Proteins, their stability and dynamics

1
Proteins, their stability and dynamics

• Lecture 4
• September 13th

2
Homework for Tuesday, Sep 13th
Can ions penetrate in between the bases in DNA or
RNA polymers? Please respond this for either
Na, K, and Cl- ions.
3
Outline

• What are the proteins?

• What are the elementary secondary structures?

• What are the classes of molecular interactions
  in proteins?

• What is the relationship between enthalpy and
  entropy with
• the structural changes in proteins

4
Central Dogma of Molecular Biology
DNA
Transcription
Transcription
Translation
mRNA
Translation
Protein
5
Protein Engineering
1. Change DNA Sequence 2. Change RNA Sequence 3.
Change Amino Acid Sequence
6
mRNA Reading Direction Corresponds to Protein
Chemical Directionality
3
5
mRNA
NH2-terminus
COOH-terminus
7
Proteins -- The monomers are amino acids

• In a cell, gt60 a.a. in a protein, 20 a.a.
• The basic structure a carboxyl groups, an amino
  group, a hydrogen group, and a R group.
• Stereochemistry of amino acids L- D- and only L
  type amino acids occur in proteins.
• R group defines amino acids chemical nature.

8
(No Transcript)
9
The polymers of amino acids are polypetides and
proteins

• The dehydration (condensation) reaction form
  peptide bond
• N-terminus and C-terminus
• Polypeptides vs. proteins monomeric proteins and
  multimeric proteins (homomeric and heteromeric)

Hemoglobin is a heteromeric protein with 4 heme
group for each subunit that is capable to carry
an oxygen molecule
10
Peptide Bond
11
Important bonds for protein folding and stability
Weak (2-5 kcal/mol vs. covalent 70-100
kcal/mol), but massive
The oxidization of the sulfhydryl groups of two
cystein residues (intramolecule ribonuclease
intersubunit dimeric protein insulin)
Weak (3 kcal/mol), affected by pH value
Dipole molecules attract each other by van der
Waals force (transient and weak 0.1-0.2
kcal/mol) Hydrophobic interaction, a tendency of
hydrophobic groups or molecules being excluded
from interact with hydrophilic environment
12
Protein structure depends on amino acid sequence
and interactions
13
(No Transcript)
14
?-helix Secondary Structure
http//www.expasy.ch/swissmod/course/text/chapter1
.htm
15
?-Sheet Secondary Structure
Anti-Parallel b-sheet
Parallel b-sheet
16
Motifs often consist of small segments of alpha
helix and/or beta sheet connected together by
looped regions (random coil) of varying length
17
(No Transcript)
18
(No Transcript)
19
Protein Tertiary Structure Tied to Function
20
Enthalpy and Entropy
DGo DHo - TDSo
Typically, DG and DH are measurable and DS
calculated
DH and DS Provide Mechanistic Insight
In very rough generalities DH related to bond
formation/breaking DS related to configurational
freedom and water ordering
dDH DCpdT Gives Thermal Dependence of K
21
Basic Thermodynamics
Gibb Free Energy

A B
A-B
KBAAB/ A B e -DGBA/RT
DG work required to convert molecules A and B
to A-B or free energy of conversion
between 2 standard states
G H - TS, where H is enthalpy and S is entropy
dG dH - TdS - SdT
At constant T (and p) dG dH - TdS
22
Biology and Free Energy
Because K exp (DGo/RT)
Small changes in DG give large changes in
products/reactants
KU/Na/1-a
Unfolded Protein
Folded Protein
95
5
5
95
23
Molecular Interactions Determine Chemical
Potential
State 1

State 2
How are DG, DH, DS related to specific atoms
24
Classes of Molecular Interactions
Ion-Ion q1q2/er
Coulombic
van der Waals Aij/r12 - Bij/r6
Hydrogen Bonds
Hydrophobic Effect
Hydration Effects
25
van der Waals Interactions
Dispersion Forces
26
van der Waals Potential Function
The van der Waals energy for carbon-carbon
interactions calculated as a Lennard-Jones 6,12
potential. The interaction energy is plotted as a
function of distance for two atom centers. Note
that the folding free energy is only between 15
to 50 kJ/mol for typical proteins - corresponding
to a handful of optimal interactions, or a single
close approach to 3 Å.
27
Hydrophobic Effect
Fundamental 1 The Hydrophobic Effect is a
Solvent Effect
28
Hydrogen Bonding Energetics
DH - negative, bond making
DS - negative, lose entropy
DG ranges widely in biochemistry
29
Hydrophobic Effect in Protein Folding
30
Hydrophobic Effect in Biomolecular Interactions
Exposed Surface Area

DCpo large, negative
Buried Surface Area
31
Hydrophobic Effect in Biomolecular Interactions

From Structures, calculate DAnp, DAp
DCpo ( aDAnp)- (bDAp) cal/mol oK
Good Correlation for Protein Folding,
Protein-ligand, Protein-DNA
32
Biomolecular Energetics
Electrostatic Interactions
COO-
H3N
Hydrophobic/van der Waals Interactions
CH3 H3C
OH N
Hydrogen Bonding Interactions
33
Gibbs Free Energy
DG
DGfold
34
Protein Folding Energetics
Folded
Unfolded
K exp (DGo/RT)
Optical Signal
Denaturant
35
Case Study Protein Stability
Chemical Denaturant
Urea Guanidinium HCl
Unfolded
Folded
KU/Na/1-a
DG -RTlnK
36
Measuring Protein Stability
100
Midpoint

a
Denaturing Condition, e.g. Temp.
How to determine KU/N ? Need signal specific
to each species
37
Determining Equilibrium Parameters
100
Unfolded
New KN--gtU At Each Temperature
Temperature
Need to quantitate unfolded percentage
Need Optical Signal Proportional to 2 Species
Change in folded state change in optical signal
38
Engineering Protein Stability
Destabilize Unfolded State Stabilize Folded State
39
Molecular Interactions Determine Chemical
Potentials
40
Molecular Interactions Determine Chemical
Potential
41
Summary of Energetics Intro
Hydrophobic Effect is Driving Force - Dominant
Free Energy Term van der Waals stabilize in
bound state Hydrogen Bond Electrostatic
Interactions - Favorable, Specificity
42
References
1. Proteins. Structures and Molecular Properties.
2nd Edition Thomas E. Creighton, (W. H.
Freeman and Company, New York, 1993). 2.
Introduction to Protein Structure. 2nd Edition
Carl Branden, John Tooze (Garland Publishing,
Taylor Francis Group, New York,
1999). 3. Principles of Physical Biochemistry,
chapter 1.6 Kensal E. van Holde, W. Curtis
Johnson, P. Shing Ho (Prentice Hall, Upper
Saddle River, New Jersey, 1998). 4. Introduction
to Protein Science. Architecture, Function, and
Genomics Arthur M. Lesk (Oxford University
Press, Oxford, 2004) 5. Molecular Biophysics.
Structures in Motion. Michel Daune (Oxford
University Press, Oxford, 1992)