Protein are polymers of -amino acidsThe amino acids used to
make proteins are2-aminocarboxylic acids.The (alpha) carbon is the
carbon to which afunctional group is attached.
Properties of amino acids:structure and chemical
functionalitychiralityacid-base propertiescapacity to
polymerize
Proteins in the Diet9 of the 20 amino acids must be obtained
from thediet. These are referred to as the essential aminoacids.
Histidine Isoleucine Leucine Lysine Methionine Phenylalanine
Threonine Tryptophan Valine Proteins are also the major source of
nitrogen in thediet
Properties of amino acids:Aliphatic chains: Gly, Ala, Val, Leu,
and Ile HydrophibicityHydroxyl or sulfur side chains: Ser, Thr,
Cys, MetAromatic: Phe, Trp, TyrBasic: His, Lys, ArgAcidic and their
amides: Asp, Asn, Glu, Gln
Amino acids Classified according to their capacity to interact
with water 4 classes: NON POLAR, POLAR, ACIDIC AND BASIC Non polar
amino acids contain hydrocarbon R groups R groups do not have (+)
or (-) charges and interact poorly with water 2 types of
hydrocarbon chains: aliphatic and aromatic
Non-Polar Side Chains: Side chains which have pure hydrocarbon
alkyl groups (alkane branches) or aromatic (benzene rings) are
non-polar. Examples include valine, alanine, leucine, isoleucine,
phenylalanine. The number of alkyl groups also influences the
polarity. The more alkyl groups present, the more non-polar the
amino acid will be. This effect makes valine more non-polar than
alanine; leucine is more non-polar than valine.
Polar Side Chains:Side chains which have various functional
groups such asacids, amides, alcohols, and amines will impart a
more polarcharacter to the amino acid.The ranking of polarity will
depend on the relative ranking ofpolarity for various functional
groupsIn addition, the number of carbon-hydrogens in the alkane
oraromatic portion of the side chain should be considered alongwith
the functional group.
>Example: Aspartic acid is more polar than serine because
anacid functional group is more polar than an alcohol group.
>Example: Serine is more polar than tyrosine, since
tyrosinehas the hydrocarbon benzene ring.
Acid - Base Properties of Amino Acids: Acidic Side Chains: If
the side chain contains an acid functional group, the whole amino
acid produces an acidic solution. Normally, an amino acid produces
a nearly neutral solution since the acid group and the basic amine
group on the root amino acid neutralize each other in the
zwitterion. If the amino acid structure contains two acid groups
and one amine group, there is a net acid producing effect. The two
acidic amino acids are aspartic and glutamic
Basic Side Chains: If the side chain contains an amine
functional group, the amino acid produces a basic solution because
the extra amine group is not neutralized by the acid group.Amino
acids which have basic side chains include:lysine, arginine, and
histidine.
Hydrophobic Amino Acids (aliphatic)
Hydrophobic Amino Acids (aromatic) all very hydrophobic Some
contain aromatic group Absorb UV at 280 nm
Sulfur Containing Amino AcidsMethionine (Met, M) startamino
acid, very hydrophobicCysteine (Cys,C) sulfur inform of
sulfhydroyl, importantin disulfide linkages, weak acid,can form
hydrogen bonds.
Charged Amino Acids Asp and Glu are acidic amino acids Contain
carboxyl groups Negatively charged at physiological pH, present as
conjugatebases Hydrophillic nitrogenous bases Carboxyl groups
function as Positively charged at nucleophiles in some enzymatic
physiological pH reactions Histidine imidazole ring
protonated/ionized, only amino acid that functions as buffer in
physiol range. Lysine - diamino acid, protonated at pH 7.0 Arginine
- guianidinium ion always protonated, most basic amino acid
Polar Amino Acids 1
Polar Amino Acids 2
Classification of Amino Acids by Polarity POLAR Acidic Neutral
Basic Asp Asn Ser Arg Tyr Cys His Glu Gln Thr Lys Gly POLAR Ala Ile
Phe Trp NON- Val Leu Met Pro Polar or non-polar, it is the bases of
the amino acid properties. Juang RH (2003) Biochemistry
Functional significanceHydrophobic amino acids: encountered in
the interiorof proteins shielded from direct contact with
waterHydrophillic amino acids: generally found on theexterior of
proteins as well as in the active centers ofenzymesImidazole group:
act as either proton donor oracceptor at physiological pH Reactive
centers of enzymesPrimary alcohol and thiol groups: act as
nucleophilesduring enzymatic catalysis Disulfide bonds
Stereochemistry Note that the R group means that the -carbon is
a chiral center. All natural amino acids are L-amino acids.
L-Form Amino Acid StructureCarboxylic group - COO Amino group +
H3 N H H = Glycine R group CH3 = Alanine Juang RH (2004)
BCbasics
Mirror Images of Amino Acid Mirror image Same chemical
properties Stereo isomers Juang RH (2004) BCbasics
THE ACID-BASE BEHAVIOUR OF AMINO ACIDS Amino acids are
zwitterions: An amino acid has both a basic amine group and an
acidic carboxylic acid group.
There is an internal transfer of a hydrogen ion from the -COOH
group to the -NH2 group to leave an ion with both a negative charge
and a positive charge. This is called a zwitterion.
Adding an alkali to an amino acid solution increase the pH of a
solution of an amino acid by adding hydroxide ions, the hydrogen
ion is removed from the -NH3+ group The amino acid would be found
to travel towardsthe anode (the positive electrode).
Adding an acid to an amino acid solution decrease the pH by
adding an acid to a solution of an amino acid, the -COO- part of
the zwitterion picks up a hydrogen ion. the amino acid would move
towards thecathode (the negative electrode).
Shifting the pH from one extreme to the other Suppose you start
with the ion weve just produced under acidic conditions and slowly
add alkali to it. That ion contains two acidic hydrogens - the one
in the -COOH group and the one in the -NH3+ group. The more acidic
of these is the one in the -COOH group, and so that is removed
first - and you get back to the zwitterion.
So when you have added just the right amountof alkali, the
amino acid no longer has a netpositive or negative charge. That
means that it wouldnt move towards either the cathode oranode
during electrophoresis. The pH at which this lack of movement
duringelectrophoresis happens is known as theisoelectric point of
the amino acid. This pHvaries from amino acid to amino acid.
If you go on adding hydroxide ions, you will get the reaction
weve already seen, in which a hydrogen ion is removed from the
-NH3+ group.
You can, of course, reverse the whole processby adding an acid
to the ion weve just finishedup with. That ion contains two basic
groups - the -NH 2group and the -COO- group. The -NH2 group isthe
stronger base, and so picks up hydrogenions first. That leads you
back to the zwitterionagain.
. . . and, of course, you can keep going by then adding a
hydrogen ion to the -COO- group.
Proton Is Adsorbed or Desorbed Proton abundant and small,
affects the charge of a molecule lone pair High Low electrons pKa
H+Amino N H H+ N H H H Low pKa High O H OCarboxylic C C H+ O O
Ampholyte contains both positive and negative groups on its
molecule Juang RH (2004) BCbasics
Amino Acids Have Buffering EffectpH 12 pK2 9 NH2 H+ 6 H-C-R
Isoelectric point = pI COO- pK1 + pK2 3 2 pK1 0 [OH] Juang RH
(2004) BCbasics
Buffer pHEnvironment pH vs Protein Charge 10 9 8 7 Isoelectric
point, pI 6 5 4 3 + 0 - - Net Charge of a Protein Juang RH (2004)
BCbasics
H first Aspartic acid HOOC-CH2-C-COOH +1 NH3+ Isoelectric point
is the average pK1 = 2.1 of the two pKa flanking the zero
net-charged formsecond H 2.1 + 3.9 HOOC-CH2-C-COO- 0 2 = 3.0 NH3+
Isoelectric point pK2 = 3.9 H -2 - OOC-CH2-C-COO- -1 pK3 NH3+ third
-1 pK2 pK3 = 9.8 0 H pK1 +1 - OOC-CH2-C-COO- -2 [OH] NH2 Juang RH
(2004) BCbasics
Peptide bond formation: Polypeptides are linear polymers
composed of amino acids linked together by peptide bonds Peptide
bonds are amide linkages formed when unshared electron pair of
-carboxyl of another amino acid When 2 amino acids reacted with one
another, the product is called a dipeptide. Therefore tripeptide
contain 3 amino acid residues, tetrapeptide 4 and so forth
Formation of Peptide Bonds by Dehydration Amino acids are
connected head to tailNH2 1 COOH NH2 2 COOH Carbodiimide
Dehydration -H2O O NH2 1 C N 2 COOH H Juang RH (2004) BCbasics
By convention, amino acid residue with free NH2 group is called
N terminal residue and is written to the left Free COOH on
C-terminal is written on the right. Peptides are named by using
their amino acid sequences beginning from N-terminal residues,
E.g:H2N----Tyr----Ala----Cys----Gly----COOHAbove is a tetratpeptide
named tyrosylalanylcysteinylglycine
Polypeptide backbone: Polypeptides are polymers composed of
amino acids linked together by peptide/amide bonds Order of amino
acids in polypeptide is called amino acid sequence Disulphide
bridges formed by oxidation of Cys residues are an important
structural element in polypeptides and proteins
Peptides: Less complex than larger protein molecules have
significant biological activities E.g: Glutathione, Oxytocin,
Vasopressin, substance P and bradykinin Peptides are found in
almost all organisms, involved in many important biological
processes: -protein DNA synthesis -Drug and environment toxin
metabolism -amino acid transport -reducing agent (-SH group of cys)
protects cells from destructuve effects of oxidation by reacting
with substances such as peroxidase
Disulphide bond 2 cysteine - cystine ; 2 R-SH- R-S-S-R
(+2H)(Oxidation reaction)- Intracellular conditions are maintained
sufficiently reducing to inhibit formation of most disulfide bonds-
Extracellular conditions (as well as those found in some
organelles) are more oxidizing, favouring disulphide formation-
Thus, extracellular proteins containing cysteines often have
disulfides, while intracellular (cytosolic) proteins rarely have
disulfides.
Detection, identificationand quantificaton of amino acids and
proteins Reaction between the thiol group of cysteine and Ellmans
reagent Produce nitrothiobenzoate anion and since this product
adsorbs light at 410nm it provides a route for quantifying protein
concentration. Other reagents for estimating protein concentration
are: ninhydrin, fluorescamine, dansyl chloride, nitrophenols and
fluorodinitrobenzene (all react with functional groups)
Protein quantitation:Quick and simple way of estimating protein
cncentration1. Spectrophotometric method at 280 nm using quartz
cuvettes, absorption mainly due to Trp and Tyr2. Biuret reaction3.
Bradford method: widely used4. BCA (Bichinchoninic acid)5. Modified
lowry assay6. fluorescamine protein assay Note: to understand the
principle behind the reaction used to determine protein
concentration, also sensitivity of method used (eg: detection
limits of protein assay)Extracts containing protein should be
treated with care
1. Absorbance at 280 nm:Principle: Proteins in solution absorb
ultraviolet light with absorbance maxima at 280 and 200 nm. Amino
acids with aromatic rings are the primary reason for the absorbance
peak at 280 nm. Peptide bonds are primarily responsible for the
peak at 200 nm. Secondary, tertiary, and quaternary structure all
affect absorbance, therefore factors such as pH, ionic strength,
etc. can alter the absorbance spectrum. Advantage: Quick
estimation, protein not consumed, no additional reagent,incubation
needed, no protein standard needed
Historically use biuret reaction: solutionofcopper(II) sulphate
in alkaline tartarate solution reacts with peptide bonds to form
purple complex absorbing light at540 nm
Disadvantage: considerable error due to varying absoprtion
characteristics of protein samples2. Bradford method:Principle: The
assay is based on the observation that the absorbance maximum for
an acidic solution of Coomassie Brilliant Blue G-250 shifts from
465 nm to 595 nm when binding to protein occurs. Both hydrophobic
and ionic interactions stabilize the anionic form of the dye,
causing a visible color change. Advantage: relatively fast, fairly
accurate
Disadvantage: -The dye reagent reacts primarily with arginine
residues and less so with histidine, lysine, tyrosine, tryptophan,
and phenylalanine residues. Obviously, the assay is less accurate
for basic or acidic proteins. -The Bradford assay is rather
sensitive to bovine serum albumin, more so than "average" proteins,
by about a factor of two.
3. BCAPrinciple: BCA serves the purpose of the Folin reagent in
the Lowry assay, namely to react with complexes between copper ions
and peptide bonds to produce a purple end product. The advantage of
BCA is that the reagent is fairly stable under alkaline conditions,
and can be included in the copper solution to allow a one step
procedure. A molybdenum/tungsten blue product is produced as with
the Lowry Disadvantage: greater variability among proteins and the
assay is less sensitive
4. Modified lowry assayPrinciple: Under alkaline conditions the
divalent copper ion forms a complex with peptide bonds in which it
is reduced to a monovalent ion. Monovalent copper ion and the
radical groups of tyrosine, tryptophan, and cysteine react with
Folin reagent to produce an unstable product that becomes reduced
to molybdenum/tungsten blue Advantage: fairly accurate
Disadvantage: proteins are consumed and proteins with an abnormally
high or low percentage of tyrosine, tryptophan, or cysteine
residues will give high or low errors, respectively.
5. Fluorescamine protein assay:Principle: Fluorescamine react
with amino acids containing primary amines such as lysine and
n-terminal amino acid to yield a highly fluorescent product.
Fluoresence measure using a standard fluorometer with the
excitation wavelenght at 390 nm and emission at 475nm Advantage:
sensitive (nano gram range), fast, reaction is instantaneous
Disadvantage: reagents hydrolyzed very rapidly therefore rapid
mixing is required to produce reproducible results as fluorescamine
react with primary amine, primary amine buffer eg: tris and glycine
cant be used
Secondary stucture: Secondary structure of polypeptides
consists of several repeating structures most common types: -helix
and - pleated sheet -helix and -pleated sheet stabilize by H bonds
between carbonyl and NH groups (interactions with other amino acids
in close proximity) in polypeptide backbone -helix : rigid, rodlike
structure that forms when a polypeptide chain twists into
right-handed or left-handed helical conformation.
Nonstandard amino acidschemically modified after they have been
incorporated into aprotein (termed a posttranslational
modification)- -carboxyglutamic acid, a calcium-binding amino acid
residuefound in the blood-clotting protein prothrombin (as well as
inother proteins that bind calcium as part of their
biologicalfunction).- collagen: Significant proportions of the
amino acids incollagen are modified forms of proline and lysine:
4-hydroxyproline and 5-hydroxylysine.- Phosphate molecule to the
hydroxyl portion of the R groups ofserine, threonine, and tyrosine.
This event is known asphosphorylation and is used to regulate the
activity of proteinsin the cell. Serine is the most common in
proteins, threonine issecond, and tyrosine is third.
- Glycoproteins are widely distributed in nature and providethe
spectrum of functions already discussed for unmodifiedproteins. The
sugar groups in glycoproteins are attached toamino acids through
either oxygen (O-linked sugars) ornitrogen atoms (N-linked sugars)
in the amino acid residues.- Selenocysteine: Although it is part of
only a few knownproteins, there is a sound scientific reason to
consider this the21st amino acid because it is in fact introduced
during proteinbiosynthesis rather than created by a
posttranslationalmodification. Selenocysteine is actually derived
from theamino acid serine (in a very complicated fashion), and
itcontains selenium instead of the sulfur of cysteine.
A helix has the following features:every 3.6 residues make one
turn,the distance between two turns is 0.54 nm,the C=O (or N-H) of
one turn is hydrogen bonded to N-H (orC=O) of the neighboring
turn.Hydrogen bonds play a role in stabilizing the a
helixconformation. However, the size and charges of sidechainsare
also important factors. Alanine has a greater propensityto form a
helices than proline.
The hydrogen bonds that stabilize the helix are parallel to the
long axis of the helix.
Beta strandIn a beta strand, the torsion angle of N-Ca-C-N in
thebackbone is about 120 degrees. The following figure showsthe
conformation of an ideal b strand. Note that thesidechains of two
neighboring residues project in theopposite direction from the
backbone
Beta sheetA beta sheet consists of two or more hydrogen bonded
bstrands. The two neighboring b strands may be parallel ifthey are
aligned in the same direction from one terminus (Nor C) to the
other, or anti-parallel if they are aligned in theopposite
direction.
Structural motif (supersecondary structure):a structural motif
is a three-dimensional structural element or fold within the chain,
which appears also in a variety of other molecules. In the context
of proteins, the term is sometimes used interchangeably with
"structural domain," although a domain need not be a motif nor, if
it contains a motif, need not be made up of only one.
Rossman fold: Decarboxylase enzyme
What are domains of proteins? A domain is a basic structural
unit of a protein structure-distinct from those that make up the
conformation Part of protein that can fold into a stable
structureindependently different domains can impart different
functions to proteinsProteins can have one too many domains
depending onprotein sizeIn an unbranched chain-like biological
molecule, such as aprotein or RNA, a structural motif is the three
dimensionalstructural element within the chain, which appears also
inavariety of other molecules.
Pyruvate kinase
Tertiary structure: 3D conformation as a result of interactions
betweeen side chains in their primary structure Hydrophobic
intercations: as polypeptide folds, R groups are brought into close
proximity Electrostatic interactions: strongest electrostatic
interaction between ionic groups of opposite charge H bonds:
significant number of H bonds forms within interior of protein,
polar amino acids interact with water or with polypeptide backbone
Covalent bonds: most important, covalent bonds in tertiary
structure are disulfide bridges found in many extracellular
proteins
Quaternary structure: Proteins esp high M.W composed of several
polypeptide chains Each polypeptide is called a subunit Subunits in
a protein complex may be identical or quite different Multisubunit
proteins in which some or all subunits are identical are called
oligomers Polypeptide units assemble and held together by
noncovalent interactions such as -hydrophobic interactions
-electrostatic interactions -H bonds -covalent cross links
Hydrophobic interactions play an important role in protein
folding as well as covalent crosslinks help stabilize multisubunit
proteinsEg: disulfide bridges in immunoglobulins, the desmosine and
lysinonorleucine linkages in certain connective tissuesEg:
desmosine cross links connects 4 polypepide chains in the
rubberlike connective tissue called elastinLysinonorleucine:
crosslink structure found in elastin and collagenInteractions
between subunit are also affected by binding of ligands
In allostery, control of protei fundtion through ligand binding
to specific site in protein triggers conformational change that
alters its affinity for other ligandsLigand induced corformational
changes in such proteins are called allosteric transitions, ligands
which trigger them are called effectors or modulatorsLoss of
protein structure: Protein sensitive to environmental factors
Disruption of native conformation is called denaturation Factors:
physical and chemicalDenaturing agents:1. Strong acids or base2.
Organic solvents
Hydrogen bonding in a protein
3. Detergents4. Reducing agents5. Salt concentrations6. Heavy
metal ions7. Temperature changes8. Mechanical stress
Antibody family: A family of proteins that can be created to
bind almost any molecule Ntibodies (imminoglobulin) are made in
response to a foreign molecule i.e: bacteria, virus,
pollen..callled and antigen Bind together tightly and therefore
inactivates the antigen or marks it for destruction
Protein folding: The peptide bond allows for rotation around it
and therefore the protein can fold and orient the R groups in
favourable positions Weak non covalent interactions will hold the
protein in its functional shape-these are weak and will take many
to hold the shape. H bonds form between 1) atoms involved in the
peptide bonds 2) peptide bond atoms and R groups, 3) R
groupsProtein folding: Protein shape is determined by the sequence
of the amino acids The final shape is called the conformation and
has the lowest free energy possible
3 main classes of protein folding accessory proteins:Allow
protein to fold within few minutes in cell (in vivo)a) Protein
disulfide isomerasesb) Peptidyl prolyl ci-trans isomerasesc)
Molecular chaperones Denatured proteins may renature or refold if
chemical compound that causes denaturation can be removed Molecular
chaperons are small proteins that help guide the folding and help
keep the new protein from associating with the wrong partner
Useful protein: There are many diferent combinations of amino
acids that can make up proteins and that would increase if each one
had multiple shape Proteins usually have one useful conformation
because otherwise it would not be efficient use of energy available
to the system Natural selection has elimited proteins that donot
perform a specific function in the cell Have similarities in amino
acid sequence and 3d structure Have similar functions such as
breakdown proteins but do it differently
Proteins multiple peptides non covalent bonds can form
interactions betweenindividual polypeptide chains binding
site-where proteins interact with one anotherSubunit-each
polypeptide chain of large protein dimer protein made of 2
subunits
Oxygen binding protein: Hemoglobin: Carry O2 in blood from
lungs to other tisues in body; function is to supply O2 to cells
for oxidative phosphorylation Myoglobin stores O2 in tissues of
body, available when cells reuire it; highest concentration of
myoglobin in skeletal and cardiac muscle which require large
amounts of energyMyoglobin: small protein, 17.8 Kda, made up of 153
amino acids in a single polypeptide
Globular protein have a highly folded compact structure with
most of the hydrophobic residues found in the interior while polar
residues on surfaces Structure of hemoglobin determined by Max
Perutz was the first protein structure determined via x-ray
crystallography Secondary structure: -helix, 8 -helices, heme
prosthetic group is found in hydrophobic crevice formed by folding
of polypeptide chains Hemoglobin made up of 4 polypeptide chains
Each have similar 3D of single polypeptide chain in myoglobin even
though aino acid sequences differ at 83 % of residues This
highlight relatively common theme in protein structure: different
primary sequence can specify very similar 3D structures
Major tyoe of hemoglobin found in adults (HbA): Made of 2
diferent polypeptide chains: - -chain: 141 amino acid --chain: 146
amino acid Each chain has 8 -helices, each containing heme
prosthetic group; therfore hemoglobin can bind 4 molecules of O2 4
polypeptide chains are 22, consists of 2 and 2 packed tightly
together ina tetrahedral array to form spherical shaped molecule
held together by multiple noncovalent interactions
Important fibrous proteins: Intermediate filaments of the
cytoskeleton -structural scaffold inside the cells -keratin in
hair, horns and nails Extracellular matrix -binds cells together to
make tissues -secreted from cells and assemble in long fibers
-collagen: fiber with a glycine every third amino acid in the
protein -Elastin: unstructured fibers that give tissues an elastic
characteristic
Fibrous proteins: Typically contain high proportion of regular
secondary structures such as -helices and -pleated sheets E.g:
alpha-keratin, collagen, silk fibroin alpha-keratin:bundles of
helical polypeptides twisted together into large bundles
Alpha-keratin found in hair wool, skins, horns, fingernails are
alpha-helical polypeptides.
Globular protein:Stabilization of cross linkages Cross linkages
can be between 2 parts of a protein or between 2 subunits
Disulphide bonds (-S-S-) form between adjacent SH groups on the
amino acid cystein
Proteins at work: Conformation of a protein gives it a unigque
function To work proteins must interact with other molecules,
usually 1 or a few molecules from the thousands . Ligand: the
molecule that a protein can bind Binding site -part of a protein
that interacts with the ligand -consists of a cavity formed by a
specific arrangment of amino acids The binding site forms when
amino acids from within the protein come together The remaining
sequence may play a role in regulating the proteins activity
Chemical characteristic of proteins: Proteins have ionic and
hydrophobic sites both internally (within folds of tertiary
structure) and on surfaces where primary structures come in contact
with the environment Ionic sites are provided by charged amino
acids at physiological pH and by covalentl attached modifying group
(eg: carbohydrates and phosphate) Net charge on protein contributed
by free alpha-amino of N- terminal residue, free alpha-carbonyl
group of c terminal residue, ionizable R groups and unique array of
modifications attached to proteins At isoelectric point (pI): no of
(+) and (-) charges on protein are equal. Protein is electrically
neutral Protein has net (+) at pH values below its pI and (-)
charge above is pI