Principles of protein structure and stability.

1
Principles of protein structure and stability.
2
Polypeptide bond is formed between two amino
acids.
3
Backbone conformation is described by f and ?
angles.
Picture from T. Przytycka, 2002
4
Hierarchy of protein structure.

1. Amino acid sequence
2. Secondary structure
3. Tertiary structure
4. Quaternary structure

Picture from Branden Tooze Introduction to
protein structure
5
Right-handed alpha-helix.

• Helix is stabilized by HB between backbone NH
  and backbone carbonyl atom.
• Geometrical characteristics
• 3.6 residues per turn
• translation of 5.4 Å per turn
• translation of 1.5 Å per residue

6
?-strand and ß-sheet.
7
Loop regions are at the surface of protein
molecules.
Adjacent antiparallel ß-strands are joined by
hairpin loops. Loops are more flexible than
helices and strands. Loops can carry binding and
active sites, functionally important sites.
Branden Tooze Introduction to protein
structure
8
Protein classification based on the secondary
structure content.

• Class a - proteins with only a-helices
• Class ß proteins with only ß-sheets
• Class aß - proteins with a-helices and ß-sheets

9
Protein stability.Anfinsens experiments
10
Native proteins have low stability

• Scale of interactions in proteins
• - Interactions less than kT0.6 kcal/mol
• are neglected.
• - Interactions more than ?G 10 kcal/mol
• are too large
• Potential energy Van der Waals
  Electrostatic Hydrophobic

G
U
F
?G
Reaction coordinate
11
Electrostatic force.
Coulombs law for two point charges in a vacuum
q point charge, e dielectric constant
e 2-3 inside the protein, e 80 in water
Na
Cl-
d 2.76 Å, E 120 kcal/mol
12
Dipolar interactions.
- 0.42
Dipole moment
O
0.42
C
Interaction energy of two dipoles separated by
the vector r
-0.20
N
Peptide bond µ 3.5D, Water molecule µ 1.85D.
0.20
H
13
Van der Waals interactions.
Lennard-Jones potential
E (kcal/mol)
0.2
repulsion
London dispersion energy
0
d
d-
attraction
d
d-
- 0.2
12
10
8
6
4
2
Distance between centers of atoms
14
Hydrogen bonds
d-
d

• NH OC N
• 
  H
• 
  O
• 
  H
• 
  N

3 ?
D
A
D
A

HOH
OHH
HOHOHH
15
Hydrogen bonding patterns in globular proteins.

• 1. Most HB are local, close in sequence.
• 2. Most HB are between backbone atoms.
• 3. Most HB are within single elements of
  secondary structure.
• 4. Proteins are almost equally saturated by HB
  0.75 HB per amino acid.

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Disulfide bonds.

• PROTEIN GS-SG ?PROTEIN GSH?PROTEIN 2GSH

SH
HS
SH
S-SG
- Breakdown and formation of S-S bonds are
catalyzed by disulfide isomerase. - In the cell
S-S bonds are reversible, the energetic
equilibrium is close to zero. - Secreted proteins
have a lot of S-S bonds since outside the cell
the equilibrium is shifted towards their
formation.
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Hydrophobic effect.
H

• Hydrophobic interaction tendency of
• nonpolar compounds to transfer from an
• aqueous solution to an organic phase.
• The entropy of water molecules decreases when
  they make a contact with a nonpolar surface, the
  energy increases.
• As a result, upon folding nonpolar AA are burried
  inside the protein, polar and charged AA
  outside.

O
H
H
O
H
18
Hydrophobicities of amino acids.
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Cooperativity of protein interactions

• Protein denaturation is a first
• order (all-or-none) transition.
• As T increases
• 1. Globule expansion, loose packing.
• 2. As expansion crosses the barrier,
• liberation of side chains and
• increase in enthropy.

E
T1
T2
T
W(E)
T2
T
T1
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Summary

• Hydrophobic effect is mostly responsible for
  making a compact globule. Final specific tertiary
  structure is formed by van der Waals
  interactions, HB, disulfide bonds.
• Secret of stability of native structures is not
  in the magnitude of the interactions but in their
  cooperativity.

21
Classwork I CN3D viewer.

• Go to http//ncbi.nlm.nih.gov
• Select alpha-helical protein (hemoglobin)
• Select beta-stranded protein (immunoglobulin)
• Select multidomain protein 1I50, chain A
• View them in CN3D

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PDB databank.

• Archive of protein crystal structures was
  established in 1971 with several structures
• in 2002 17000 structure including NMR
  structures
• Data processing data deposition, annotation and
  validation
• PDB code nXYZ, n integer, X, Y, Z -characters

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Content of Data in the PDB.

• Organism, species name
• Full protein sequence
• Chemical structure of cofactors and prosthetic
  groups
• Names of all components of the structure
• Qualitative description of the structural
  characteristics
• Literature citations
• Three-dimensional coordinates

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Protein secondary structure prediction.

• Assumptions
• There should be a correlation between amino acid
  sequence and secondary structure. Short aa
  sequence is more likely to form one type of SS
  than another.
• Local interactions determine SS. SS of a residues
  is determined by their neighbors (usually a
  sequence window of 13-17 residues is used).
• Exceptions short identical amino acid sequences
  can sometimes be found in different SS.
• Accuracy 65 - 75, the highest accuracy
  prediction of an a helix

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Methods of SS prediction.

• Chou-Fasman method
• GOR (Garnier,Osguthorpe and Robson)
• Neural network method

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Chou-Fasman method.

• Analysis of frequences for all amino acids to be
  in different types of SS.
• Ala, Glu, Leu and Met strong predictors of
  alpha-helices,
• Pro and Gly predict to break the helix.

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GOR method.

• Assumption formation of SS of an amino acid is
  determined by the neighboring residues (usually a
  window of 17 residues is used).
• GOR uses principles of information theory for
  predictions.
• Method maximizes the information difference
  between two competing hypothesis that residue
  a is in structure S, and that a is not in
  conformation S.

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Neural network method.
Input layer
Input sequence window
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Output layer
Predicted SS
Hidden layer
L A W P G E V G A S T Y P
a
Si
Hj
Oi
1
ß
0
coil
0
Wij Sj
Hj Oi
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PHD neural network program with multiple
sequence alignments.

• Blast search of the input sequence is performed,
  similar sequences are collected.
• Multiple alignment of similar sequences is used
  as an input to a neural network.
• Sequence pattern in multiple alignment is
  enhanced compared to if one sequence used as an
  input.

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Classwork

• Go to http//ncbi.nlm.nih.gov, search for protein
  flavodoxin in Entrez, retrieve its amino acid
  sequence.
• Go to http//cubic.bioc.columbia.edu/predictprotei
  n and run PHD on the sequence.

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Definition of protein domains.

• Geometry group of residues with the high contact
  density, number of contacts within domains is
  higher than the number of contacts between
  domains.
• - chain continuous domains
• - chain discontinous domains
• Kinetics domain as an independently folding
  unit.
• Physics domain as a rigid body linked to other
  domains by flexible linkers.
• Genetics minimal fragment of gene that is
  capable of performing a specific function.

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Domains as recurrent units of proteins.

• The same or similar domains are found in
  different proteins.
• Each domain performs a specific function.
• Proteins evolve through the duplication and
  domain shuffling.
• The total number of different types of domains is
  small (1000 3000).

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The Conserved Domain Architecture Retrieval Tool
(CDART).

• Performs similarity searches of the NCBI Entrez
  Protein Database based on domain architecture,
  defined as the sequential order of conserved
  domains in proteins.
• The algorithm finds protein similarities across
  significant evolutionary distances using
  sensitive protein domain profiles. Proteins
  similar to a query protein are grouped and scored
  by architecture.