Understanding Biology through structures Course work 2009 Principles that Govern Protein Structure and Stability.

Understanding Biology through structures Course work 2009

Principles that GovernProtein Structure and

Stability


Forces that stabilize protein structure

• Interactions between atoms within the protein chain• Interactions between the protein and the solvent


Bond types in proteins

• Covalent bonds• Hydrogen bonds• Metal ligands• Ionic interactions• Disulfide bonds• Non-bond interactions


Favourable conformations in polypeptides

• Covalent interactions establish the structural framework of the protein molecule, the chemical expression of primary structure

• Backbone conformation constrained by steric restrictions on and torsions

• Sidechain conformations are also constrained

• Favourable sidechain conformations depend on the sidechain and also on its neighbours.

• S-S Bonds between cysteine residues can form within proteins



• Charged side chains in protein can interact favorably with an opposing charge of another side chain according to Coulomb’s law:

• Examples of favorable electrostatic interaction include that between positively charged lysine and negatively charged glutamic acid.

• Salts have the ability to shield electrostatic interactions.

Electrostatic Interactions

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Charge-charge interactions

• Coulomb interaction between two ions• At close range, Coulomb interactions are as strong as

covalent bonds• Their energy decreases with 1/r and fall off to less than kT at

about 56 nm separation between charges• In practice, charge-charge interactions have been shown to

be chemically significant at up to 15 Å in proteins• Small charged metal ions can act as positive charge in an ion

pair

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+

Mg-ATPSalt Bridge



Hydrogen bonds

• Noncovalent chemical bond in which an electronegative atom (a hydrogen-bond acceptor) shares a hydrogen atom with an electronegative atom with a bound hydrogen

• Energy : 10-40 kJ/mol

• Approximately 1.7-3 Å in length

• Strength varies with angle of hydrogen-bond interaction

• Individually, not very strong, but the large numbers of hydrogen bonds in regular secondary structures stabilize the framework of the protein

C O H N


Hydrogen bonds in protein structure


• van der Waals forces are also known as London forces.

• They are weak interactions caused by momentary changes in electron density in a molecule.

• They are the only attractive forces present in nonpolar compounds.Even though CH

4 has no net dipole, at any

one instant its electron density may not be

completely symmetrical, resulting in a

temporary dipole. This can induce a

temporary dipole in another molecule. The

weak interaction of these temporary dipoles

constituents van der Waals forces.

The surface area of a molecule determines

the strength of the van der Waals

interactions between molecules. The larger

the surface area, the larger the attractive

force between two molecules, and the

stronger the intermolecular forces.

van der Waals forces


Dispersion

A neutral atom: argon. It is like a large spherical jelly

with a golf ball at the centre. The golf ball is the nucleus

carrying a large positive charge and the jelly

represents the clouds of electrons.

At a point external to the atom the net average field will be zero because

the positively-charged nucleus' field will be exactly balanced by the

electron clouds.

However, atoms vibrate (even at 0K)

and so that at any instant the cloud

is likely to be slightly off centre.

This disparity creates an "instantaneous

dipole":


Dispersion

Suppose that we have another argon atom close to the first. This atom will

see the electric field resulting from the instantaneous dipole.

This field will effect the the electron and induce a dipole

The two dipoles attract one another - producing an attractive interaction.

The Dispersion interaction can be shown to vary according to the inverse

sixth power of the distance between the two atoms:

B depends on the polarizability of the atoms

r is the distance between them


Repulsion

When two atoms are brought increasing close together there is a large

energetic cost as the orbitals start to overlap.

The Pauli exclusion principle states that no two electrons can share the

same state so that in effect half the electrons of the system would have

to go into orbitals with an energy higher than the valence state.

For this reason the repulsive core is sometimes termed a

"Pauli exclusion interaction".

The Hard Sphere Model: atoms have a characteristic radius

(below the van der Waals radius) and cannot overlap.

Can represent the energy costs of close approach using a term


van der Waals forces

• Nonspecific forces between like or unlike atoms• Decrease with r6

• approximately 1 kJ/mol• If r0 is the sum of van der Waals radii for the two atoms. Van der

Waals forces are attractive forces when r> r0 and repulsive when r< r0.


Approximate Strengths of Interactions between atoms

Bond Type kJ/mol

Covalent Bond 250

Electrostatic 5

van der Waals 5

Hydrogen bond 20


The hydrophobic force

Observation: • Hydrophobic residues are buried while hydrophilic residues are

on the outside. • The exterior surface area of proteins can be up to 60% polar

atoms• Proteins fold to maximize their effectiveness as hydrogen-

bonding partners to water Explanation: • When hydrophobic residues are exposed to solvent, the extended

hydrogen bonding structure of water is disrupted• Breaking hydrogen bonds in water is energetically unfavourable• Water molecules reorient around the hydrophobic molecule, so

that the least number of hydrogen bonds are sacrificed to accommodate it

• Burying hydrophobic residues releases water and increases entropy.


Protein Interior and Exterior

Internal packing of atoms in a protein can be analysed by depicting every

atom in the protein as a sphere with the appropriate van der Waals radius.

Overlapping regions (regions of covalent bonds) are truncated. This

surface is called the van der Waals surface.

Cannot measure a van der Waals surface of a protein because any chemical

probe will have some dimension greater than zero.

A more realistic representation is the solvent accessible surface that is

defined by the center of a water molecule (sphere with radius 1.4 Å) as it

moves over the surface of a protein.


Protein Interior and Exterior

A more realistic representation is the solvent accessible surface that is

defined by the center of a water molecule (sphere with radius 1.4 Å) as it

moves over the surface of a protein.

Protein-protein interactions, which

form the basis for most cellular

processes, result in the formation

of protein interfaces.

The protein-protein docking

problem is the prediction of a

complex between two proteins

given the three-dimensional

structures of the individual proteins


Packing of Globular Proteins

• Secondary structures pack closely to one another and also intercalate with extended polypeptide chains

• Most polar residues face the outside of protein and interact with solvent but may be buried if H-bonding and charge is satisfied

• Most hydrophobic residues face the interior of the protein and interact with each other thereby minimizing contact with water

• van der Waal’s volume is about 72-77% of the total protein volume; about 25% is not occupied by protein atoms. These cavities provide flexibility in protein conformation and dynamics

• Random coil or loops maybe of importance in protein function (interacting with other molecules, enzyme reactions)


The Thermodynamics of Folding

• Folding of a globular protein is a thermodynamically favored process, i.e. G must be negative.

• The folding process involves going from a multitude of random-coil conformations to a single folded structure.

• The folding process involves a decrease in randomness and thus a decrease in entropy -S and an overall positive contribution to G. This decrease in entropy is termed “conformational entropy”.

• An overall negative G : a result of features that yield a large negative H or some other increase in entropy on folding.

G = H - TS


Protein Folding : No Net Enthalpic Contribution

• Formation of secondary structure is an enthalpy driven process – Energy derived from the formation of many van der waals and H-

bonding interactions as well as the alignment of dipoles overcomes the loss of entropy associated with the formation of the peptide backbone conformation.

• Formation of tertiary structure is enthalpically unfavorable– Energy loss in the burying of ion-pairs (~1 kcal/mol) and the breaking

of shorter, stronger H2O bonds. – Though some energy is gained from van der waals packing, very little

is gained from the formation of internal h-bonds because as many h-bonds with water are broken as are formed in the process of folding a protein.

• Free energy associated with solvation of an ion is ~-60 kcal/mol– An ion will NOT be buried in the hydrophobic interior of a protein.


Protein Folding : Entropy Driven Process

• Upon protein folding – hydrophobic residues move to the interior of the protein

– caged H2O molecules are released

– Enthalpy is gained : unfavorable (H +)– entropy is also gained (S +) : extremely favorable

• Increase in entropy of water compensates for the loss of conformational entropy of the protein and drives the protein folding process


Free energy of folding

• Difference in energy (free energy) between folded (native) and unfolded (denatured) state is small, 5-15 kcal/mol

• Enthalpy and entropy differences balance each other, and G is a small positive number.

• Small G is necessary because too large a free energy change would mean a very stable protein, one that would never change

• However, structural flexibility is important to protein function, and proteins need to be degraded


Protein Folding

• What are the forces that guide this process?

• What are the Steps Involved?

• How Fast Can this Happen?

Random Coil Native conformation


The Thermodynamic Hypothesis

“The native, folded structure of a protein, under optimal conditions, is the most energetically stable conformation possible” Christian Anfinsen, 1972

•Most of the information for determining the three-dimensional structure of a protein is carried in its amino acid sequence

Anfinsen, C.B. Principles that govern the folding of protein chains. Science 181, 223-30 (1973).


Levinthal’s Paradox

Consider a protein of 100 amino acids. Assign 2 conformations to each amino acid. The total conformations of the protein is 2100=1.27x1030. Allow 10-

13 sec for the protein to sample through one conformation in search for the overall energy minimum. The time it needs to sample through all conformations is

(10-13)(1.27x1030)=1.27x1017sec = 4x109 years!

Levinthal’s paradox illustrates that proteins must only sample through limited conformations, or fold by “specific pathways”. Much research efforts are devoted in searching for the principles of the “specific pathways”.


Protein folding

• For any given protein, there is one conformation that has significantly lower free energy than any other state

• Achieved through kinetic pathway of unstable intermediates (not all intermediates are sampled)

• Assisted by chaperones and protein disulfide isomerases so intermediates are not trapped in a local low energy state


The Kinetic Theory of Protein Folding

-Folding proceeds through a definite series of steps or a Pathway. A protein does not try out all possible rotations of conformational angles, but only enough to find the pathway

- The final state may NOT be the most stable conformation possible, but it could be the most stable conformation that is accessible in a reasonable amount of time. This is also the biologically important time frame


Folding Pathways

• Protein folding is initiated by reversible and rapid formation of local secondary structures

• Secondary structures then form domains through the cooperative aggregation of folding nuclei

• Domains finally form the final protein through “Molten globule” intermediates.


Models for Protein Folding

1. Hydrophobic collapse. Formation of a 'molten globule'

2. Framework model. Secondary structure forms first, perhaps including supersecondary structure.

3. Nucleation. Domains fold independently, and sub-domains serve as 'structural kernels’.



Framework model

• Local interactions are the main determinants of protein structures

• Interactions as the ones responsible for forming secondary structural elements, -helices and -sheets

• Isolated helices/sheets form early in the protein folding pathway, then assemble in the native tertiary structure


What's really happening?

• Hydrophobic side chains are being buried

• Secondary structure formation insulates the polar protein backbone from the nonpolar protein interior

• Hydrogen bonds, disulfide bonds and salt bridges begin to form and stabilize structure

• van der Waals interactions bring protein substructures into stable contact


What's most important in folding?

• Nonspecific interactions (hydrophobic effect, van der Waals) are required to bring the protein together into a globular conformation

• Steric interactions (restraints on the backbone) limit the ways in which the globular conformation can form

• Chemically specific interactions (hydrogen bonds, ionic interactions, dipolar interactions) determine the fine detail of the protein structure


Enzymes that speed folding

• Protein disulphide isomerase Facilitates formation of correct disulphide bridges

• Peptidyl proline isomerase Catalyses cis-trans isomerisation of peptide bonds involving proline

• Molecular chaperones Help folding, especially of large proteins, by preventing interaction with other proteins


What is the molecular basis of the observation that proteins of

thermophiles are more stable than their homologues in

mesophiles?

Understanding Biology through structures Course work 2009 Principles that Govern Protein Structure and Stability.

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