Download B2 Protein structure

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, primarysecondary
 tertiary  quaternary, prosthetic groups
Domains, motif, and family
Function
B3 Protein analysis
Purification  Determine sequence, mass, and
structure (X-ray crystallography and NMR)
B1 氨基酸 basic structure
COOH3N
Ca
R
Common
H structure
of 19 AAs
Proline
脯氨酸
亚氨基
1. a-carbon is chiral (不对称性) except glycine (R is H)
2. Both D- and L- stereoisomers(异构体), but only Lisomers are found in proteins
3. 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
chemical reactivity
5. Nonstandard amino acids (> proline脯氨酸and lysine赖氨酸)
B1 Amino acids- charged (5)
Can form salt bridges, are hydrophilic (亲水)
1. “Acidic” amino acids (2): containing additional
carboxyl羧基 groups which are usually ionized
aspartic acid (Asp, D，天冬氨酸)
glutamic acid (Glu, E，谷氨酸)
2. “Basic” amino acids (3): containing positively charged groups
e
d
Lysine (Lys, K, 赖氨酸)
Arginine (arg, R,精氨酸)
a guanidino group (胍基）
Histidine (His, H，组氨酸)
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.
B1 Amino acids- polar uncharged (5)
Contain groups that form hydrogen bonds with
water, hydrophilic亲水的
Serine (Ser, S，丝氨酸)
Threonine (Thr, T，苏氨酸)
Asparagine (Asn, N，天冬酰氨)
Glutamine (Gln, Q，谷氨酰氨)
Contain
hydroxyl
groups.
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
B1 Amino acids- nonpolar aliphatic脂肪族的(7)
(hydrophobic 疏水)
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
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)
B1 Amino acids- aromatic芳香族(3)
Accounts for most of UV absorbance of proteins at 280 nm
hydrophobic （疏水的)
Phenylalanine (Phe, F，苯丙氨酸)
Tyrosine (Tyr, Y，酪氨酸)
Tryptophan (Trp, W，色氨酸)
Non-standard amino acids(稀有氨基酸):
e.g. 4-hydroxyproline(4-羟基脯氨酸), 5hydroxylysine(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, primarysecondary
 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.
B2 Protein structure －Shapes
Globular proteins: enzymes
chymotrypsin
(糜蛋白酶)
Complementary fit of a substrate molecule to the catalytic site
(groove-like) on an enzyme molecule.
Fibrous proteins: important structural proteins
(silk fibroin, keratin in hair and wools )
Keratin (角蛋白)
Protofibril (初原纤维)
keratin in hair
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
cytoskeleton, 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
B2 Protein structure －Primary
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.
N terminus
C terminus
Structure of the pentapeptide Ser-Gly-Tyr-Ala-Leu.
B2 Protein structure －Secondary
a-helix
• right-handed
• 3.6 aa per
turn
• hydrogen
bond
N-H···O=C
A stereo, space-filling representation Collagen胶原质triple helix:
three polypeptide
intertwined
b-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
Parallel b sheet: sections run
in the same direction
Antiparallel b sheet: sections
run in the opposite direction
A stereo, space-filling
representation of the sixstranded antiparallel b sheet.
fibroin蚕丝蛋白
B2 Protein structure －
Domains (结构域):, motifs （基序）
and families（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
bab motif
Protein families （家族） : structurally and functionally
related proteins from different sources
Motif
The primary structures of c-type cytochromes from
different organisms
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 －Tertiary
The different sections of a-helix, b-sheet, other minor
secondary structure and connecting loops of a polypeptide
fold in three dimensions
B2 Protein structure －Quaternary
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
a1-yellow; b1-light blue; a2-green; b2-dark blue; heme亚铁血红素-red
back
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>pI
+
+
+
+
pH=pI
+
pH<pI
Ion-exchange chromatography
Sample mixture
++
+
Protein
binding
Column + anions
Ion
displacing
Column + proteins
Column + anions
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
•
Enzyme-substrate binding
Substrate analogs: competitive inhibitors
ding
d
• Receptor-ligand
binding
• Antibody-antigen
binding
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
V8
AWLV
V1 HLMGSHLVDALE
V2
V3
LVMGDRGFE
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 (matrixassisted laser desorption/ionization) can measure the mass
of proteins smaller than 100 KDa
• Protein sequencing: relying on the protein data base
• Helpful to detect post-translational modification
X-ray crystallography and NMR
Determing the tertiary structure (3-D) of a protein
X-ray crystallography:
• Measuring the pattern of diffraction of a beam of Xrays 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
C1 Nucleic Acid Structure
Comparisons of names of bases, nucleosides and nucleotides
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
Deoxy-guanosine
Cytosine (C) Cytidine胞苷
Deoxy-cytidine
Uridine尿苷
Uracil (U)
Thymine (T) Thymidine/胸苷
deoxythymidine
Guanosine 5’-triphosphate (GTP)
Deoxy-guanosine 5’-triphosphate (dGTP)
Cytidine 5’-triphosphate (CTP)
Deoxy-cytidine 5’-triphosphate (dCTP)
Uridine 5’-triphosphate (UTP)
Thymidine/deoxythymidine
5’-triphosphate (dTTP)
Purine: A & G; Pyrimidine: C & T/U; (deoxy)-ribose,
C1 Nucleic Acid Structure
Nitrogenous bases
含氮碱基
Bicyclic purines:
Monocyclic pyrimidine:
Thymine (T) is 5-methyluracil (U)
C1 Nucleic Acid Structure
Nucleosides
In nucleic acids, the bases are covalently attached to the 1’ position
of a pentose sugar ring, to form a nucleoside
Glycosidic (glycoside, glycosylic) bond
(糖苷键)
R
Ribose or 2’-deoxyribose
Adenosine, guanosine, cytidine, thymidine, uridine
C1 Nucleic Acid Structure
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.
Phosphate diester二酯bonds
7
9
Deoxynucleotides
(deoxyribose containing)
5
4
6
1
2
5
4
1 2
Ribonucleotides
(ribose containing)
C1 Nucleic Acid Structure
5’end: not always has attached phosphate groups
DNA/RNA sequence:
From 5’ end to 3’ end
Example:
5’-UCAGGCUA-3’
= UCAGGCUA
Phosphodiester bonds
3’ end: free hydroxyl (-OH) group
C1 Nucleic Acid Structure
DNA double helix
•Watson and Crick , 1953.
•Two separate strands
Antiparellel (5’3’ direction)
Complementary (sequence)
Base pairing: hydrogen
bonding that holds two strands
together
• Sugar-phosphate backbones
(negatively charged): outside
• Planar bases (stack one above
the other): inside
back
Essential for replicating DNA and transcribing RNA
5
4
5
4
6
3
7
8
9
2
6
5
4
6
3
1
7
9
3
5
8
1
1
4
2
3
A:T
2
6
2
G:C
Base pairing via
hydrogen bonds
1
C1 Nucleic Acid Structure
•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, lefthanded helical, one
groove
C1 Nucleic Acid Structure
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)
rRNA
Ribozyme RNA
C1 Nucleic Acid Structure
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
Fibrous protein
Globular protein
Structural proteins • Enzymes,
• antibodies,
• receptors etc
Nucleic Acids
Helical DNA Globular RNA
Genetic
information
maintenance
•Ribozymes
•Transfer RNA (tRNA)
•Signal recognition e.g.
7SL.
C1 Nucleic Acid Structure
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).
C2 Chemical and Physical Properties of Nucleic Acids
1. Stability of Nucleic Acids
2. Effect of Acid & applications
3. Effect of alkali & applications
Chemical properties
4. Chemical denaturation
5. Viscosity & applications
6. Buorant density & application
Physical properties
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C2 Chemical and Physical Properties of Nucleic Acids
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
C2 Chemical and Physical Properties of Nucleic Acids
Effect of Acid & applications
Strong acid + high temperature  completely hydrolyzed to
(perchloric acid+100°C) bases, riboses/deoxyribose, and phosphate
脱嘌呤核酸
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)
C2 Chemical and Physical Properties of Nucleic Acids
Effect of Alkali & Application
DNA denaturation at high pH
keto form
keto form
enolate 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
C2 Chemical and Physical Properties of Nucleic Acids
Effect of Alkali & Application
RNA hydrolyzes at higher pH because of 2’-OH groups in RNA
2’, 3’-cyclic phosphodiester
alkali
OH
RNA is unstable at higher pH
free 5’-OH
C2 Chemical and Physical Properties of Nucleic Acids
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
C2 Chemical and Physical Properties of Nucleic Acids
Viscosity粘性
Reasons for the DNA high viscosity
1. High axial ratio
2. 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
C2 Chemical and Physical Properties of Nucleic Acids
Buoyant density
1.7 g cm-3, a similar density to 8M CsCl. Rho=1.66+0.098 (GC)%
Purifications of DNA: equilibrium density gradient centrifugation
Protein floats
RNA pellets at the bottom
back
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
RNA is 260 nm (lmax = 260 nm)
• Application: detection, quantitation, assessment of purity
(A260/A280)
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 acids
Extinction coefficient (e): 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 (e :
purines > pyrimidines)
(2) The absorbance values also depend on the amount of secondary structures
due to hypochromicity.
4. Purity of DNA
A260/A280:
dsDNA--1.8
pure RNA--2.0
protein--0.5
5. Thermal denaturation/melting: heating leads to the destruction of doublestranded hydrogen-bonded regions of DNA and RNA.
RNA: the absorbance increases gradually and irregularly
DNA: 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 decreased
Slow cooling: Whole complementation of dsDNA.
Absorbance decreases greatly and cooperatively.
Annealing退火: Base paring of short regions of
complementarity within or between DNA strands.
(example: annealing step in PCR reaction)
Hybridization: Renaturation of complementary sequences
between different nucleic acid molecules.
(examples: Northern or Southern hybridization)
C4 DNA Supercoiling
(General understanding but not details are required)
1. Almost all DNA molecules in cells are on average negatively
supercoiled.
2. Supercoiled DNA has a higher energy than relaxed DNA.
Negative supercoiling may thus facilitate cellular processes
which require the unwinding of the helix, such as
transcription initiation or replication
3. Topoisomerases异构酶exist in the cell regulate the level of
supercoiling of DNA molecules. (important to know in the
sense of gene expression)
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.
2.
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).
For a given isomer of a circular closed DNA (Lk = 0), the increase in
twist will cause a corresponding decrease in writhe.
Relaxed
closed circular
Lk = Lko
Break the circular DNA
twist 4 x 360o
Untwist 4 x 360o
Re-join the DNA
Lk = Lko + 4
Lk = + 4
Lk = Lko – 4
Lk = -4
positively
supercoiled
Negatively
supercoiled
DNA isolated from cell negatively supercoiled by
~ 6 turns per 100 turns of the helix. Lk / Lk = -0.06
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)