Section B 蛋白质结构 Protein Structure. Central dogma 中心法则 : DNA > RNA > Protein Nucleic acids: (DNA, RNA): polymer of nucleotides (4 for each) Protein: Polymers

Section B 蛋白质结构

Protein Structure

Central dogma 中心法则 :

DNA > RNA > Protein

Nucleic acids: (DNA, RNA): polymer of nucleotides (4 for each)

Protein: Polymers of amino acids (20 aa)

B1 Amino acids: structure, side chains (charged, polar uncharged, nonpolar aliphatic, aromatic)

B2 Protein structure and function: Structure: size and shapes, primarysecondary tertiary quaternary, prosthetic groups Domains, motif, and family Function

B3 Protein analysis Purification Determine sequence, mass, and structure (X-ray crystallography and NMR)

H3N

COO-

R

HC

1. -carbon is chiral ( 不对称性 ) except glycine (R is H)2. Both D- and L- stereoisomers( 异构体 ), but only L-iso

mers are found in proteins3. Amino acids are dipolar ions (zwitterions [ 两性离子 ])

in aqueous solution and are amphoteric ( 酸碱两性的 )4. The side chains (R) differ in size, shape, charge and che

mical reactivity5. Nonstandard amino acids (> proline 脯氨酸 and lysine 赖氨

酸 )

亚氨基

B1 氨基酸 basic structure

Proline脯氨酸Proline脯氨酸

Common structure of 19 AAs

Common structure of 19 AAs

1. “Acidic” amino acids (2): containing additional carboxyl 羧基 groups which are usually ionized

Can form salt bridges, are hydrophilic ( 亲水 )

aspartic acid (Asp, D ，天冬氨酸 )

glutamic acid (Glu, E ，谷氨酸 )

B1 Amino acids- charged (5)

2. “Basic” amino acids (3): containing positively charged groups

Lysine (Lys, K, 赖氨酸 )

The imidazole group ( 咪唑基 ) has a pKa near neutrality. This group can be reversibly protonated under physiological conditions, which contribute to the catalytic mechanism of many enzymes.

Arginine (arg, R, 精氨酸 )

a guanidino group ( 胍基）

Histidine (His, H ，组氨酸 )

Contain groups that form hydrogen bonds with water, hydrophilic 亲水的

Contain hydroxyl groups.

Asparagine (Asn, N ，天冬酰氨 )

B1 Amino acids- polar uncharged (5)

Serine (Ser, S ，丝氨酸 )

Threonine (Thr, T ，苏氨酸 )

Glutamine (Gln, Q ，谷氨酰氨 )

B1 Amino acids- polar uncharged (5)

Cysteine (Cys, C ，半胱氨酸 ) has a thiol ( 巯醇 ) group ， which is often oxidizes to cystine 胱氨酸

x-S-S-x

Glycine (Gly, G ，甘氨酸 )

Proline (Pro, P ，脯氨酸 ): imino acid ( 亚氨基酸 )

Methionine (Met, M ，甲硫氨酸 ): contains a sulfur atom

B1 Amino acids- nonpolar aliphatic 脂肪族的 (7) (hydrophobic 疏水 )

Alkyl ( 烷基 ) side chains

Alanine (Ala, A ，丙氨酸 )

Valine (Val, V ，缬氨酸 )

Leucine (Leu, L ，亮氨酸 )

Isoleucine (Ile, I ，异亮氨酸 )

Phenylalanine (Phe, F ，苯丙氨酸 )

Tyrosine (Tyr, Y ，酪氨酸 )

Tryptophan (Trp, W ，色氨酸 )

B1 Amino acids- aromatic 芳香族 (3)

Accounts for most of UV absorbance of proteins at 280 nm hydrophobic （疏水的 )

Non-standard amino acids( 稀有氨基酸 ):

e.g. 4-hydroxyproline(4- 羟基脯氨酸 ), 5-hydroxylysine(5- 羟基赖氨酸 ) in collagen( 胶原质 )

- not encoded, formed by post-translational modification( 翻译后修饰 )

B2 蛋白质的结构与功能

Structure: size and shapes, primarysecondary tertiary quaternary, prosthetic groups ( 辅基，nonprotein molecules of conjugated proteins ( 共轭蛋白 )

Domains 结构域 , motif 基序 , and family 家族

Protein function

B2 Protein structure － Sizes

1. A few thousands Daltons (x 103) to more than 5 million Daltons (x 106)

2. Some proteins contain bound nonprotein materials (prosthetic groups 辅基 or other macromolecules), which accounts for the increased sizes and functionalities of the protein complexs.

Globular proteins: enzymes

Complementary fit of a substrate molecule to the catalytic site (groove-like) on an enzyme molecule.

B2 Protein structure － Shapes

chymotrypsin( 糜蛋白酶 )

Fibrous proteins: important structural proteins (silk fibroin, keratin in hair and wools )

keratin in hair

Keratin ( 角蛋白 ) Protofibril ( 初原纤维 ) microfibril ( 微管 )

蛋白质的功能• 1 催化功能 ----enzyme 酶• 2 信号传递 ---- cell membrane protein• 3 转运与贮存 ---- hemoglobin transports oxygen• 4 结构与运动 ---- collagen, keratin, tubulin in cyt

oskeleton, actin and myosin for muscle contraction

• 5 营养 ----casein ( 酪蛋白 ) and ovalbumin( 卵清蛋白 )

• 6 免疫 ---- antibodies• 7 调节 ---- transcription factors• 8 抗癌药物 ---- 毒蛋白• 9 支持与保护作用 ----- 毛发的角蛋白

Polypeptides 多肽 contain N- and C- termini and are directional, usually ranging from 100-1500 aa

Formation of a peptide bond (shaded in gray) in a dipeptide.

B2 Protein structure － Primary

Structure of the pentapeptide Ser-Gly-Tyr-Ala-Leu.

N terminus

C terminus

-helix• right-handed• 3.6 aa per turn• hydrogen bond N-H···O=C

Collagen 胶原质 triple helix:

three polypeptide intertwined

A stereo, space-filling representation

B2 Protein structure － Secondary

-sheet: hydrogen bonding of the peptide bond N-H and C=O groups to the complementary groups of another section of the polypeptide chain x

A stereo, space-filling representation of the six-stranded antiparallel sheet.

Parallel sheet: sections run in the same directionAntiparallel sheet: sections run in the opposite direction

fibroin 蚕丝蛋白

B2 Protein structure － Domains ( 结构域 ):, motifs （基序） and fa

milies （家族）

Domains( 结构域 ): structurally independent units of many proteins, connected by sections with limited higher order structure within the same polypeptide. (Figure)

They can also have specific function such as substrate binding

Structural motifs （基序） : • Groupings of secondary structural elements that frequently occur in globular proteins• Often have functional significance and represent the essential parts of binding or catalytic sites conserved among a protein family

motif

Protein families （家族） : structurally and functionally related proteins from different sources

The primary structures of c-type cytochromes from different organisms

Motif

趋异进化 ---- 直系同源 / 共生同源。趋同进化 ---- 无关基因进化至产生具有相似结构和催化活性蛋白质。如细菌蛋白水解酶与人的糜蛋白酶。

B2 Protein structure － TertiaryThe different sections of -helix, -sheet, other minor secondary structure and connecting loops of a polypeptide fold in three dimensions

Many proteins are composed of two or more polypeptide chains (subunits). These subunits may be identical or different. The same forces which stabilize tertiary structure hold these subunits together. This level of organization called quaternary structure.

A stereo, space-filling drawing showing the quaternary structure of hemoglobin 血色素

1-yellow; 1-light blue; 2-green; -dark blue; heme 亚铁血红素 -redback

B2 Protein structure － Quaternary

B3 Protein analysis

1. Purification: to obtain enough pure sample for study

2. Sequencing: determine the primary structure of a pure protein sample

3. Mass determination: determine the molecular weight (MW) of an interested protein.

4. X-ray crystallography and NMR: determine the tertiary structure of a given sample.

The principal properties of proteins used for purification

1. Size: gel filtration chromatography

2. Charge: ion-exchange chromatography, isoelectric focusing electrophoresis

3. Hydrophobicity: hydrophobic interaction chromatography

4. Affinity: affinity chromatography

5. Recombinant techniques: involving DNA manipulation and making protein purification so easy

1. gel filtration chromatography 凝胶过滤色谱 ( 法 )

2. Charge: ion-exchange chromatography( 离子交换层析 ), isoelectric focusing( 等电聚焦 ), electrophoresis( 电泳 )

Isoelectric point (pI): the pH at which the net surface charge of a protein is zero

--

--

-- -

-

++

+

+

+

+ ++

pH=pIpH>pI pH<pI

Ion-exchange chromatography

Column + anions

+++

Sample mixture

Protein binding

Column + proteins Column + anions

Ion displacing

Purified protein

+

Electrophoresis

Protein migrate at different position depending on their net charge

Isoelectric focusing

A protein will stop moving at position corresponding to its isoelectric point (pI) in a pH gradient gel.

3. Hydrophobicity( 疏水性 ): hydrophobic interaction chromatography

Similar to ion-exchange chromatography except that column material contains aromatic( 芳香族的 ) or aliphatic alkyl( 脂肪烷基 ) groups

4. Affinity chromatography 亲合色谱法

ding

d

• Enzyme-substrate binding

• Receptor-ligand binding

• Antibody-antigen binding

Substrate analogs: competitive inhibitors

5. Recombinant techniques:

•Clone the protein encoding gene of interest in an expression vector with a purification tag( 纯化标签 ) added at the 5’- or 3’ end of the gene

•Protein overexpression in a cell

•Protein purification with affinity chromatography.

Determine the primary structure of a protein: p

Amino acid composition:

1. Acid treatment to hydrolyze peptide bonds: 6M HCl, 110°C for 24 hrs.

2. Chromatographic analysis 色谱 [ 层 ] 分析

However, you cannot get the sequence!

Protein sequence analysis (1)

Sequence: HLMGSHLVDALELVMGDRGFEYTPKAWLV

Trypsin T1 HLMGSHLVDALELVMGDR

T2 GFEYTPK

T3 AWLV

V8 V1 HLMGSHLVDALE

V2 LVMGDRGFE

V3 YTPKAWLV

Mass Determination

Gel filtration chromatography and SDS-PAGE

•Comparing of the unknown protein with a proper standard

•Popular SDS-PAGE: cheap and easy with a 5-10% error

•SDS: sodium dodecyl sulfate, makes the proteins negatively charged and the overall charge of a protein is dependent on its mass.

Mass Determination

Mass spectrometry 质谱分析 :

• Molecules are vaporized and ionized (by Xe/Ar beam), and the degree of deflection (mass-dependent) of the ions in an electromagnetic field is measured

• Extremely accurate (0.01% error), but expensive

• ESI (electrospray ionization) and MALDI (matrix-assisted laser desorption/ionization) can measure the mass of proteins smaller than 100 KDa

• Helpful to detect post-translational modification

• Protein sequencing: relying on the protein data base

X-ray crystallography and NMRDeterming the tertiary structure (3-D) of a protein

X-ray crystallography:

• Measuring the pattern of diffraction of a beam of X-rays as it pass through a crystal. The first hand data obtained is electron density map, the crystal structure is then deduced.

• A very powerful tool in understanding protein tertiary structure

• Many proteins have been crystallized and analyzed

基因组 (genome): 指单倍体细胞中包括编码序列和非编码序列在内的全部 DNA 分子。

转录组 (transcriptome) ：一个细胞在它的生存期或它生存的任何一个时间内基因组转录的全部 mRNA 。

蛋白质组 (proteome): 一个细胞在它的生存期或它生存的任何一个时间内转录表达的全套蛋白。

蛋白质组学（ proteomics): 阐明生物体各种生物基因组在细胞中表达的全部蛋白质的表达模式及功能模式的学科。包括鉴定蛋白质的表达、存在方式 ( 修饰形式 ) 、结构、功能和相互作用等。

Section C 核酸的性质

C1 Nucleic Acid Structure

C2 Chemical and Physical Properties of Nucleic Acids

C3 Spectroscopic and Thermal Properties of Nucleic Acids

C4 DNA Supercoiling

BASES 碱基 NUCLEOSIDES核苷

NUCLEOTIDES 核苷酸

Adenine (A) Adenosine 腺苷 Adenosine 5’-triphosphate (ATP)

Deoxyadenosine Deoxyadenosine 5’-triphosphate (dATP)

Guanine (G) Guanosine 鸟苷 Guanosine 5’-triphosphate (GTP)

Deoxy-guanosine Deoxy-guanosine 5’-triphosphate (dGTP)

Cytosine (C) Cytidine 胞苷 Cytidine 5’-triphosphate (CTP)

Deoxy-cytidine Deoxy-cytidine 5’-triphosphate (dCTP)

Uracil (U) Uridine 尿苷 Uridine 5’-triphosphate (UTP)

Thymine (T) Thymidine/ 胸苷deoxythymidine

Thymidine/deoxythymidine

5’-triphosphate (dTTP)


Comparisons of names of bases, nucleosides and nucleotides

Purine: A & G; Pyrimidine: C & T/U; (deoxy)-ribose,


Nitrogenous bases含氮碱基

Bicyclic purines:

Monocyclic pyrimidine:

Thymine (T) is 5-methyluracil (U)


Nucleosides

In nucleic acids, the bases are covalently attached to the 1’ position of a pentose sugar ring, to form a nucleoside

Adenosine, guanosine, cytidine, thymidine, uridine

Glycosidic (glycoside, glycosylic) bond ( 糖苷键 )

R Ribose or 2’-deoxyribose


Nucleotides

A nucleotide is a nucleoside with one or more phosphate groups bound covalently to the 3’-, 5’, or ( in ribonucleotides only) the 2’-position. In the case of 5’-position, up to three phosphates may be attached.

Deoxynucleotides (deoxyribose containing)

Ribonucleotides (ribose containing)

Phosphate diester 二酯 bonds

4

5 12

67

9 21

54


Phosphodiester bonds

DNA/RNA sequence:From 5’ end to 3’ endExample:5’-UCAGGCUA-3’= UCAGGCUA

3’ end: free hydroxyl (-OH) group

5’end: not always has attached phosphate groups


DNA double helix

•Watson and Crick , 1953.

•Two separate strands Antipa

rellel (5’3’ direction)

Complementary (sequence)

Base pairing: hydrogen bondin

g that holds two strands together

Essential for replicating DNA and transcribing RNA

• Sugar-phosphate backbones (negatively charged): outside• Planar bases (stack one above the other): inside

back

Base pairing via hydrogen bonds

A:TG:C

1

234

567

8

9

12

3

4 5

6

2

1

34

5 67

89

2 13

4 5

6

•Double helix

•B form:

Right-handed

10 base pairs/turn

0.34nm /turn

Diameter: ca. 2.0nm Å

Other forms:

A: 11 bases/turn, base plate 20° slant

Z: 12 bases/turn, left-handed helical, one groove



RNA Secondary Structure

Single stranded, no long helical structure like double-stranded DNA

Globular conformation with local regions of helical structure formed by intramolecular hydrogen bonding and base stacking.

tRNA(clover-like)

Ribozyme RNArRNA

Conformational variability of RNA is important for the much more diverse roles of RNA in the cell, when compared to DNA.

Structure and Function correspondence of protein and nucleic acids

Protein Nucleic Acids

Fibrous protein Globular protein Helical DNA Globular RNA

Structural proteins • Enzymes,

• antibodies,

• receptors etc

Genetic information maintenance

•Ribozymes

•Transfer RNA (tRNA)

•Signal recognition e.g. 7SL.



Modified Nucleic Acids

Modifications correspond to numbers of specific roles. We will discuss them in some related topics. For example, methylation of A and C to avoid restriction digestion of endogenous DNA sequence (Topic G3).


1. Stability of Nucleic Acids

2. Effect of Acid & application

s

3. Effect of alkali & applicatio

ns

4. Chemical denaturation

5. Viscosity & applications

6. Buorant density & applicati

on

Chemical properties

Physical properties

back


Stability of Nucleic Acids

1. Hydrogen bonding • Contribute to specificity, not overall stability of DNA helix• Stability lies in the stacking interactions between base pairs

2. Stacking interaction/hydrophobic interaction between aromatic base pairs/bases contribute to the stability of nucleic acids.• It is energetically favorable for the hydrophobic bases to exclude waters and stack on top of each other (base stacking & hydrophobic effect). • This stacking is maximized in double-stranded DNA


Effect of Acid & applications

Strong acid + high temperature completely hydrolyzed to (perchloric acid+100°C) bases, riboses/deoxyribose, and phosphate

pH 3-4 apurinic nucleic acids [glycosylic bonds attaching purine (A and G) bases to the ribose ring are broken ]

Maxam and Gilbert chemical DNA sequencing:A DNA sequencing technique based on chemical removal and modification of bases specifically and then cleaving the sugar-phosphate backbone of the DNA and RNA at particular bases (J2)

脱嘌呤核酸


Effect of Alkali & Application

DNA denaturation at high pH

keto form enolate formketo form enolate form

Base pairing is not stable anymore because of the change of tautomeric ( 异构 ) states of the bases, resulting in DNA denaturation变性


RNA hydrolyzes at higher pH because of 2’-OH groups in RNA

Effect of Alkali & Application

RNA is unstable at higher pH

OH free 5’-OH

2’, 3’-cyclic phosphodiester

alkali


Chemical Denaturation

Urea (H2NCONH2) ( 尿素） : denaturing PAGE

Formamide (HCONH2) （甲酰胺） and formaldehyde ( 甲醛 ): Northern blot

Disrupting the hydrogen bonding of the bulk water solution

Hydrophobic effect (aromatic bases) is reduced

Denaturation of strands in double helical structure


Viscosity 粘性

Reasons for the DNA high viscosity 1. High axial ratio2. Relatively stiff 僵硬的

Applications:1. Long DNA molecules can easily be shortened by

shearing force.2. When isolating very large DNA molecule, always avoid

shearing problem


Buoyant density1.7 g cm-3, a similar density to 8M CsCl. Rho=1.66+0.098 (GC)%

Purifications of DNA: equilibrium density gradient centrifugation

back

RNA pellets at the bottom

Protein floats

C 3 Spectroscopic and Thermal Properties of Nucleic Acids

1. UV absorption: • Nucleic acids absorb UV light due to the aromatic bases• The wavelength of maximum absorption by both DNA and RN

A is 260 nm (max = 260 nm)• Application: detection, quantitation, assessment of purity (A260/

280)

2. Hypochromicity: fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption. dsDNA, ssDNA/RNA, nucleotide

3. Quantitation of nucleic acidsExtinction coefficient (): 1 mg/ml dsDNA has an A260 of 20 (OD1=50ug/ml)

ssDNA and RNA=25 (OD1=40ug/ml)

The values for ssDNA and RNA are approximate

(1) The values are the sum of absorbance contributed by the different bases ( : p

urines > pyrimidines)

(2) The absorbance values also depend on the amount of secondary structures du

e to hypochromicity.

4. Purity of DNA

A260/280:

dsDNA--1.8

pure RNA--2.0

protein--0.5

5. Thermal denaturation/melting: heating leads to the destruction of double-stranded hydrogen-bonded regions of DNA and RNA.

RNA: the absorbance increases gradually and irregularlyDNA: the absorbance increases cooperatively.Melting temperature (Tm): the temperature at which 40% increase in

absorbance is achieved.

6. Renaturation: 复性

Rapid cooling: Only allow the formation of local base paring. Absorbance is slightly decreasedSlow cooling: Whole complementation of dsDNA. Absorbance decreases greatly and cooperatively.

Annealing 退火 : Base paring of short regions of compleme

ntarity within or between DNA strands. (example: anneali

ng step in PCR reaction)

Hybridization: Renaturation of complementary sequences

between different nucleic acid molecules.

(examples: Northern or Southern hybridization)

C4 DNA Supercoiling

1. Almost all DNA molecules in cells are on average negatively

supercoiled.

2. Supercoiled DNA has a higher energy than relaxed DNA. Ne

gative supercoiling may thus facilitate cellular processes whi

ch require the unwinding of the helix, such as transcription i

nitiation or replication

3. Topoisomerases 异构酶 exist in the cell regulate the level of su

percoiling of DNA molecules. (important to know in the sense

of gene expression)

(General understanding but not details are required)

Linker number (Lk, 连接数 , 连环数 ): a topological property of a closed-circular DNA, which can be changed only if one or both of the DNA backbones are broken.

Topoisomer ( 拓扑异构体 ) : A molecule of a given linking number is known as a topoisomer. Topoisomers of the same molecule differ from each other only in their linker number.

The conformation (geometry) of the DNA can be altered while the linking number remains constant. Writhe (wrap around, 缠绕 ) and Twist ( 扭转 ) changes are to measure the conformational change of a DNA molecule (Lk = Tw + Wr).

1. The topological change (Lk) in supercoiling of a DNA molecule is partitioned into a conformational change of twist (Tw )and/or a change of writhe (Wr).

2. For a given isomer of a circular closed DNA (Lk = 0), the increase in twist will cause a corresponding decrease in writhe.

Lk = Lko

Relaxed closed circular

Re-join the DNA

Lk = Lko – 4

Lk = -4

Break the circular DNA

Untwist 4 x 360otwist 4 x 360o

Lk = Lko + 4

Lk = + 4

DNA isolated from cell negatively supercoiled by~ 6 turns per 100 turns of the helix. Lk / Lk = -0.06

Negatively supercoiled

positively supercoiled

Lk = -4

Ethidium bromide (intercalator 插入物 ): locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe.

Topoisomerases 异构酶

Type I: break one strand of the DNA (via P-tyrosine 酪氨酸 bond) , and change the linking number in steps of ±1.Type II: break both strands of the DNA , and change the linking number in steps of ±2. (ATP)Bacterial gyrase ( 旋转酶 ):introduce negative supercoiling. ATP.

Summary:

1. Nucleic acid structure: bases > nucleosides (base+sugar) > nucleotides (nucleoside+phosphate) > polynucleotides /DNA/RNA (via 3’,5’-phosphodiester bond) > DNA double helix/RNA secondary structure

2. Chemical and physical properties: stability support, effect of acid and alkali, chemical denaturation, viscosity, buoyant density

3. Spectroscopic and thermal properties

4. DNA supercoiling: Linking number (twist and writhe)

Documents

Section B 蛋白质结构 Protein Structure. Central dogma 中心法则 : DNA > RNA > Protein Nucleic acids: (DNA, RNA): polymer of nucleotides (4 for each) Protein: Polymers