1. Chapter 5. Protein Structure
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)

2. I. Introduction
Milestones in the research of protein structure (蛋白质结构研究的里程碑)
Overview of protein structure
Important forces stabilizing the protein structure (稳定蛋白质结构的重要作用力)
Four levels of protein structure (四级蛋白质结构)

3. 1. Milestones in the research of protein structure
When?
What?
Who?
1920s
Crystallization of several proteins
1948
-Helix structure
Linus Pauling
1953
Amino acid sequence of bovine insulin
Frederick Sanger
1950s
3-dimensional structure of myoglobin
John Kendrew
1959
3-dimensional structure of hemoglobin
Max Perutz & John Kendrew
I. Introduction

4. 2. Overview of protein structure
An isolated protein has a unique structure.
Amino acid sequence  3-dimensional structure  biological functions
Protein structure is stabilized largely by:
Covalent bonds (peptide bonds, disulfide bonds)
4 noncovalent interactions
Water
There are some common structural patterns.
Each protein has a flexible structure.
I. Introduction

5. 3. Important forces stabilizing the protein structure
I. Introduction

6. 4. Four levels of protein structure

7. II. Primary Structure (一级结构)
Importance of amino acid sequence
(氨基酸序列的重要性)
Determination of amino acid sequence (氨基酸序列的测定)

8. 1. Importance of amino acid sequence
Relationship between amino acid sequence and biological function
(氨基酸序列和生物功能之间的关系)
Protein homology among species
(蛋白质的同源性)
Molecular disease
II. Primary structure

9. 1) Relationship between amino acid sequence and biological function
3-dimensional structure  biological function
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 amino acid sequences.
e.g. Ubiquitin (?)
II. Primary structure
Human, pig, dog, rabbit, sperm whale
Cow, pig, dog, goat, horse
Bovine insulin (牛胰岛素)

10. 2) Protein homology among species (蛋白质的同源性)
Homologous proteins
(同源蛋白质)
Variant residues (可变残基)
Invariant residues
(不变残基)
Conservative
Nonconservative
II. Primary structure
MW ~12,500
~100 amino acid residues
28 invariant residues
Cytochrome c

11. Phylogenetic tree (进化树) – cytochrome c
II. Primary structure

12. 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)

13. 3) Molecular disease – Sickle-cell anemia (镰刀形红细胞白血病)
neutral
Gene mutation
基因突变
(base substitution)
Protein mutation
蛋白质突变
(amino acid substitution)
nonfunctional
Inherited blood disorder
Molecular disease
Caused by mutant hemoglobin
(突变型血红蛋白)
Product of gene alteration
Symptoms
Excruciating pain
Eventual organ damage
Earlier death
Segments of -chain in HbA and HbS
Human adult hemoglobin (HbA):
Sickle-cell hemoglobin (HbS):
Val-His-Leu-Thr-Pro-Glu-Glu-Lys-
Val-His-Leu-Thr-Pro-Val-Glu-Lys-

15. Gene mutation
Hemoglobin: HbA
HbS
(lower O2-carrying capacity)
-O2
Deoxy-HbS
Red blood cells: cup-shaped
Sickled-shaped
(shorter lifetime)
Blockage
Anemia

16. 2. Determination of amino acid sequence
1）General steps (一般步骤): P
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)
II. Primary structure

17. 1) General steps II. Primary structure
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
Repeat step e to generate a different and overlapping set of fragments
Reconstruct the overall amino acid sequence of the protein
Locate disulfide bonds
II. Primary structure

18. 2) Dissociation of multimeric proteins (多聚蛋白质亚基的解离)
Subunits associated by non-covalent forces:
-- Exposure to:
pH extremes
6M guanidine hydrochloride (盐酸胍)
8M urea (尿素)
Subunits associated by covalent S-S bridges:
-- Cleavage of S-S bonds
II. Primary structure

19. Mercaptoethanol (巯基乙醇):
Cleavage of disulfide bridges
P.171
HCOOOH (过甲酸):
II. Primary structure
Mercaptoethanol (巯基乙醇):

20. 3) Identification of the N-terminal (N端的确定)
P.169
3) Identification of the N-terminal (N端的确定)
DNFB – Sanger’s method
DNS
PITC – Edman’s method
Amino peptidase (氨肽酶)
II. Primary structure

21. II. Primary structure Identification of N-terminal residues
DNP-amino acid
(yellow)
Sanger’s method
(DNFB法)
DNS method
(丹黄酰氯法)
DNS-amino acid
(fluorescent)
II. Primary structure
Edman’s method
(PITC法)
PTH-amino acid
(colorless)
Can also be used for sequencing

22. 4) Identification of the C-terminal (C端的确定)
P.170
4) Identification of the C-terminal (C端的确定)
NH2NH2 (肼解)
LiBH4 (还原)
Carboxypeptidase (羧肽酶)
II. Primary structure

23. II. Primary structure Identification of C-terminal residues
Hydrazinolysis (肼解法)
II. Primary structure
Reduction (还原法)

24. 5) Fragmenting the polypeptide chain (部分断裂多肽链)
Methods
R1-amino acid
R2-amino acid
Enzymatic
Trypsin (胰蛋白酶)
Lys, Arg
Chymotrypsin
(胰凝乳蛋白酶)
Phe, Trp, Tyr
Thermolysin
(嗜热菌蛋白酶)
Leu, Ile, Phe, Trp, Val, Tyr, Met
Pepsin (胃蛋白酶)
Phe, Leu, Trp, Tyr
Staphylococcal protease
Glu, Asp
Clostripain
Arg
Chemical
CNBr
Met
NH2OH
Asn --
Gly

25. Amino acid composition
6) An example
Amino acid composition 水解
N-terminal
DNFB, 定N-末端
Sanger’s method
Cleavage of S-S bridge
Edman degradation
胰蛋白酶
II. Primary structure
Fragmentation
CNBr
Sequencing

26. 7）Other methods for sequencing
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
II. Primary structure

27. III. Secondary Structure
Peptide plane (肽平面)
Common patterns of secondary structure (常见二级结构元件)
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)

28. 1. Peptide plane (肽平面) III. Secondary structure P.204
Fundamental structural unit in all proteins
Formed by peptide bond
Partial double bond character
trans-Configuration:
C vs C
Free rotation: Co-C, C-N
Rigid and planar
Each C is a joining point for two adjacent peptide planes
-carbon
III. Secondary structure
-carbon

29. - +      

30. Dihedral angles:  and  (二面角)
P.204 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

31. Definitions about  and  (二面角的定义)
P.204 C
 and 
 -- C-N bond
 -- C-Co bond 0o
For both  and , 0o corresponds to an orientation with the amide plane bisecting the H-C-R plane and a cis-configuration of the main chain around the rotating bond in question.
“+” and “”
For both  and , when looking from C along the bond
Clockwise rotation – “+”
Anti-clockwise rotation – “” Co N C

32. =  = 0o
Prohibited
=  = 180o
Fully extended

33. Ramachandran plot (拉氏构象图)
允许区
III. Secondary structure
不完全允许区
不允许区

34. 2. Common protein secondary structure (常见二级结构元件)
-Helix (螺旋)
-Pleated sheet (折叠片)
-Turn (转角)

35. 1) -Helix (螺旋) III. Secondary structure P.207
A. Structural parameters (结构参数)
Each turn: 0.54 nm, 3.6 residues, 13 atoms – helix
Each residue: 100o, 0.15 nm
 ~ -57o,  ~ -47o
Right-handed helix (右手螺旋)
B. Chirality and optical activity: Right-handed helix
Stability: Optimal use of internal H-bonds (充分利用链内氢键)
Dipole
(偶极)
III. Secondary structure
Myoglobin
(肌红蛋白)

36. -Helix C O Each turn: 0.54 nm H 3.6 residues 13 atoms Each residue: N

38. III. Secondary structure
H-bonding in an -Helix
III. Secondary structure
3.613-helix

39. Chirality & optical activity (手性和旋光性) Dipole & polarity (偶极和极性)
Always right-handed
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.
(右手螺旋和左手螺旋不是对映体)

40. Amino acid sequence affects stability of the -Helix
P.208
Electrostatic repulsion between successive R groups (R基的电荷)
Bulkiness of adjacent R groups (R基的大小)
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)
Pro and Gly – both rarely found in  helices
Interactions between amino acid residues at the terminals (肽链末端残基的相互作用)

41. Poly(Lys):
Poly(Glu):

42. -Pleated sheet / -conformation (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)

43. -Pleated sheets: parallel & antiparallelC
III. Secondary structure
平行式
反平行式

44. Antiparallel -sheets are more stable than parallel -sheets.
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.

45. Parallel & antiparallel -pleated sheets: A comparison
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
Structure
(结构)
Larger (>5 strands)
Smaller (~2 strands)
More regular
Less regular
Hydrophoblic side chains
(疏水侧链)
On both sides of the sheet
(在折叠片的两边)
On one side of the sheet
(在折叠片的同一边)

46. 3) -Turn (转角) III. Secondary structure P.211
Common, 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.
III. Secondary structure

47. 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)

48. IV. Tertiary and quaternary structures
Higher levels of protein structure: tertiary and quaternary structures
Fibrous proteins
Globular proteins
Two important terms related to protein structural patterns: motif and domain
Classification of globular proteins
Quaternary structure

49. 1. Higher levels of protein structure
Tertiary structure
 overall three-dimensional structure of a protein, especially when it is composed of a single polypeptide chain.
Quaternary structure
 three-dimentional structure of a multisubunit protein
Fibrous and globular proteins

50. Fibrous and globular proteins (纤维状蛋白和球状蛋白)
Polypeptide Chain
(多肽链)
Secondary Structure
(二级结构)
Solubility in water
(水溶性)
Biological Functions
(生物功能)
Fibrous protein
(纤维状蛋白)
Long strands/sheets
Single type
Insoluble
Support, shape, protection
Globular protein
(球状蛋白)
Folded in a spherical shape
Several types
Soluble
Catalysis, regulation, etc.

51. 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

52. Examples of Fibrous proteins
-Keratin (-角蛋白) – -helix
Fibroin (丝心蛋白) – antiparalle -pleated sheet
Collagen (胶原蛋白) – triple helix

53. Left-handed supertwist
Structure of hair
Right-handed helix
Left-handed supertwist
1 intermediate filament = 4 protofibrils
= 8 protofilaments
= 16 coiled coils
= 32 strands of -keratins

54. Collagen
Gly

55. 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)

56. Permanent waving is biochemical engineering
Rearrangement of H bonds:
Moist heat
-conformation
(-keratin)
-Helix
(-keratin)
Cooling
Rearrangement of S-S bonds and H bonds:

57. 3. Globular proteins (球状蛋白)
Containing several types of secondary /supersecondary structures and domains (含多种二级/超二级结构和结构域)
Compact globular structure due to efficient packing (紧密球状结构)
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)

58. 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

59. 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
Lysozyme (溶菌酶) 40 12
Myoglobin (肌红蛋白) 78

60. Myoglobin (肌红蛋白) 1 heme group 8 -helices MW 16,700
Show tertiary structure of myoglobin
MW 16,700
1 single polypeptide chain
153 amino acid residues
1 heme group
8 -helices

61. 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.

62. Supersecondary structures (超二级结构)
Supersecondary structures are particularly stable arrangements of several elements of secondary structure and the connections between them. 
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)

Two parallel -pleated sheets are connected by an -helix segment 
Antiparallel -pleated sheets are connected by -turns.

63. Supersecondary structures
Rossman折叠
 

: -hairpin
(发夹)
: -meander
(曲折)
: Greek key (希腊钥匙拓扑结构)

64. 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

65. Immunoglobulin structure
P.222

66. 5. Classification of globular proteins (分类)
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)

67. 6. Quaternary Structure (四级结构)
Hemoglobin (血红蛋白)
Symmetry of quaternary structure (四级结构的对称性): P.246
Forces driving quaternary association (四级缔合的驱动力): P.243
Structural and functional advantages of quaternary association (四级缔合在结构和功能上的优越性): P.247

68. Hemoglobin (血红蛋白)  subunit Heme Ribbon presentation
MW 64,500
4 Subunits
2 + 2
4 Heme groups
 subunit
Heme
Ribbon presentation
Space-filling model
 subunit

69. Symmetry of quaternary structure (四级结构的对称性)
P.246
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

70. Rotational symmetry in proteins
二十面体对称
二面角对称

71. Forces driving quaternary association (四级缔合的驱动力)
P.243
Subunit association is accompanied by both favorable and unfavorable energy changes.
Favorable :
Van der Waals interactions
H bonds
Ionic bonds
Hydrophobic interactions
Disulfide bonds
Unfavorable:
Entropy loss (熵的减少)
Immunoglobulin (免疫球蛋白)

72. Structural and functional advantages of quaternary association (四级缔合在结构和功能上的优越性)
P.247
Structural stability – decreased surface/volume ratio
Increasing interactions within the protein
Shielding hydrophobic residues from water
Genetic economy and efficiency
Genetic code capacity
Accuracy of the protein biosynthesis process
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)
Cooperativity (协同性) & allosteric effect (别构效应)

73. Tryptophan synthase (色氨酸合酶): 22
Indoleglycerol phosphate
Indole + L-Serine
-subunit
-subunit
Glyceraldehyde-3-phosphate + Indole
L-Tryptophan

74. Cooperativity (协同性) & allosteric effect (别构效应)
Positive cooperativity (正协同性)
Positive – activator (激活剂)
Cooperativity
(协同性)
Effector
Negative – inhibitor (抑制剂)
Negative cooperativity (负协同性)
Homotropic effect
(同促效应)
Substrate = Effector
(底物 = 效应物)
Allosteric effect
(别构效应)
Heterotropic effect
(异促效应)
Substrate  Effector
(底物  效应物)

75. Subunit interactions in an allosteric enzyme

76. V. Protein Denaturation and Folding (变性和折叠)
Denaturation and renaturation
(变性与复性)
Denaturing conditions (变性条件)
The Anfinsen experiment
The mad cow disease

77. 1. Denaturation and renaturation (蛋白质的变性和复性)
Native, folded state
(天然折叠状态)
Denatured, unfolded state
(变性的解折叠状态)
Renaturation (复性)
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)

78. 2. Denaturing conditions (变性条件)
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)
V. Denaturation and Folding

79. Denaturing conditions (1): pH extremes (强酸强碱的变性作用)
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
V. Denaturation and Folding

80. Denaturing conditions (2): Organic solvents
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
V. Denaturation and Folding

81. 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
SDS 胍 尿素

82. Denaturing conditions (4): Reducing agents
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
V. Denaturation and Folding

83. Denaturing conditions (5): Heavy metal ions
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
V. Denaturation and Folding

84. 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.

85. 3. The Anfinsen experiment
加入尿素，巯基乙醇
VI. Denaturation and Folding
Christian B. Anfinsen
( )
Denaturation and redenaturation of ribonuclease
(核糖核酸酶的变性与复性)
除去尿素，巯基乙醇
An early evidence that the 3-dimensional structure of a protein is determined by its amino acid sequence

86. 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

87. Bovine spongiform encephalopathy
Mad cow disease
(疯牛病)
Bovine spongiform encephalopathy
(海绵状脑软化症)
Prion protein (PrP, MW 28,000)
Infectious agent
Misfolding
Rich in -helix
Rich in -sheets
PrPsc
PrPc
PrPsc

88. VI. Structure-function relationship (蛋白质的结构与功能的关系)
Overview
Oxygen-binding proteins: Myoglobin & hemoglobin
Myoglobin (肌红蛋白)
Structure
Effect of protein structure on O2 binding
O2-binding curve
Hemoglobin (血红蛋白)
Structural change upon O2 binding
Cooperative O2 binding
Effect of H+, CO2 and BPG on O2 binding

89. 1. Overview
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)
VI. Structure-function relationship

90. 2. Oxygen-binding proteins (载氧蛋白) – Myoglobin & hemoglobin
1) Both are oxygen-binding heme proteins (氧合血红素蛋白质)
-- Conjugated proteins (缀合蛋白质)
2) 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)
3) 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)
Red blood cells
O2 transport

91. Heme Prosthetic group Oxygen Binding to Heme (血红素辅基)
Fe2+ (sp3d2) -- reversible binding to O2
Fe3+ (sp3d) -- no binding to O2
Arterial blood – O2-rich, bright red
Venous blood – O2-depleted, dark purple

92. Amino acid sequences of whale myoglobin and ,  chains of human hemoglobin

93. Structural Similarities Between Myoglobin and Hemoglobin
VI. Structure-function relationship
肌红蛋白
血红蛋白

94. 3. Myoglobin Structure Effect of protein structure on O2 binding
O2-binding curve
VI. Structure-function relationship

95. 1). Structure P.252 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)
C-terminal
N-terminal

96. 2). Effect of protein structure on Oxygen binding (蛋白结构对结合氧的影响)
O2-binding site (氧结合位点)
Molecular motions (分子运动)
Cavity produced by Val E11 & 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
Fe2+ is protected from being 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)
Distal His
(远侧)
Proximal His
(近侧)

97. 3) O2-binding curve (氧结合曲线)
P.255
MbO2
Mb + O2
[O2]
Dissociation constant
(解离常数)
[Mb][O2]
K =
[O2] + K
[MbO2] 
[MbO2]
Fractional saturation
(氧分数饱和度)
Binding sites occupied
Y = =
Total binding sites
[MbO2] + [Mb]
[O2]
pO2
pO2
O2-binding curve
(氧合曲线)
Y = = =
[O2] + K
pO2 + K
pO2 + P50
When Y = 0.5, pO2 = K = P50 Y
Hill plot
log pO2 – log K
log =
1-Y

98. Hill plot for the binding of O2 to myoglobin
log =
log pO2 – log K
log
1-Y
1-Y
Slope = 1.0
Y = 0.5 Y
log P50
log pO2
Oxygen binds tightly to myoglobin with a P50 = 0.26 kPa

99. 4. Hemoglobin (血红细胞) Structure (结构)
Structural change upon oxygen binding (氧结合引起的结构变化)
Cooperative oxygen binding (协同性的氧结合)
Effect of H+, CO2 and BPG on oxygen binding (H+, CO2, BPG对氧结合的影响)
VII. Structure-function relationship

100. 1). Structure  chain: 141 residues  chain: 146 residues
P.257
MW 64,500
4 subunits (2 + 2)
 chain: 141 residues
 chain: 146 residues
Structural similarity with myoglobin (与血红蛋白结构相似)
Allosteric interactions (别构作用)
Responsible for O2 transport
Hemoglobin
O2 transport:
Arterial blood (动脉血): 96% saturated with O2
Venous blood (静脉血): 64% saturated with O2

101. 2). Structural change upon oxygen binding (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

102. The T – R Transition +O2 –O2 T state (tense, 紧张态) Deoxyhemoglobin
H: His146 or His HC3
D: Asp94 or Asp FG1
+O2
–O2
T state (tense, 紧张态)
Deoxyhemoglobin
(去氧血红蛋白)
R state (relaxed, 松弛态)
Oxyhemoglobin
(氧合血红蛋白)

103. Changes in conformation near heme on O2 binding
Hemoglobin

104. 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

105. Asp FG1 (Asp94)
His HC3 (His146)
Lys C5

106. 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

107. Oxygen binding curves O2: R state Y T state Hemoglobin A ligand (配体)
An activator (激活剂)
A positive homotropic effector (正同促效应物)
R state Y
T state
Hemoglobin
Myoglobin – Hyperbolic curve (双曲线)
Hemoglobin – Sigmoid curve (S形曲线)

108. Quantitative Measurement of O2 binding
P.261
MbO2
Mb + O2
Hb(O2)4
Hb + 4O2
Dissociation constant
(解离常数)
[Mb][O2]
[Hb][O2]4
K =
K =
[MbO2]
[Hb(O2)4]
[MbO2]
[Hb(O2)4]
Fractional saturation
(氧分数饱和度)
Y =
Y =
[MbO2] + [Mb]
[Hb(O2)4] + [Hb]
pO2
(pO2)n
O2-binding curve
(氧合曲线)
Y =
Y =
pO2 + K
(pO2)n + K
Hyperbolic (双曲线)
Sigmoidal (S形)
K = P50
K = (P50)n Y Y
Hill equation
(Hill方程)
log
= log pO2 – log K
log
= n log pO2 – log K
1 – Y
1 – Y
Slope = 1
Slope = n

109. Hill Plot Hill equation: n  nH  1 Y log n log(pO2) – logK = 1 – Y
n: Number of binding sites
nH: Hill coefficient (Hill系数)
 a measure of degree of cooperativity
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)
Hemoglobin
n  nH  1

110. Hill plots for the binding of oxygen to myoglobin and hemoglobin
log
1-Y
Hemoglobin

111. 4). Effect of H+, CO2 and BPG on oxygen binding (H+, CO2, BPG对氧结合的影响)
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).
Heterotropic allosteric modulation (异促别构调节)
Negative cooperativity (负协同性)
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)
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)

112. 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.
His146 of the  chain makes a major contribution:
[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 Y
(in lungs)
(in tissues)

113. Amino-terminal residue
Bohr Effect – CO2
CO2 and O2 are bound to hemoglobin with inverse affinity (血红蛋白对CO2和O2的亲和力相反).
When [CO2] is high，
CO2 + Hb-NH2
Hb-NH-COO- + H+
Amino-terminal residue
Carbaminohemoglobin
(氨甲酸血红蛋白 )
Carbonic anhydrase
CO2 + H2O
H+ + HCO3-
(碳酸酐酶)
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
Hemoglobin

114. 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
2,3-二磷酸甘油酸
BPG is bound
BPG is released
+ charge groups
+O2
-O2
T state
R state

115. BPG plays an important role in physiological adaptation to the lower pO2 available at high altitudes
Sea level
Mountain
BPG
5 pm
8 pm
Lung A C
Tissue B D Y
0.38
0.37
(Sea level)
(4500 m)

116. 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 Hb, and a correspondingly higher affinity for O2.

117. VIII. Methods for studying protein conformation (研究蛋白质构象的方法)
X-ray diffraction (X射线衍射)
UV/Vis spectrophotography (紫外可见分光光度测定)
Fluoresence and fluoresence polarization (荧光和荧光偏振)
Circular dichroism (CD, 圆二色性)
Nuclear magnetic resonance (NMR, 核磁共振)