrepresented in the Y2H
and Krogan datasets, but not in the Gavin dataset. This could be due to
differences in detection and handling of the samples between the two TAP
setups, or indicate that disordered regions not only are important for
transient interactions, but also for interactions between proteins of lower
abundance. (4) Concerning the highly interactive domains, the ten most
promiscuous ones seem to be involved in nearly half of all interactions.
We conclude that longer proteins with promiscuous domains, disordered
54 Chapter 8 • Summary of the papers
regions and repeat domains are prone to interact frequently. However, before
deeper analysis of these properties can be carried out, the effect of protein
abundance on detection of protein interactions needs to be better understood.
Also, to ensure interaction assignments of high quality, the analysis should
be based on data stemming from a combination of different experimental
methods.
55
8.5 Final thoughts
Not too long ago, transmembrane helices were regarded as simple anchors
made up of hydrophobic residues. Then biochemical studies indicated that it
may not be that simple. Now, with more solved three-dimensional structures,
pictures of proteins frozen in one of their conformations, we see more and
more anomalies. So many in fact, that they cannot be considered anomalies
anymore, but rather proof that we yet do not fully understand the process by
which proteins integrate into the membrane and find their functional fold.
However, the field is in steady progress.
In paper I, we searched the known structures for segments of low
hydrophobicity that still are transmembrane. We found that almost one fifth
of the helices available today are predicted not to integrate well into the
membrane, and that of the ones we investigated experimentally, most indeed
did not. This raises the interesting question as to how they are inserted.
Literature, including paper II, has some suggestions: post-translational
rearrangement, strong topological signals in loops or the previous and
subsequent helices, high concentration of polypeptides in the biological
membrane, direct interactions with neighboring helices (ion bonds, hydrogen
bonds, tight packing), etc. The last example would require the neighboring
helices to be retained in close proximity to the lateral gate, or even inside the
translocon pore. Some of the examples presented in paper I seem to be
explained by positively charged residues in the flanking loops, complying
with the strong topological “positive inside” rule. For others, the flanking
helices improved insertion (three out of five examples). For our examples
where local sequence context did not improve insertion, one TMH is known
to become integrated after a major structural rearrangement requiring the
presence of the entire protein chain, and the other one has not been reported
before. It will be interesting to follow further investigations of the exact
procedure behind the integration of these TMHs.
My belief is that the integration into the bilayer of marginally
hydrophobic helices of co-translationally integrated proteins can take many
routes and that we are now in the beginning of understanding how this
works. All the mechanisms/factors mentioned above are likely to be
involved, and it will be a challenge to decipher the “rules” for what
mechanism/factor that is decisive for each segment. The translocon
accessory proteins may also play a greater role than we presently are aware
of. With the much different mechanisms for post-translational translocation
(not integration) of proteins in eukaryotes (chaperones) and bacteria
(chaperones, SecA) in mind, it seems plausible that the mechanisms for co-
translational integration into the membrane differ at least somewhat between
different organisms, even if the protein sequence requirements seem to be
surprisingly similar. The core of the translocon, the pore, is conserved
throughout the three kingdoms of life. However, the variation in accessory
56 Chapter 8 • Summary of the papers
proteins seems to be greater. To figure out the details of the
translocation/integration processes, more light needs to be shed on the
proteins assisting the translocon pore and how they interact with the
translocating/nascent protein chain, especially the marginally hydrophobic
segments. Post-translational rearrangements can also be expected to play a
substantial part in the integration of the latter kind of segment. The findings
that we present in paper II indicate that many segments of low
hydrophobicity could become transmembrane after the initial insertion of an
overlapping, more hydrophobic segment. It will be exciting to see if other
examples of repositioned TMHs will be presented, and also to see if the
repositioning process involves any chaperones, yet unidentified structural
signals, or just a series of spontaneous re-formations of molecular bonds due
to known factors.
One thing that I’ve learnt to appreciate during my work is the number of
databases collecting available data and presenting it in an apprehensible
form. However, when using these databases, one must always be aware of
the features of the dataset. For instance, you may have access to 100
proteins, but if all of them are homologues, you cannot really draw any
conclusions that are valid for proteins in general. For instance, we always
work with homology-reduced datasets for statistics on protein structures and
sequences. Also, what is the quality of the data, does one need to filter it
somehow? What was the method used to gain the information, is it biased
and if so, how? In paper IV we analyzed the data on protein-protein
interactions from two similarly executed large-scale investigations together
with results gained using an altogether different technique. We found that the
methods indeed were biased and that our knowledge on this was highly
useful for the interpretation of the results. We also found, as many others
have stated before, that conclusions drawn from a combination of data,
preferably stemming from orthogonal methods, are the most reliable ones.
57
Acknowledgements
No man is an island, John Donne wrote almost four hundred years ago. We
all need others to thrive. A large number of people helped me thrive during
my years in the Arrhenius Laboratories. You know who you are and there are
many of you, but the following people deserve a special thank you:
My supervisors Arne Elofsson and Susana Cristobal, for all your ideas and
your support and dedication during these years and for the relaxed and
permissive environment you create. All these things mean a lot to me, and I
have enjoyed being a family-run venture.
Ingmarie Nilsson and Gunnar von Heijne, for your great expertise and ideas,
and for always having time to listen to problems and come with suggestions.
It has been great collaborating with you.
Erik Lindahl, for always being so enthusiastic and for sharing good advice
on both science and life in general. I always enjoy talking to you.
Anni Kauko and Yana Vereshchaga, for stimulating collaborations. Anni, you
do have an expert eye for three-dimensional structures.
Mirjam Lerch-Bader and Karl Enquist for teaching me the tricks of the trade
of rough microsomes and in vitro glycosylation. I could not have done any
of it without you.
Karin Öjemalm, for all of those clones and assays. We finally did it! It was
great working with you.
Kristoffer Illergård and Aron Hennerdal, for five years of studying, hanging
out and playing Settlers, followed by five years of PhD studies, hanging out
and publishing a paper. Who would have guessed? It will be weird working
without you. Kristoffer also for the “rolled out” structures of LacY and GltPh
in figure 14.
Åsa Björklund, for being a mentor as well as a colleague for the new girl
five years ago. We may not have a paper to show for it, but working with
you in the lab was still a rewarding experience.
Acknowledgements 58
The lab rat gang in the crowded office. Itxaso Apraiz, for being a great
colleague and teaching me a lot about myself. Anya Amelina, for being the
sweet person that you are and for being with me since the very beginning.
Minttu Virkki, I’m so happy that Arne and Susana agreed with me that we
needed some extra hands in the lab that summer. Proteins, promenades and
parties, it has all been great fun! Maria Bendz, our very first post-doc, thank
you for having great ideas both in the lab and for whatever festivity that is
coming up and for going upstairs with me to buy coffee. Narges Bayat, the
nicest daffodil I have ever met, for teaching me that people from Iran eat
stew. Daniel Nilsson, for arguing for positive thinking and making me do it.
Gabriella Danielsson, for your awesome recipes and for all your help.
Sara Light, Niklas Barkå and Stefan Nordlund for proofreading this thesis.
Stefan also for being a rock to lean on for all us PhD Students at DBB.
Christine Sophie Schwaiger, for the pymol pictures of water and lipids used
in figures 2 and 3.
Hanna Eriksson, for all those hours discussing biochemistry in preparation
for the Big Exam. What would I have done without you?
All not previously mentioned friends, co-workers, administrators and
technical staff at the department. Thank you for making my life easier, both
in the scientific field and outside. I could not have wished for better
company at lunch breaks, cake clubs, three o’clock “fika”, late evenings at
the department, after work events, DBB pubs, conferences, dinners and so
on.
Josefin Illergård, Sandra Siljeström and Cecilia Århammar, non-DBB PhD
Students and dear friends, for lending listening ears even to work-related
babble.
My family, both original and extended. You are with me in everything I do
and I am lucky to have you in my life. Especially my parents, always
encouraging me to pursue what I want and reminding me to slow down from
time to time. Your love is my solid foundation.
Jonas, because even after having to deal with me while I wrote this thesis,
you still want to marry me. Where you are, that is home for me.
59
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