Chapter 5. Protein Structure
I.
II.
III.
IV.
V.
VI.
VII.
Introduction
Primary structure (一级结构)
Secondary structure (二级结构)
Tertiary and quaternary structure (三、四级结构)
Protein denaturation and folding (变性和折叠)
Structure-function relationship (结构与功能的关系)
Methods for studying protein conformation (研究蛋
白质构象的方法)
1
I. Introduction
1. Milestones in the research of protein
structure (蛋白质结构研究的里程碑)
2. Overview of protein structure
3. Important forces stabilizing the protein
structure (稳定蛋白质结构的重要作用力)
4. Four levels of protein structure (四级蛋白
质结构)
2
1. Milestones in the research of protein structure
I. Introduction
When?
What?
1920s
Crystallization of several
proteins
1948
-Helix structure
Linus Pauling
1953
Amino acid sequence of
bovine insulin
3-dimensional structure of
myoglobin
3-dimensional structure of
hemoglobin
Frederick Sanger
1950s
1959
Who?
John Kendrew
Max Perutz &
John Kendrew
3
2. Overview of protein structure
I. Introduction
1) An isolated protein has a unique structure.
Amino acid sequence  3-dimensional structure
 biological functions
2) Protein structure is stabilized largely by:
 Covalent bonds (peptide bonds, disulfide
bonds)
 4 noncovalent interactions
 Water
3) There are some common structural patterns.
4) Each protein has a flexible structure.
4
3. Important forces stabilizing the
protein structure
I. Introduction
5
4. Four levels of protein structure
6
II. Primary Structure (一级结构)
1. Importance of amino acid sequence
(氨基酸序列的重要性)
2. Determination of amino acid sequence
(氨基酸序列的测定)
7
1. Importance of amino acid sequence
1) Relationship between amino acid
sequence and biological function
II. Primary structure
(氨基酸序列和生物功能之间的关系)
2) Protein homology among species
(蛋白质的同源性)
3) Molecular disease
8
1) Relationship between amino acid sequence
and biological function
Amino acid sequence 
3-dimensional structure 
biological function

II. Primary structure
Proteins with different functions
always have different amino acid
sequences.
 Thousands of genetic diseases are
traced to the production of defective
proteins.
 Functionally similar proteins from
different species often have similar
Cow, pig, dog,
amino acid sequences.
goat, horse
e.g. Ubiquitin (?)
Bovine insulin (牛胰岛素)
Human, pig,
dog, rabbit,
sperm whale
9
2) Protein homology among species
(蛋白质的同源性)
P.181
Homologous proteins
(同源蛋白质)
II. Primary structure
Invariant residues
(不变残基)
Cytochrome c
Variant residues (可变残基)
Conservative
MW ~12,500
~100 amino acid residues
28 invariant residues
Nonconservative
10
P.182
Phylogenetic tree (进化树) – cytochrome c
II. Primary structure
11
What can we get from the study of protein homology?


Invariable residues -- more critical to the structure and function
of a protein than variable ones.
Variable residues:
 No. of difference  phylogenetic difference between species (氨基酸差异
数与物种间的系统发生差异成正比)
 No. of difference  Divergence history (氨基酸差异数与物种进化的分
歧时间成正比)

Relations between sequence homology and biological activities of
proteins from different species:
 Proteins with related functions often show a high degree of sequence
similarity. (有相关生物功能的蛋白质经常表现出高度的序列同源性)
e.g. the globin family, P.185
 Proteins with strong sequence homology may show divergent biological
functions. (序列同源性很高的蛋白质会显示出趋异的生物功能)
e.g. the serine protease family, P.185

Different proteins may share a common ancestry
 Lysozyme (溶菌酶) vs. -lactalbumin (乳白蛋白)
 Actin (肌动蛋白) vs. hexokinase (己糖激酶)
12
3) Molecular disease
– Sickle-cell anemia (镰刀形红细胞白血病)
Gene mutation
基因突变
(base substitution)
Protein mutation
蛋白质突变
(amino acid substitution)
 Inherited blood disorder
 Molecular disease
 Caused by mutant hemoglobin
(突变型血红蛋白)
 Product of gene alteration
neutral
nonfunctional
Symptoms
Excruciating pain
Eventual organ damage
Earlier death
Segments of -chain in HbA and HbS
Human adult hemoglobin (HbA): Val-His-Leu-Thr-Pro-Glu-Glu-LysSickle-cell hemoglobin (HbS):
13
Val-His-Leu-Thr-Pro-Val-Glu-Lys-
14
Gene mutation
HbS
(lower O2-carrying capacity)
Hemoglobin: HbA
-O2
Deoxy-HbS
Red blood cells: cup-shaped
Sickled-shaped
(shorter lifetime)
Blockage
Anemia
15
2. Determination of amino acid sequence
II. Primary structure
1）General steps (一般步骤): P.168-181
2）Separating polypeptide chains（分离多肽链）
3）Identifying the N-terminal (确定N端)
4）Identifying the C-terminal (确定C端)
5）Fragmenting the polypeptide chain（部分断裂
多肽链）
6）An example of sequencing a polypeptide (肽链
测序举例)
7）Other methods for sequencing (测序的其他方法)
16
P.168-181
1) General steps
II. Primary structure
a.
b.
c.
d.
e.
Determine the No. of polypeptide chains
Separate each polypeptide chain
Determine amino acid composition of each chain
Identify amino acid residues at C- and N-terminals
Cleave the polypeptide chain into shorter fragments
and determine amino acid composition and sequence
of each fragment
f. Repeat step e to generate a different and overlapping
set of fragments
g. Reconstruct the overall amino acid sequence of the
protein
h. Locate disulfide bonds
17
2) Dissociation of multimeric proteins
(多聚蛋白质亚基的解离)
 Subunits
associated by non-covalent forces:
-- Exposure to:
II. Primary structure
 pH extremes
 6M guanidine hydrochloride (盐酸胍)
 8M urea (尿素)
H2N
C
NH2
NH
Guanidine
 Subunits
H2N
C
NH2
O
Urea
associated by covalent S-S bridges:
-- Cleavage of S-S bonds
18
Cleavage of disulfide bridges
P.171
HCOOOH (过甲酸):
H
N
CH
O
O
H
N
C
CH2
HCOOOH
(performic acid)
SO3SO3-
S
II. Primary structure
H
N
CH
C
CH2
S
H2C
CH
O
H2C
H
N
C
O
CH
C
Mercaptoethanol (巯基乙醇):
O
O
H
N
CH
C
H
N
HSCH2CH2OH
(2-mercaptoethanol)
H2C
H
N
CH
ICH2COOH
(iodoacetic acid)
H2C
O
C
H
N
CH
CH
C
CH2
S-CH2-COO-
SH
SH
S
H
N
C
CH2
CH2
S
CH
O
Br(CH2)3NH2
3-bromopropylamine
S-CH2CH2CH2-NH2
O
C
H2C
H
N
CH
O
C
19
P.169
3) Identification of the N-terminal
(N端的确定)
II. Primary structure
a.
b.
c.
d.
DNFB – Sanger’s method
DNS
PITC – Edman’s method
Amino peptidase (氨肽酶)
20
Identification of N-terminal residues
NO2
NO2
NO2
Sanger’s method
(DNFB法)
NH2
R1 CH
C O
NO2
F
NO2
NH
H+
R1 CH
NH
R1 CH
C O
R2 CH
COOH
NH
C O
DNP-amino acid
(yellow)
NO2
NH
+
Mixture of free amino acids
R2 CH
C O
H3C
H3C
II. Primary structure
DNS method
(丹黄酰氯法)
H3C
O S O
Cl
DNS-amino acid
(fluorescent)
N
C O
NH
C O
C O
CH3
O S O
C O
H+
NH
R1 CH
R2 CH
N
CH3
N
R1 CH
NH
CH3
O S O
NH2
C O
N
N
NH
R2 CH
R1 CH
COOH
+
Mixture of free amino acids
C O
Edman’s method
(PITC法)
NH2
R1 CH
C O
NH
R2 CH
Can also be used
for sequencing
C O
NH
N
C
S
O
C S
NH
R1 CH
C O
NH
R2 CH
C O
CF3COOH
C
N
CH
R1
C
S
PTH-amino acid
(colorless)
NH
+
NH3+
R2 CH
C O
Shortened peptide
21
P.170
4) Identification of the C-terminal
(C端的确定)
II. Primary structure
a. NH2NH2 (肼解)
b. LiBH4 (还原)
c. Carboxypeptidase (羧肽酶)
22
Identification of C-terminal residues
Hydrazinolysis (肼解法)
O
H
N
O
H
N
CH C
O
H
N
CH C
R3
CH C
R2
OH
100oC
R1
O
+ H2N CH C
NH2NH2
NHNH2
O
O
+ H2N CH C
NHNH2 + H2N CH C
II. Primary structure
R3
R2
OH
R1
Reduction (还原法)
O
H
N
CH C
O
H
N
R3
CH C
H
N
R2
O
H
N
O
CH C
CH C
OH
LiBH4
R1
O
H
N
R3
CH C
R2
+ H2N CH COOH
R3
H
N
CH CH2OH
H2O
R1
+ H2N CH COOH + H2N CH CH2OH
R2
R1
23
P.173
5) Fragmenting the polypeptide chain
O
(部分断裂多肽链)
H
N
CH C
H
N
R1
Methods
CH C
H
N
R2
R1-amino acid
Trypsin (胰蛋白酶)
Lys, Arg
Chymotrypsin
(胰凝乳蛋白酶)
Phe, Trp, Tyr
Thermolysin
Enzymatic (嗜热菌蛋白酶)
Pepsin (胃蛋白酶)
O
R2-amino acid
Leu, Ile, Phe, Trp,
Val, Tyr, Met
Phe, Leu, Trp, Tyr
Phe, Leu, Trp, Tyr
Staphylococcal protease Glu, Asp
Chemical
Clostripain
Arg
CNBr
Met
NH2OH
Asn -- Gly
24
6) An example
Amino acid
composition
N-terminal
水解
DNFB, 定N-末端
Sanger’s method
Cleavage of
S-S bridge
II. Primary structure
胰蛋白酶
Edman degradation
Fragmentation
CNBr
Sequencing
25
7）Other methods for sequencing
II. Primary structure
 Exopeptidases (用外切酶的酶降解法): P.177
 Mass spectroscopy (质谱): P.177
 DNA sequencing method (根据DNA序列的
推定法): P.177
 Protein database (蛋白质数据库): P.181
26
III. Secondary Structure
1. Peptide plane (肽平面)
2. Common patterns of secondary structure (常见
二级结构元件)
3. Characteristic bond angles and amino acid
probabilities for common secondary structures
(常见二级结构元件的二面角和各氨基酸出现
的几率)
27
P.204
1. Peptide plane (肽平面)
-carbon
III. Secondary structure
1) Fundamental structural unit in
all proteins
2) Formed by peptide bond
 Partial double bond
character
 trans-Configuration:
C vs C
 Free rotation: Co-C, C-N
3) Rigid and planar
4) Each C is a joining point for
two adjacent peptide planes
-carbon
28
-
O
C
C
C
O-
O
N
H
C
C
+
N
H
C
C
C
+
C
N
H
29
P.204
Dihedral angles:  and  (二面角)
C
 Definition about the two angles:
P.204
 Peptide backbone structure is
determined by specified  and
.
 Varying range for both angles:
180o ~ +180o
 Some values of  and  are not
sterically allowed. For example,
  =  = 0o
  = 0o,  = 180o
  = 180o,  = 0o
Co
N
C
30
P.204
Definitions about  and  (二面角的定义)
C
  and 
 -- C-N bond
 -- C-Co bond
 0o
Co
For both  and , 0o corresponds to an
orientation with the amide plane
bisecting the H-C-R plane and a cisconfiguration of the main chain around
the rotating bond in question.
N
 “+” and “”
For both  and , when looking from C
along the bond
 Clockwise rotation – “+”
 Anti-clockwise rotation – “”
C
31
 =  = 0o
Prohibited
 =  = 180o
Fully extended
32
Ramachandran plot (拉氏构象图)
允许区
III. Secondary structure
不完全允许区
不允许区
33
2. Common protein secondary structure
(常见二级结构元件)
1) -Helix (螺旋)
2) -Pleated sheet (折叠片)
3) -Turn (转角)
34
P.207
1) -Helix (螺旋)
III. Secondary structure
A. Structural parameters (结构参数)
 Each turn: 0.54 nm, 3.6 residues, 13 atoms – 3.613 helix
 Each residue: 100o, 0.15 nm
  ~ -57o,  ~ -47o
 Right-handed helix (右手螺旋)
B. Chirality and optical activity: Right-handed helix
C. Stability: Optimal use of internal H-bonds (充分利用链内
氢键)
D. Dipole
(偶极)
Myoglobin
(肌红蛋白)
35
-Helix
C
O
H
N
Each turn:
0.54 nm
3.6 residues
13 atoms
Each residue:
100o
 0.15 nm
36
37
H-bonding in an -Helix
III. Secondary structure
3.613-helix
O
C
H
H
N
H
C
C
N
3
R
O
38
Chirality & optical activity
(手性和旋光性)
Dipole & polarity
(偶极和极性)
Always
righthanded
蛋白质中的
螺旋几乎
都是右手的
Right- and left-handed -helices are not enantiomers to each other.
39
(右手螺旋和左手螺旋不是对映体)
Amino acid sequence affects stability of the -Helix
P.208
a) Electrostatic repulsion
between successive R groups
(R基的电荷)
b) Bulkiness of adjacent R
groups (R基的大小)
c) Interactions between amino
acid side chains spaced 3-4
residues apart (相隔3-4个残
基的R基之间的相互作用)
d) Pro and Gly – both rarely
found in  helices
e) Interactions between amino
acid residues at the terminals
(肽链末端残基的相互作用)
40
Poly(Lys):
O
H
N
H
C
O
pKa = 10.5
C
n
H
N
H
C
C
(CH2)4
(CH2)4
NH3+
NH2
n
Poly(Glu):
O
H
N
H
C
C
O
pKa = 4.2
n
H
N
H
C
C
(CH2)2
(CH2)2
COOH
COO-
41
n
-Pleated sheet / -conformation
P.209
(折叠片)
Common features:
•
 -pleated sheets form when two or more polypeptide chain
segments line up side by side (由两条或两条以上肽链片段
并排而成).
 Each such segment is referred to as a -strand (折叠片中每
条肽链称为折叠股).
 Each -strand is fully extended to make a zigzag pattern (每条
折叠股充分伸展成锯齿状).
 -pleated sheets are stabilized by interstrand (but not
intrastrand) H-bonds (起稳定作用的氢键是在股间而不是在
股内形成).
 Location of individual segments that form a -pleated sheet
(形成折叠片的肽链片段可以位于不同肽链上或同一条肽
链的相邻或相距很远的位置)
 The R groups of adjacent amino acid residues (每条折叠股
上相邻的氨基酸残基的R基团交替出现)
42
-Pleated sheets: parallel & antiparallel
C
III. Secondary structure
反平行式
平行式
43
反平行式
平行式
Antiparallel -sheets are more stable than parallel -sheets.
When two or more βsheets are layered within a protein, the R groups of the amino
acid residues on the touching surfaces must be relatively small.
e.g. β-Karatins have a very high content of Gly and Ala.
44
Parallel & antiparallel -pleated sheets:
A comparison
Parallel (平行)
Antiparallel (反平行)
NC direction of the strands (股取向)
Same direction
(同向)
Opposite direction
(反向)
Stability (稳定性)
Less stable
More stable
Repeat period (重复周期)
Shorter (0.325 nm)
Longer (0.347 nm)
&
(二面角)
Smaller
Bigger
 = 119o
 = 139o
 = +113o
 = +135o
Larger (>5 strands)
Smaller (~2 strands)
More regular
Less regular
Structure
(结构)
Hydrophoblic side chains
(疏水侧链)
On both sides of the sheet On one side of the sheet
(在折叠片的两边)
(在折叠片的同一边)
45
3) -Turn (转角)
P.211
 Common,
III. Secondary structure
especially in
globular proteins
 180o turn involving 4
amino acid residues and 3
peptide planes
 Amino acids that often
occur in -turns:
Gly – small & flexible
Pro – cyclic structure
with a fixed 
 Commonly connecting the
ends of two adjacent
segments of an antiparallel
 sheet
 -Turns are often found
near the surface of a protein.
46
3. Characteristic bond angles and amino acid probabilities
for common secondary structures
(常见二级结构元件的二面角和各氨基酸出现的几率)
47
IV. Tertiary and quaternary structures
1. Higher levels of protein structure:
tertiary and quaternary structures
2. Fibrous proteins
3. Globular proteins
4. Two important terms related to protein
structural patterns: motif and domain
5. Classification of globular proteins
6. Quaternary structure
48
1. Higher levels of protein structure
① Tertiary structure
 overall three-dimensional structure of a protein,
especially when it is composed of a single polypeptide
chain.
② Quaternary structure
 three-dimentional structure of a multisubunit protein
③ Fibrous and globular proteins
49
Fibrous and globular proteins
(纤维状蛋白和球状蛋白)
Polypeptide Secondary
Chain
Structure
(多肽链)
(二级结构)
Fibrous protein
Long
Single
type
(纤维状蛋白) strands/sheets
Globular protein
(球状蛋白)
Folded in a
spherical
shape
Several
types
Solubility Biological
in water
Functions
(水溶性) (生物功能)
Insoluble
Support,
shape,
protection
Soluble
Catalysis,
regulation,
etc.
50
2. Fibrous proteins
 Containing polypeptide chains organized





approximately parallel along a single axis
Producing long fibers or large sheets
Mechanically strong
Resistant to solubilization in water
Playing a structural role in nature
Containing single type of secondary structure
51
Examples of Fibrous proteins
-Keratin (-角蛋白) – -helix
 Fibroin (丝心蛋白) – antiparalle -pleated sheet
 Collagen (胶原蛋白) – triple helix

52
Structure of hair
Right-handed helix
Left-handed supertwist
1 intermediate filament = 4 protofibrils
= 8 protofilaments
= 16 coiled coils
= 32 strands of -keratins
53
Collagen
Gly
54
Structure of Collagen

Rod-shaped molecule with a MW of 300,000:



3000 Å in length (~1000 amino acid residues)
15 Å in thickness
Three-stranded superhelix made up of 3 -chains
(-chain  -helix)


Each -chain – left-handed (3 AA per turn)
Triple helix – right-handed
Amino acid sequence in the -chain repeating as
Gly-X-Pro, Gly-X-HyPro
 Amino acid contents:

35% Gly, 11% Ala, 21% Pro & HyPro

Tight wrapping  tensile strength (greater than a
steel wire of equal cross section)
55
Permanent waving is biochemical engineering
Rearrangement of H bonds:
-conformation
-Helix
Cooling
(-keratin)
(-keratin)
Rearrangement of S-S bonds and H bonds:
Moist heat
56
P.228
3. Globular proteins (球状蛋白)
1) Containing several types of secondary /supersecondary
structures and domains (含多种二级/超二级结构和结构域)
2) Compact globular structure due to efficient packing (紧密球
状结构)
3) Hydrophobic side chains (疏水侧链) – inside
Hydrophilic side chains (亲水侧链) – outside
4) Cavities/clefts on the surface allow for substrate or ligand
binding. (分子表面的空穴/裂沟是底物等配体与蛋白质结
合的场所)
5) Residues distant from each other in the primary structure
come into close proximity. (在一级结构中彼此远离的基团
在球状蛋白结构中互相靠近)
57
Surface contour (表面轮廓图解)
Ribbon representation (带式图解)
细胞色素 c
溶菌酶
核糖核酸酶
58
Approximate amounts of -helix and -pleated sheet
in some single-chain proteins
Protein
Residues (%)
-Helix
-Pleated sheet
Chymotrypsin (胰凝乳蛋白酶)
14
45
Ribonuclease (核糖核酸酶)
26
35
Carboxypeptidase (羧肽酶)
38
17
Cytochrome c (细胞色素 c)
39
0
Lysozyme (溶菌酶)
40
12
Myoglobin (肌红蛋白)
78
0
59
Myoglobin (肌红蛋白)
MW 16,700
1
single polypeptide chain
153 amino acid residues
1
heme group
8 -helices
60
4. Motif and domain
 2 important terms that describe protein
structural patterns or elements in a polypeptide
chain
 Motif / supersecondary structure / fold
 a recognizable folding pattern involving two or more
elements of secondary structure and the connection(s)
between them.
 Domain
 a part of a polypeptide chain that is independently
stable or could undergo movements as a single entity
with respect to the entire protein.
61
Supersecondary structures (超二级结构)
P.220
Supersecondary structures are particularly
stable arrangements of several elements of secondary
structure and the connections between them.
1) 




A bundle of -helices (螺旋束)
Each -helix -- Right-handed (右手螺旋)
Final coiled coil -- Left-handed (左手卷曲螺旋)
Major structure pattern for fibrous proteins such as keratin (是纤维状蛋白质的主要结构模式)
2) 

Two parallel -pleated sheets are connected by an -helix
segment
3) 

Antiparallel -pleated sheets are connected by -turns.
62
Supersecondary structures


Rossman
折叠

: -hairpin
(发夹)
: Greek key (希腊钥匙拓扑结构)
: -meander
(曲折)
63
Domains
 Polypeptides often fold into two or more stable, globular
units called domains.
 A domains usually retains its 3-dimensional structure even
when separated from the remainder of the polypeptide
chain.
 Different domains often have distinct functions.
 An enzyme’s active site is usually located at a position
between two domains. (see Fig.5-35, P.224)
Troponin C with two
separate Ca-binding domains
64
P.222
Immunoglobulin structure
65
P.224
5. Classification of globular proteins (分类)
1)
2)
3)
4)
All  (全-结构蛋白质) – Myoglobin
All  (全 -结构蛋白质)
/ or + (, -结构蛋白质)
Metal- and disulfide-rich proteins
(富含金属或二硫键蛋白质)
66
6. Quaternary Structure
(四级结构)
① Hemoglobin (血红蛋白)
② Symmetry of quaternary structure (四级结
构的对称性): P.246
③ Forces driving quaternary association (四级
缔合的驱动力): P.243
④ Structural and functional advantages of
quaternary association (四级缔合在结构和
功能上的优越性): P.247
67
①Hemoglobin (血红蛋白)
 subunit
Hemoglobin:
MW 64,500
4 Subunits
2 + 2
4 Heme groups
Heme
Ribbon presentation
 subunit
Space-filling model
68
P.246
②Symmetry of quaternary structure
(四级结构的对称性)
1) Rotational symmetry (旋转对称) – closed structure
The subunits are arranged around a single
rotation axis: e.g. C2, C3, C5
 Dihedral symmetry (二面体对称) : A structure
possesses at least one 2-fold rotation axis
perpendicular to another n-fold rotation axis.
 Tetrahedral symmetry (四面体对称)
 Octahedral symmetry (八面体对称)
 Icosahedral symmetry (二十面对称)
2） Helical symmetry (螺旋对称) – open structure
69
Rotational symmetry
in proteins
二十面体对称
二面角对称
70
P.243
③Forces driving quaternary association
(四级缔合的驱动力)
1) Subunit association is accompanied by both favorable
and unfavorable energy changes.
2) Favorable :
 Van der Waals interactions
 H bonds
 Ionic bonds
 Hydrophobic interactions
 Disulfide bonds
3) Unfavorable:
 Entropy loss (熵的减少)
Immunoglobulin (免疫球蛋白)
71
④Structural and functional advantages of
quaternary association
(
四级缔合在结构和功能上的优越性
)
P.247
1) Structural stability – decreased surface/volume ratio
 Increasing interactions within the protein
 Shielding hydrophobic residues from water
2) Genetic economy and efficiency
 Genetic code capacity
 Accuracy of the protein biosynthesis process
3) Bringing catalytic sites together
 Bringing necessary catalytic groups together to form an active
enzyme (e.g. glutamine synthetase)
 Carrying out different but related reactions on different subunits
(e.g. tryptophan synthase)
4) Cooperativity (协同性) & allosteric effect (别构效应)
72
Tryptophan synthase (色氨酸合酶): 22
Indoleglycerol phosphate
-subunit
Glyceraldehyde-3-phosphate + Indole
Indole + L-Serine
-subunit
L-Tryptophan
73
P.247
Cooperativity (协同性) & allosteric effect (别构效应)
Positive cooperativity (正协同性)
Cooperativity
(协同性)
Positive – activator (激活剂)
Effector
Negative – inhibitor (抑制剂)
Negative cooperativity (负协同性)
Allosteric effect
(别构效应)
Homotropic effect
(同促效应)
Substrate = Effector
(底物 = 效应物)
Heterotropic effect
(异促效应)
Substrate  Effector
(底物  效应物) 74
Subunit interactions in an allosteric enzyme
75
P.233
V. Protein Denaturation and
Folding (变性和折叠)
1. Denaturation and renaturation
(变性与复性)
2. Denaturing conditions (变性条件)
3. The Anfinsen experiment
4. The mad cow disease
76
1. Denaturation and renaturation
(蛋白质的变性和复性)
Native, folded state
(天然折叠状态)
Denaturation (变性)
Renaturation (复性)
Denatured, unfolded state
(变性的解折叠状态)
Denaturation leads to the change in:
Structure (结构)
Biological functions (生物活性)
Physical/chemical properties (物理化学性质)
Biochemical properties (生物化学性质)
Attention:
Denatured state  completed unfolded state (蛋白质变性不等于其结构完全解折叠)
Denaturation does not involve the cleavage of the primary structure (蛋白质变性不涉
及一级结构共价键的破裂)
77
2. Denaturing conditions (变性条件)
V. Denaturation and Folding
1)
2)
3)
4)
5)
6)
7)
pH extremes (强酸强碱)
Organic solvents (有机溶剂)
Denaturing agents (变性剂)
Reducing agents (还原剂)
Heavy metal ions (重金属离子)
Heat (热变性)
Mechanical stress (机械应力)
78
Denaturing conditions (1): pH extremes
(强酸强碱的变性作用)
V. Denaturation and Folding
 Use
of pH extremes results in a change in
the protein structure due to:
 Protonation/deprotonation (质子化/去质子化)
of some protein side groups
 Alteration of H-bonding and salt bridge
patterns
 Control
of pH to approach the protein’s pI
results in precipitation of the protein
79
Denaturing conditions (2): Organic solvents
V. Denaturation and Folding
 Interacting with
polar and nonpolar R
groups of the protein
 Forming H bonds with water and polar
protein groups
 Disrupting hydrophobic interactions
within the protein molecule
80
Denaturing conditions (3): Denaturing agents

Urea and guanidine HCl:
 Competing with protein for H bonds
– disrupting the secondary structure
 Minimizing hydrophobic interactions
– disrupting the tertiary structure

SDS:
 Disrupting hydrophobic interactions –
inside out
 Causing the protein to unfold into
extended polypeptide chains
H2N
C
NH2
H2N
C
NH2
O
NH
Guanidine
胍
O
Urea
尿素
O
S
+
O- Na
O
SDS
81
Denaturing conditions (4): Reducing agents
V. Denaturation and Folding
Reducing agents (e.g. -mercaptoethanol) are
used along with denaturating agents (e.g. urea).
 Urea – to unfold the protein so that the S-S
bonds inside the protein are exposed
 -mercaptoethanol – to cleave the S-S bonds
82
Denaturing conditions (5): Heavy metal ions

V. Denaturation and Folding
Heavy metal ions (e.g. Hg2+, Pb2+) affect protein
structure by:
Disrupting salt bridges by forming ionic bonds with
negatively charged groups
Bonding with –SH groups

Anemia is one symptom of lead poisoning
83
Denaturing conditions (6): Heat
Melting temperature (Tm)
Higher temperature results in the disruption of weak interactions such as
H-bonds and in turn the unfolding of the protein.
84
P.234
3. The Anfinsen experiment
VI. Denaturation and Folding
加入尿素，巯基乙醇
Christian B. Anfinsen
(1916-1995)
除去尿素，巯基乙醇
Denaturation and
redenaturation of
ribonuclease
(核糖核酸酶的变性与复性)
An early evidence that the 3dimensional structure of a
protein is determined by its
amino acid sequence 85
4. The mad cow
disease
The Nobel Prize in
Physiology/Medicine 1997
“for his discovery of Prions – a new
biological principle of infection”
Stanley B. Prusiner
University of California
School of Medicine
San Francisco, CA, USA
86
Mad cow disease
(疯牛病)
Bovine spongiform encephalopathy
(海绵状脑软化症)
Infectious agent
Prion protein (PrP, MW 28,000)
Misfolding
Rich in -helix
PrPc
Rich in -sheets
PrPsc
PrPsc
87
VI. Structure-function relationship
(蛋白质的结构与功能的关系)
1. Overview
2. Oxygen-binding proteins: Myoglobin &
hemoglobin
3. Myoglobin (肌红蛋白)
1)
2)
3)
Structure
Effect of protein structure on O2 binding
O2-binding curve
4. Hemoglobin (血红蛋白)
1)
2)
3)
4)
Structure
Structural change upon O2 binding
Cooperative O2 binding
Effect of H+, CO2 and BPG on O2 binding
88
1. Overview
VI. Structure-function relationship
1. Protein functions involve reversible binding of
ligands (蛋白质与配体的可逆结合)
 Ligand
(配体) and binding site (结合位点)
 Complementary (互补): size, shape, charge, polarity
 Specific (专一)
2. Proteins have flexible conformations (构象易变)
 Essential to
protein functions
 Induced fit (诱导契合)
3. Protein-ligand interactions can be regulated (蛋白质配体相互作用可被调节)
 Allosteric effect
(别构效应)
89
2. Oxygen-binding proteins (载氧蛋白)
– Myoglobin & hemoglobin
1)
2)
3)
Both are oxygen-binding heme proteins (氧合血红素蛋白质)
-- Conjugated proteins (缀合蛋白质)
Both belong to the globin family (珠蛋白家族)
-- Homologous proteins (同源蛋白质)
-- Similar in secondary and tertiary structure (结构相近)
Differences (区别)
MW
Subunits
No. of AA
Existence
Function
Myoglobin
(肌红蛋白, Mb)
16,700
1
153
Muscle tissue
O2 storage
Hemoglobin
(血红蛋白, Hb)
64,500
4
(2 + 2)
141 + 146
Red blood cells O2 transport
90
Heme Prosthetic group
(血红素辅基)
Oxygen Binding to Heme
Fe2+ (sp3d2) -- reversible binding to O2
Fe3+ (sp3d) -- no binding to O2
Arterial blood – O2-rich, bright red
Venous blood – O2-depleted, dark purple
91
Amino acid sequences of whale myoglobin and , 
chains of human hemoglobin
92
Structural Similarities
Between Myoglobin and Hemoglobin
VI. Structure-function relationship
肌红蛋白
血红蛋白
93
3. Myoglobin
VI. Structure-function relationship
1) Structure
2) Effect of protein structure
on O2 binding
3) O2-binding curve
94
P.252
1). Structure
C-terminal
N-terminal
Found in muscle tissues of
almost all mammals
MW 16,700
1 subunit of 153 amino
acid residues
1 heme group
8  helix segments (A-H)
78% of amino acid
residues involved in 
helices
Important amino acid
residues:
His93 – His F8 –
Proximal (近侧)
His64 – His E7 –
Distal (远侧)
95
2). Effect of protein structure on Oxygen binding
(蛋白结构对结合氧的影响)
P.254
 O2-binding site (氧结合位点)
 Molecular motions (分子运动)
Distal His
 Cavity produced by Val E11
(远侧)
& Phe CD1 (Val E11 和Phe
CD1之间产生的空穴)
 H bonding with His E7 (与
His E7产生的氢键)
 Affinity for different ligands (与
不同配体结合的亲合度): CO/O2
 Free heme: 20,000
 Heme in myoglobin: 200
Proximal His  Fe2+ is protected from being
(近侧)
oxidized (蛋白分子的疏水环境
使Fe2+不容易被氧化成Fe3+ )
 O2 binding leads to a minor
change in protein conformation
(结合氧使蛋白构象发生轻微变化96
)
P.255
3) O2-binding curve (氧结合曲线)
Mb + O2
MbO2
Dissociation constant
(解离常数)
K=
[O2]
[O2] + K

[Mb][O2]
[MbO2]
Fractional saturation Y =
(氧分数饱和度)
Binding sites occupied
Total binding sites
[MbO2]
=
[MbO2] + [Mb]
O2-binding curve
(氧合曲线)
pO2
[O2]
=
pO2 + K
[O2] + K
pO2
Y=
=
pO2 + P50
When Y = 0.5, pO2 = K = P50
Hill plot
Y
log
1-Y
= log pO2 – log K
97
Hill plot for the binding of O2 to myoglobin
log Y
1-Y
log
Y
= log pO2 – log K
1-Y
Slope = 1.0
Y = 0.5
Y
0
log P50
log pO2
Oxygen binds tightly to myoglobin with a P50 = 0.26 kPa
98
4. Hemoglobin (血红细胞)
VII. Structure-function relationship
1) Structure (结构)
2) Structural change upon oxygen binding
(氧结合引起的结构变化)
3) Cooperative oxygen binding (协同性的
氧结合)
4) Effect of H+, CO2 and BPG on oxygen
binding (H+, CO2, BPG对氧结合的影响)
99
P.257
1). Structure
 MW 64,500
 4 subunits (2 + 2)
 chain: 141 residues
 chain: 146 residues
Hemoglobin
 Structural similarity with
myoglobin (与血红蛋白结构
相似)
 Allosteric interactions (别构
作用)
 Responsible for O2 transport
O2 transport:
Arterial blood (动脉血): 96% saturated with O2
Venous blood (静脉血): 64% saturated with O2
100
2). Structural change upon oxygen binding
P.259



(氧结合引起的蛋白质结构变化)
Two major conformations of hemoglobin (两种主要构象态):
T state  R state
O2 has significantly higher affinity for hemoglobin in the R state
than in the T state (O2对R态比对T态更亲和).
Oxygen binding triggers the T  R transition (氧结合引发TR
的构象转变):




Shift of Fe (II), His F8 and Helix F towards the porphyrin plane (Fe (II),
His F8 and Helix F 向卟啉环平面的移动)
Disruption of old stabilizing interactions (一些稳定T态的相互作用被断裂)
e.g. Asp FG1(D) – His HC3 (H) salt bridge
Formation of new stabilizing interactions (一些稳定R态的新的相互作用
形成)
Narrowing of the pocket between the  chains (链之间形成的口袋变窄)
to release BPG
101
The T – R Transition
H: His146 or His HC3
D: Asp94 or Asp FG1
+O2
–O2
T state (tense, 紧张态)
Deoxyhemoglobin
R state (relaxed, 松弛态)
Oxyhemoglobin
(去氧血红蛋白)
(氧合血红蛋白)
102
Changes in conformation near heme
on O2 binding
Hemoglobin
103
Some ion pairs that stabilize the T state of deoxyhemoglobin
A close-up view of a portion
of a deoxyhemoglobin
molecule in the T state
Interactions between
ion pairs
104
O
HN
HN
CH
C
CH
O
HN
CH2
CH2
C
O
COOCH
C
O
(CH2)4
O
O-
Asp FG1 (Asp94)
NH
NH3+
+
HN
His HC3 (His146)
Lys C5
105
3). Cooperative oxygen binding
(协同性氧结合)

Sigmoidal O2-binding curve
(S形氧结合曲线)


Hill plot
Allosteric effect (别构效应)
Hemoglobin
106
Oxygen binding curves
O2: A ligand (配体)
An activator (激活剂)
A positive
homotropic effector
(正同促效应物)
R state
Y
Hemoglobin
T state
Myoglobin – Hyperbolic curve (双曲线)
Hemoglobin – Sigmoid curve (S形曲线)
107
Quantitative Measurement of O2 binding
P.261
Mb + O2
MbO2
Dissociation constant
(解离常数)
Fractional saturation
[Mb][O2]
K=
[MbO2]
Y=
(氧分数饱和度)
O2-binding curve
[MbO2]
[MbO2] + [Mb]
pO2
Y=
pO2 + K
(氧合曲线)
Hb(O2)4
Hb + 4O2
[Hb][O2]4
K=
[Hb(O2)4]
Y=
[Hb(O2)4]
[Hb(O2)4] + [Hb]
(pO2)n
Y=
Hyperbolic (双曲线)
(pO2)n + K
Sigmoidal (S形)
K = (P50)n
K = P50
Hill equation
(Hill方程)
log
Y
1– Y
= log pO2 – log K
Slope = 1
log
Y
= n log pO2 – log K
1– Y
Slope = n
108
Hill Plot
Hill equation: log Y
1–Y
= n log(pO2) – logK
n: Number of binding sites
nH: Hill coefficient (Hill系数)
 a measure of degree of cooperativity
Hemoglobin
nH = 1 Noncooperative binding (非协同性)
-- e.g. Myoglobin
nH > 1 Positive cooperativity (正协同性)
-- e.g. Hemoglobin
nH < 1 Negative cooperativity (负协同性)
nH = n Complete cooperativity (完全协同)
n  nH  1
109
P.262
Hill plots for the binding of oxygen to
myoglobin and hemoglobin
log Y
1-Y
Hemoglobin
110
4). Effect of H+, CO2 and BPG on oxygen binding
(H+, CO2, BPG对氧结合的影响)
P.263

H+, CO2 and BPG affect the oxygen binding properties of
hemoglobin via allosteric effect (通过别构效应影响氧结合).



Different ligands affect O2 binding of hemoglobin in different ways
(不同的配体对血红蛋白的氧结合有不同的影响机制)



O2 – Homotropic effector/activator (同促效应物/正协同性)
H+, CO2 and BPG – heterotropic effector/inhibitor (异促效应物/负协同性)
Bohr effect (Bohr效应) – Effect of pH and CO2 on the binding and
release of O2 by hemoglobin (pH和CO2对血红蛋白载氧和放氧的影响)



Heterotropic allosteric modulation (异促别构调节)
Negative cooperativity (负协同性)
In tissues: pH, [CO2]  O2 is released
In lung: pH, [CO2]  O2 is bound
O2 Binding to hemoglobin is regulated by BPG (氧结合受BPG调节)
111
Bohr Effect – pH：
HbH+ + O2
HbO2 + H+
 H+
and O2 are bound to hemoglobin
with inverse affinity (血红蛋白对H+和O2的
亲和力相反).
 In tissues: pH 7.2, low pO2  releasing O2
 In lungs: pH 7.6, high pO2  binding O2
 H+
and O2 are bound at different sites in
hemoglobin (H+和O2与血红蛋白在不同的结
合位点结合).
 O2 – binding to Fe(II) of the heme group
 H+ – binding to some amino acid residues,
particularly His146 of the  chain.
of the  chain makes a major
contribution:
Y
(in lungs)
 His146
[H+]  His146 protonated
 ion-pairing with Asp94
 stabilizing the T state
 releasing O2
 pKa of His146:
T state: pKa = 8.0, protonated at pH 7.2
R state: pKa = 6.0, unprotonated at pH 7.6
(in tissues)
112
Bohr Effect – CO2
 CO2 and O2 are bound to hemoglobin
(血红蛋白对CO2和O2的亲和力相反).
 When
with inverse affinity
[CO2] is high，
Hb-NH-COO- + H+
CO2 + Hb-NH2
Amino-terminal residue
Carbaminohemoglobin
(氨甲酸血红蛋白 )
Carbonic anhydrase
CO2 + H2O
(碳酸酐酶)
H+ + HCO3-
Hemoglobin
 Transport
of CO2 and O2 by hemoglobin:
In tissues: [CO2] is high  CO2 is bound, O2 is released
In lungs: [CO2] is low  CO2 is released, O2 is bound
113
Regulation of O2 binding by BPG
 Heterotropic
allosteric modulation (异促别构调节)
 BPG is present in erythrocytes (血红细胞).
 BPG lowers Hb’s affinity for O2 by stabilizing
the T state.
 Binding of BPG to hemoglobin:
HbBPG + O2
HbO2 + BPG
BPG is bound
+ charge
groups
2,3-二磷酸甘油酸
BPG is released
+O2
-O2
T state
R state
114
BPG plays an important
role in physiological
adaptation to the lower pO2
available at high altitudes
E
Y
Sea level Mountain
BPG
5 pm
8 pm
Lung
A
C
Tissue
B
D
0.38
0.37
Y
A
C
B
D
(Sea level)
(4500 m)
115
BPG plays an important role in fetal development
22hemoglobin
胎儿
母亲
22hemoglobin
Hemoglobin
The Hb in fetuses has a much lower affinity for BPG than normal adult
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Hb, and a correspondingly higher affinity for O2.
P.197
VIII. Methods for studying protein conformation
(研究蛋白质构象的方法)
1. X-ray diffraction (X射线衍射)
2. UV/Vis spectrophotography (紫外可见分光光
度测定)
3. Fluoresence and fluoresence polarization (荧光
和荧光偏振)
4. Circular dichroism (CD, 圆二色性)
5. Nuclear magnetic resonance (NMR, 核磁共振)
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