Transcript 蛋白质结构基础(Introduction of Protein Structure)

Molecular Biophysics
分子生物物理学
分子水平
研究生物体系物理学性质、行为
结构
功能
Molecules in Biosystem
Biopolymers:
Nucleic acid (DNA, RNA)
Protein
Saccharide
Lipid
Other
PROTEIN STRUCTURE
1965年中国在世界上首次用化学方法
人工合成的蛋白质－牛胰岛素
S—S
A链
Gly.Tle.Val.Glu.Gln.Cys.Cys.Aln.Ser.Val.Cys.Ser.Leu.Tyr.Gln.Leu.Glu.Asn.Tyr.Cys.Asn OH
6 7
1 2
20
11
S
S
B链
S
S
Phe.Val.Asn.Gln.His.Leu.Cys.Gly.Ser.His.Leu.Val.Glu.Ala.Leu.Tyr.Leu.Val.Cys.Gly.Glu.Arg.
1
2
19
7
Gly.Phe.Phe.Tyr.Thr.Pro.Lys.Ala OH
30
牛胰岛素的化学结构
Hierarchy of Protein Structure
by Linderstrøm-Lang
Primary Structure
Secondary Structure
supersecondary Structure or motif
domain
Tertiary Structure
Quaternary Structure
Property of amino acid
Chiral
COOH
NH2
CH3
H
L alanine
zwitterion
Uncharged structure
Minor component
Dipolar ion, or zwitterion
Major component
Classificatory of amino acid
based sidechains (R groups)
Non-polar
Polar
G,A,V,L,I; F,W, P,M,
neutral
S,T, N,Q
acidic
D,E; C,Y
basic
R,K, H
Histidine
?
Protein Primary Structure
Side chain
Carboxyl/C
terminus
Backbone
Peptide bond
Amine/N
terminus
Pauling & Corey
C-N, 0.149nm
C=N,0.127nm
?
=180 =180
C

C
=0
N,C
=0
Minimal Distance (Å) between nonbonding atom
(G.N.Ramachandran)
C
O
N
H
C
O
N
H
3.20
(3.0)
2.80
(2.70)
2.70
(2.60)
2.90
(2.80)
2.70
(2.60)
2.70
(2.60)
2.40
(2.20)
2.40
(2.20)
2.40
(2.20)
2.00
(1.90)
phi (), psi (Y), and omega (W)
Relation with Energy and distance
interaction
Relation with Energy and
distance
charger-charge
r -1
r -2
r -3
r -4
r -6
r -6
charger-dipole
dipole-dipole
charge-induced dipole
dipole-induced dipole
Transient dipoleinduced dipole
Van der Waals force
10 kJ·mol-1，range:0.3～0.5 nm
Lennard-Jones potential
A
B
E   6  12
r
r
Hydrogen bond
H-bond definition, H-bond location O….H-X
Hydrogen bonds can vary in strength
from very weak (1-2 kJ mol−1) to
extremely strong (40 kJ mol−1), so
strong as to be indistinguishable from
a covalent bond, as in the ion HF2−.
Typical values include:
O—H...:N (7 kcal/mol)
O—H...:O (5 kcal/mol)
N—H...:N (3 kcal/mol)
N—H...:O (2 kcal/mol)
H
O
X
Protein Secondary Structure
1951, Pauling
p= 0.54nm
P
z0= 0.15nm
Z0
= -57
= -47
Helices
repetitive secondary structure
C
Helices are the most abundant form
of secondary structure containing
approximately 32-38% of the
residues in globular proteins
(Kabsch and Sander, 1983)
a-helix
310 helix
p-helix
N
Parameters of secondary structure


n
r
P
3.613
-57
-47
3.6
0.154
0.55
310
-49
-26
3.0
0.200
0.60
p
-57
-7
4.4
0.115
0.51
Paral-
-119
+113
2.0
0.320
0.64
Antiparal-  -139
+135
2.0
0.340
0.68
[n] is the number of residues per helical turn
[r] is the helical rise per residue (nm)
[p] is the helical pitch (nm).
Parameters of secondary structure
H-bond
3.613
i, i+4
Atoms in Hbond loop
13
radius
310
i, i+3
10
1.9
p
i, i+5
16
2.8
2.3
a-helix introduction
32-38% of all residues in globular proteins
The average length of an alpha helix is
10 residues.
Found(-64 +/- 7, -41 +/- 7) /
ideal(-57.8, -47.0)
The structure repeats itself every 5.4 Å
along the helix axis, i.e. we say that the
a-helix has a pitch of 5.4 Å.
a-helices have 3.6 amino acid residues per
turn, i.e. a helix 36 amino acids long
would form 10 turns.
The separation of residues along the helix
axis is 5.4/3.6 or 1.5 Å, i.e. the a-helix
has a rise per residue of 1.5 Å
Why alpha-helix is abundant in
native globular protein?
the phi and psi angles
of the alpha helix lie in
the center of an
allowed, minimum
energy region of the
Ramachandran (phi,
psi) map.
the dipoles of
hydrogen bonding
backbone atoms
are in near perfect
alignment.
the radius (2.3 angstrom)of the helix
allows for favorable van der Waals
interactions across the helical axis
side chains are well staggered
minimizing steric interference
•
CO group toward carboxyl terminus
•
NH group toward amide terminus
•
H-bond, i-(i+4)
•
Side chain: i-(i+3); i-(i+4)
•
interactions between i and i+4 stabilize
helix
Distortions of a-helices
The majority of a-helices in globular proteins are curved or distorted
somewhat compared with the standard Pauling-Corey model. Why?
1. The packing of buried helices against other secondary structure elements
in the core of the protein
2. Proline residues induce distortions of around 20 degrees in the direction of
the helix axis
3. Solvent. Exposed
helices are often bent
away from the solvent
region. This is because
the exposed C=O
groups tend to point
towards solvent to
maximise their Hbonding capacity, i.e.
tend to form H-bonds
to solvent as well as NH groups.
310 helix introduction
Only 3.4% of the residues are
involved in 310 helices, and nearly
all those in helical segments
containing i-i+3 hydrogen bonds.
Ideal (-74.0, -4.0) / found (-71.0 and
-18.0)
CO---HN hydrogen bond: i-i+3
Standard 310 helix
Proline helix
Left handed helix
3.0 residues per turn
pitch = 9.4 Å
No hydrogen bonding in the backbone but helix
still forms.
Poly-glycine also forms this type of helix
Collagen: high in Gly-Pro residues has this
type of helical structure
p-helices introduction
The pi helix is an extremely rare secondary structural element
in proteins. the backbone C=O of residue i hydrogen bonds to
the backbone HN of residue i+5.
i- - i + 5
H-bonds
2.8angstrom
1. the phi and psi angles of the pure pi helix (
-57.1, -69.7) lie at the very edge of an allowed,
minimum energy region of the Ramachandran
(phi, psi) map.
2. the pi helix requires that the angle tau (N-CaC') be larger (114.9) than the standard
tetrahedral angle of 109.5 degrees.
3. the large radius of the pi helix means the
polypeptide backbone is no longer in van der
Waals contact across the helical axis forming
an axial hole too small for solvent water to fill.
4. side chains are more staggered than the ideal
3.10 helix but not as well as the alpha helix.
H-bond: 1-5
Helical wheel tools
alpha-helix, surface of
protein, barrier
amphiphilic
protein design projects
by Degrado, USA
Helix dipole
helix macrodipole
The partial charges on the amide hydrogen and carbonyl oxygen are
shown in units of the elementary charge contributing to an overall
dipole moment of 3.46 Debye units.
Sheet
20-28%
(Kabsch & Sander, 1983; Creighton, 1993)
a repeating secondary
structure
Parameters of secondary structure


n
r
P
3.613
-57
-47
3.6
0.154
0.55
310
-49
-26
3.0
0.200
0.60
p
-57
-7
4.4
0.115
0.51
Paral-
-119
+113
2.0
0.320
0.64
Antiparal-  -139
+135
2.0
0.340
0.68
[n] is the number of residues per helical turn
[r] is the helical rise per residue (nm)
[p] is the helical pitch (nm).
-139 and +135
Parallel sheet
Antiparallel sheet
Twists
about 30 degrees per residue in right-handed sense
Left-handed: crossover angel
Right-handed: progressive H-bond twist
Parallel sheets are less twisted
than anti-parallel and are
always buried.
Bulges
One residue backbone, two H-bonds
Strand connections
Beta-hairpin
Crossover connection:
right-handed
left-handed
Turn
1.
2.
3.
that serve to reverse the direction of the
polypeptide chain
Surface of the protein
Antibody recognition, phosphorylation,
glycosylation, hydroxylation
Gamma-turn
1. H-bond: i----i+2
2. (70, -60) and (-70, 60) for i+1 residue
Type I and I’ turn
1. H-bond: i----i+3
2. (-60, -30) and (-90, 0) for i+1, i+2 residues
2.3.3. Type II and II’ turn
The backbone dihedral angles of residue are
(-60, 120) and (80, 0) of residues i+1 and i+2,
respectively of the type II turn.
the hydrogen bond between CO of residue i and NH of residue
i+3. This is a single turn of right-handed (III) and left-handed
(III') 3.10 helix, respectively. The backbone dihedral angles of
residue are (-60, -30) and (-60, -30) of residues i+1 and i+2,
respectively of the classical type III turn.
2.3.4. Other structures
1. Loop
random coil
2. Paperclips
cap of a-helix
Identification of secondary
structure
Identification without 3D structure
CD
可信度：
a-helix, 97%; sheet 75%; 50% turn, 89% other
From Manavalan & Johnson, 1987
FTIR
amide band I 1600-1700
NMR
•
coupling constant: 3JHAHN
right-handed a-helix, phi = -57, 3JHAHN = 3.9 Hz
right handed 3.10 helix, phi = -60, 3JHAHN = 4.2 Hz
antiparallel -sheet, phi = -139, 3JHAHN = 8.9 Hz
parallel -sheet, phi = -119, 3JHAHN = 9.7 Hz
left-handed a-helix, phi = 57, 3JHAHN = 6.9 Hz
Prediction of secondary structure
(a). Homology. If sequence >25-30%, structure similarity
(b). Statistical. Chou & Fasman (1978).
(c). Stereochemical Schiffer and Edmundson (1967)
Motif
&
domain
超二级结构motif
相邻的二级结构单元组合在一起，
彼此相互作用，排列形成规则的、
在空间结构上能够辨认的二级结构
组合体，并充当三级结构的构件
（block building)，成为超二级
结构，介于二级结构与结构域之间
的结构层次。
常见的几种超二级结构形式
a.α-loop-α; b.β-α-β; c.β-loop-β; d. Rossmann折叠；
E,f,g. 回形拓扑结构
细胞色素C
α-loop-α
细胞核抗原的β-α-β结构
β-α-β
纤溶酶原的β-loop-β结构
结构域domain
多肽链在超二级结构的基础上进一
步折叠成紧密的近乎于球状的结构，
这种结构称为结构域domain
结构域的特点
• （1）结构域是球状
蛋白质的独立折叠
单位。对一些较小
的球状蛋白质分子
或亚基来说，结构
域和三级结构是一
个意思。
• 例如红氧还蛋白，
核糖核酸酶、肌红
蛋白等。
• （2）对于较大
的球状蛋白质或
亚基，其三级结
构往往由两个或
多个结构域缔合
而成也即它们是
多结构域的，例
如免疫球蛋白的
轻链含2个结构
域。
结构域有时也指功能域。功能域可以是一个
结构域，也可以是由两个结构域或两个以
上结构域组成，从功能角度看许多多结构
域的酶，其活性中心都位于结构域之间，
因为通过结构域容易构建具有特定三维排
布的活性中心。结构域之间常常只有一段
柔性的肽链连接，形成所谓铰链区，使结
构域容易发生相对运动，这是结构域的一
大特点。结构域之间的这种柔性将有利于
活性中心结合底物和施加应力。
Protein Tertiary Structure
三级结构指一个不可分的单元（分
子）的完整的三维空间结构。对于
蛋白质，此单元通常是共价连接的
一个分子。
目前已经测出三级结构的生物大分
子都储存在蛋白质数据库中
（Protein data bank,PDB)，借助
软件可查阅显示其空间结构，还可
以在不同方向旋转以获得空间结构
的细节。
嗜热菌蛋白酶与人碳酸酐酶的结构图
研究蛋白质三级结构的方法
X射线晶体衍射（X-ray crystallography)
多维核磁共振( multi-dimensional NMR)
三维电子显微镜技术（3-dimensiional EM)
扫描探针显微术( Scanning Probe Microscopy ,SPM)
Protein Quaternary Structure
独立的三级结构之间的非
共价缔合称为四级结构。这些
独立的三级结构称为亚基或亚
单位。