1. Non-covalent interactions like hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions stabilize protein structure through weak but numerous attractive forces. 2. These interactions are weak but important because they allow proteins to dynamically change shape while maintaining overall structure, which enables biochemical reactions and functions. 3. The primary non-covalent attractive forces in macromolecules are electrostatic or ionic bonds, hydrogen bonds, van der Waals forces, and hydrophobic interactions, with electrostatic being the strongest.

1. Forces that stabilize protein structure

2. 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.

3. 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

4. 1. Electrostatic or ionic
2. Hydrogen bonds
3. Van der Waals
4. Hydrophobic interactions
Primary Non-covalent attractive forces/interactions in macromolecules

5. • 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?

6. 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).

7. • 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?

8. 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
qq
F
c2
r
1
F
4D
qq
F 21
9D
qq
F 21

9. 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.

10. 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.

11. 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.

12. 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?

13. 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.

14. 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?

15. 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).

16. 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?

17. 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":

18. 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".

19. • 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.

20. 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.

21. 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.

22. 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.

23. 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.

24. 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.

25. 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.

26. 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.