Forces that stabilize protein structure

What are the different kinds of bonds?

1. Covalent bonds

2. Non-covalent bonds

What is the physical basis of making bonds?

Bonding is based on the interactions of the outer shell electrons.

How does covalent bond differ from non-covalent bonds?

Nature of bonding depends largely on electronegativity differences between individual atoms

involved in the interaction. Thus, small differences between atoms involved in interactions

lead to covalent bonding and large electronegativity differences lead to electrostatic ionic

interactions.

The typical chemical interactions that stabilize polypeptides

Interaction Distance dependence Typical

distance

Free energy (bond dissociation enthalpies for

the covalent bonds)

Covalent - 1.5 Å 356 kJ/mol (610 kJ/mol for C=C bond)

Disulfide - 2.2 Å 167 kJ/mol

Salt bridge Donor and acceptor < 3.5 Å 2.8 Å 12.5-17 kJ/mol; may be as high as 30 kJ/mol for

fully or partially buried salt bridges; less if the

salt bridge is external

Hydrogen

bond

Donor and acceptor < 3.5 Å 3.0 Å 2-6 kJ/mol in water; 12.5-21 kJ/mol if either

donor or acceptor is charged

Long-range

electrostatic

Depends on dielectric constant of

medium. Screened by water. 1/r

dependence

Variable Depends on distance and environment. Can be

very strong in nonpolar region but very weak in

water

Van der Waals Short range. Falls off rapidly

beyond 4 separation. 1/r6

dependence.

3.5 Å 4 kJ/mol (4-17 in protein interior) depending on

the size of the group (for comparison, the average

thermal energy of molecules at RT is 2.5 kJ/mol

General relative strength of each of these is typically much less than covalent bond

Electrostatic > Hydrophobic interactions > Hydrogen bonds > van der Waals

1. Electrostatic or ionic

2. Hydrogen bonds

3. Van der Waals

4. Hydrophobic interactions

Primary Non-covalent attractive forces/interactions in macromolecules

• Larger structures assemble spontaneously due to sufficient number of weak bonds

formation.

• A consequence: weak forces restrict organisms to a narrow range of environmental

conditions (temperature, ionic strength, relative acidity).

• Weak interactions can break and reform under normal, physiological conditions.

This allows conformational or shape changes in large molecules.

• These changes drive biochemical reactions, motility, etc. and are essential for many

proteins to function.

• Biomolecular recognition is performed via interplay of complementary molecules. So,

biological function is achieved through mechanisms based on structural complementarity

and weak chemical interactions.

Why are non-covalent interactions so important in biochemistry and biopolymers?

Electrostatic Interactions

What are ionic bonds ?

What kinds of biological molecules form ionic bonds?

Ionic bonds are forces of attraction between ions of opposite charge (+and -).

Any kind of biological molecule that can form ions.

An example of a functional group that can enter into ionic bonds is shown below. The

carboxyl group is shown.

Under the right conditions of pH the carboxyl group will ionize and form the negatively

charged COO ion and a positively charged H ion (or proton).

• They play an important role in determining the shapes (tertiary and quaternary structures)

of proteins.

• They are involved in the process of enzymatic catalysis.

• They are important in determining the shapes of chromosomes.

• They play a role in muscle contraction and cell shape.

• They are important in establishing polarized membranes for neuron function and muscle

contraction.

• These interactions do not majorly play role in protein stability.

What function do ionic bonds have in biology?

Electrostatic interaction depends on the distance of the two charges and the medium between

them i.e.

q1, q2: charges; r: distance; D: dielectric constant

(vacuum: 1, water: 80)

In the case of molecules where q1, q2 and D are constants:

where c: constant (=q1∗q2/D).

For example: two groups which are 2Å and 3Å apart, the interaction force between them is

distance dependent i.e.

&

The optimal distance for electrostatic interaction is 2.8 Å.

2

21

Dr

qqF

c2r

1F

4D

qqF 21

9D

qqF 21

Interaction strongest in vacuum, stronger in nonpolar solvents than in water (weakest).

Within the interior of a protein, the structure or primary amino acid sequence can lead to an

environment with a low D, under these circumstances the electrostatic bond strength can reach

significantly high levels.

Hydrogen Bond

How are H-bonds formed?

Hydrogen bonds are formed when a charged part of a molecule having polar covalent bonds

forms an electrostatic interaction with a substance of opposite charge.

What classes of compounds can form hydrogen bonds?

Under the right environmental conditions, any compound that has polar covalent bonds can

form hydrogen bonds.

What is the strength of H-bonds?

Hydrogen bonds are classified as weak bonds because they are easily and rapidly formed and

broken under normal biological conditions. The strength of a H-bond is 3-7 kcal/mol.

Hydrogen donor - holds H more tightly, has partial positive charge

Hydrogen acceptor - has partial negative charge that attracts H atom

For creating a H-bond a "lone pair" of electrons in the acceptor atom is necessary. N has one

lone pair of electrons, so it can be acceptor in one H-bond. O has 2 lone pairs of electrons and

can therefore accept 2 H-bonds.

The distance between O-N is 2.88 Å (N has 1 lone pair of electrons, thus one H-bond

possible) and distance between N-O is 3.04 Å (O has 2 lone pair of electrons, thus two H-

bonds would be possible).

Do you think that distance between O-N and N-O are different?

Which bond is stronger?

The H-bond from O-H…N is stronger than the one from N-H…O because the partial charge

at the H is greater in the case of OH.

What about the distance between N-H…O and N+-H…O?

The strength of the H-bond depends on its orientation. It is strongest if donor atom (H-atom)

and acceptor atom lie on a line.

O…H-N are in a line => stronger

O…H-N are not in a line => weaker.

Note: In antiparallel β sheets, donor H and acceptor are in a line and in parallel β sheets they

are not. That is why antiparallel β sheets are more stable and often occur in proteins.

Amino acids Hydrogen donor atoms Hydrogen acceptor atoms

Arginine (R) NE, NH1, NH2

Asparagine (N) ND2 OD1

Aspartic acid (D) OD1, OD2

Glutamine (Q) NE2 OE1

Glutamic acid (E) OE1, OE2

Histidine (H) ND1, NE2 ND1, NE2

Lysine (K) NZ

Serine (S) OG OG

Threonine (T) OG1 OG1

Tryptophan (W) NE1

Tyrosine (Y) OH OH

What about cysteine, methionine, tryptophan and tyrosine.

What are the amino acids which side chain can participate in H-bonds?

Donors or acceptors as a function of the pH: Lys, Asp, Glu, Tyr, His

At low pH, when there are more H than usual, no more H can be accepted, thus donor only.

At high pH, when there are less H than usual, no H can be donated, thus acceptor only.

Which amino acids are pH dependent to form H-bonds?

At lower pH, lysine is charged (NH3+), then it can only act as a donor. At a higher pH, when it

is not charged (NH2), it can act as donor and as acceptor.

Aspartic acid and glutamic acid are donors and acceptors at low pH, when they are not

charged (COOH). At higher pH, when they are charged, they can only act as acceptors.

Tyrosine is donor and acceptor at low pH, when it is not charged (OH). It is acceptor only at

higher pH, when it is charged (O).

Histidine is a donor only at low pH, when it is charged (NH, NH+). It is donor and acceptor at

higher pH when it is not charged (NH, N).

Van der Waals forces

Charge distribution over an atom is not uniform with time and is therefore transient. This

induces a charge on the nearby molecules. It is nonspecific attractive/repulsive forces.

What is the physical basis of Van der Waals interactions? Or

How does nonpolar molecules attain charge?

Dispersion

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":

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.

The Repulsion interaction can be shown to vary according to the inverse twelfth power of the

distance between the two atoms:

The repulsive core is sometimes termed a "Pauli exclusion interaction".

• 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. Van der Waals bonds work only when the atoms

are 3Å to 4Å apart.

Examples of Van der Waal's radii

H = 1.2 Å; C = 2.0 Å; N = 1.5 Å; O = 1.4

Å; S = 1.85 Å; P = 1.9 Å.

For the van der Walls contact distance

between 2 atoms the radii of the 2 atoms

must be added. E.g.: C and O: 2.0Å + 1.4Å

= 3.4Å.

dipole-dipole interactions

Dipole–dipole interactions are electrostatic interactions of permanent dipoles in molecules. An

example of a dipole–dipole interaction can be seen in hydrogen chloride (HCl): the positive

end of a polar molecule will attract the negative end of the other molecule and influence their

arrangement.

Repulsive: when two atoms are very close together since the electron shells overlap and the

negative-negative charge interactions are highly repulsive.

Attractive: force results from the interaction of the positively charged nucleus in one atom

and the negatively charged electrons of the second atom at the appropriate distance.

Ion-Induced Dipole Forces

An ion-induced dipole attraction is a weak attraction that results when the approach of an ion

induces a dipole in an atom or in a nonpolar molecule by disturbing the arrangement of

electrons in the nonpolar species.

What is induced dipole?

Induced dipole forces result when an ion or a dipole induces a dipole in an atom or a molecule

with no dipole.

Dipole-Induced Dipole Forces

A dipole-induced dipole attraction is a weak attraction that results when a polar molecule

induces a dipole in an atom or in a nonpolar molecule by disturbing the arrangement of

electrons in the nonpolar species.

Protein Interior and Exterior

Internal packing of atoms in a protein can be analyzed 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 and is called the Van der

Waals surface.

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.

Nonpolar molecules cluster together to minimize the

hydrophobic surface area exposed to surrounding water

molecules. This allows water to form as many hydrogen

bonds as possible and minimizes poor interactions with the

nonpolar molecule.

Hydrophobic interactions

Non-polar molecules are driven together in water not

primarily because they have a high affinity for each

other but because water bonds strongly to itself.

Hydrophobic interactions are more correctly called

hydrophobic exclusions.

Stability is defined as a net loss of free energy, a function of the

combined effects of entropy and enthalpy.

Most weak interactions release about 4–13 kJ/mole of free

energy when they occur in water and therefore contribute only a

small amount to the total stability of a protein.

Protein Stability: Weak Interactions and Flexibility

Some proteins are naturally very stable to thermal or chemical

denaturation such as thermophilic proteins retain their

structure and activity at temperatures approaching the boiling

point of water.

These proteins have more salt bridges, hydrophobic interactions,

shorter protruding loops, and so forth.