Protein Folding & Biospectroscopy

Lecture 3

F14PFBDavid Robinson

Protein Folding

1. Introduction2. Protein Structure

3. Interactions4. Protein Folding Models5. Biomolecular Modelling6. Bioinformatics

Tertiary Structure

determined by weak interactions

Steric Hydrogen bonds Electrostatic interactions (ionic

bonds) Hydrophobic interactions Van der Waals interactions

Backbone Torsion Angles• ω tends to be planar (0º - cis, or 180 º - trans)

- due to delocalization of carbonyl electrons and nitrogen lone pair

• φ and ψ are flexible rotation• but limited rotation -- due to steric hindrance• Only 10% of the {φ, ψ} combinations are

generally observed• First noticed by G.N. Ramachandran

Ramachandran Plot Used computer models of small

polypeptides to vary φ and ψ systematically, to find stable conformations

Atoms treated as hard spheres, with radius = van der Waals radius

φ and ψ angles which cause spheres to collide correspond to sterically disallowed conformations

Computed Ramachandran Plot

White = sterically disallowed conformations (atoms come closer than sum of van der Waals radii)

Blue = sterically allowed conformations

Experimental Ramachandran Plot

φ, ψ distribution in 42 high-resolution protein structures (x-ray crystallography)

Ramachandran Plot& Secondary Structure

• Repeating values of φ and ψ along the chain regular structure

• Example:repeating values of φ ~ -57° and ψ ~ -47°

-helix

Cytochrome C: many segments of helix. Ramachandran plot - tight grouping of φ, ψ angles near -60°, -40°

-helix cytochrome C Ramachandran plot

Repetitive values at φ = -110° to –140° and ψ = +110° to +135° strands. Plastocyanin: mostly sheets; Ramachandran plot shows values in the –110°, +130° region.

-sheet plastocyanin Ramachandran plot

White = sterically disallowed conformations

Red = sterically allowed regions (right-handed helix and sheet) with strict (greater) radii

Yellow = sterically allowed if shorter radii are used (i.e. atoms can be closer together; brings out left-handed helix)

Non-bonding Forces Influence Protein Structure

Amino acids of a protein are joined by covalent bonding interactions. The polypeptide is folded in three dimensions by non-bonding interactions. These interactions can easily be disrupted by extreme pH, temperature, denaturants, reducing reagents.

H-bond interactions (12-30 kJ/mol) Hydrophobic Interactions (<40 kJ/mol) Electrostatic Interactions (20 kJ/mol) Van der Waals Interactions (0.4-4 kJ/mol)

The total inter-atomic force acting between two atoms is the sum of all the forces they exert on each other.

Hydrogen bonds H-bond: a favourable interaction between a

proton bonded to an electronegative atom and an atom carrying a lone pair of electrons.

D-H + A D H A

Acceptors (A):Donors (D):

Important for maintaining backbone interactions

N OC

O

H

O

H

O

H

N

Hydrogen bonds are dependent on geometry

Hydrogen bondsExample: hydrogen bonds(white dash lines) hold a small molecule in place at the active site of an enzyme.

H-bonds in helix

Peptide bond

Thermodynamics First law: total energy of a system and its

surroundings is constant. Energy released from formation of chemical bonds must be used to generate heat or to form other new chemical bonds or both.

Second law: the total entropy of a system and its surroundings always increases for a spontaneous process.

The First Law of Thermodynamics

The 1st law can be expressed as:

“The internal energy of an isolated system is constant”

Essentially this means that energy is neither created nor destroyed, but only transformed from one form to another.

For processes occurring with no volume change, w=0 and the internal energy change is equal to the heat transferred between the system and its surroundings

However, very many processes take place under conditions of constant pressure rather than constant volume when

For constant pressure processes it is convenient to refer to changes in the enthalpy of a system.

A heat change at constant pressure equals the enthalpy change.

VqU

wqU P

HqP

Definition of entropy change

When a quantity of heat is transferred to a system at constant temperature and under reversible conditions the change in the entropy of the system is:

The Second Law of thermodynamics:

“spontaneous processes are those which increase the entropy of the universe”.

The Third Law of thermodynamics:

“the entropy of perfect crystals at zero Kelvin (absolute zero) is zero”.

T

qS rev

Gibbs energy defined

Gibbs introduced a thermodynamic function G which specifies the state of a system in terms of its enthalpy, entropy and temperature:

G = H – TS

At constant temperature

G = H - TS

and the criterion for a spontaneous change at constant temperature and pressure can be expressed as:

G < 0

i.e. spontaneous changes decrease the Gibbs energy of a system.

G is also known as the change in ‘Gibbs free energy’ because it gives the amount of energy which is free to do useful work.

J.Willard Gibbs

1839-1903

Protein folding represents a significant decrease in entropy, but it occurs spontaneously.

This is due to an increase in entropy of the water molecules surrounding the protein.

The increase in entropy of the water molecules is due to the hydrophobic nature of proteins and is termed the hydrophobic effect. The water molecules around a non polar molecule are extremely low in entropy (well ordered). “Oil and water don’t mix.”

Hydrophobic Interactions Hydrophobic interactions minimize

interactions of non-polar residues with solvent. Thus, nonpolar regions of proteins are often buried in the interior, to exclude them from the aqueous milieu.

However, non-polar residues can also be found on the surface of a protein - to participate protein-protein interactions.

This type of interaction is entropy driven.

poor solubility of non-polar groups in water is due to the ordering of the surrounding water molecules

Electrostatic Interactions

Charged side chains can interact (dis)favourably with a charge of another side chain. According to Coulomb’s law, the electrostatic force:

Favourable electrostatic interactions include that between positively charged lysine and negatively charged glutamic acid.

Salts shield electrostatic interactions.

221

Dr

qqF

Examples of Electrostatic Interactions

Intramolecular ionic bonds between charged amino acid residues in a protein:

NH3+ O

CO

CH2

H2C C

O

O- NH3+

(CH2)4

N

NN

N

NH2

O

OHOH

OPOPOP

O

O

O

O

O

OO

Mg2+

Magnesium ATP

van der Waals Interactions van der Waals interaction between two atoms is a result of

electron charge distributions of the two atoms. For atoms that have permanent dipoles:

Dipole-dipole interactions (potential energy ~r-3) Dipole-induced dipole interactions (potential energy ~r-5)

For atoms that have no permanent dipoles: Transient charge distribution induces complementary charge

distribution (also called dispersion or London dispersion force) (potential energy ~r-6)

Repulsion between two atoms when they approach each other due to overlapping of electron clouds (potential energy ~r-12)

+ - -+

+ -transientdipole

transientdipole

van der Waals Interactions In general, the permanent dipole contribution is much less

than the dispersion and repulsion forces. Thus the van der Waals potential can be expressed as 1/r12-1/r6.

r0 is the sum of van der Waals radii for the two atoms. Van der Waals forces are attractive when r> r0 and repulsive when r< r0.

Van der waals radii of common atoms:

H 0.1 nm C 0.17 nmN 0.15 nmO 0.14 nmP 0.19 nmS 0.185 nm r

Van d

er

Waals

pote

nti

al

Van d

er

Waals

Forc

e

r0

r0

Role of Sequence in Protein Structure

Weak forces operate both within the protein structure, between proteins and the water solvent;

All of the information necessary for folding the peptide chain into its “native” structure is contained in the amino acid sequence;

Certain loci along the peptide chain act as nucleation points;

Folded proteins avoid local energy minima.

Denaturation

The loss of structural order in biomolecules is called denaturation;

The central role of weak forces in biomolecular interactions restricts the folding (and thus function) of proteins to a narrow range of physical conditions, such as temperature, ionic strength, and relative acidity;

Extremes of these conditions disrupt the weak forces essential to maintaining the intricate structure and will lead to loss of their biological functions.