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Unit II


Covalent, Ionic, Hydrogen, Coordinate,
hydrophobic and Vander walls interactions in
protein structure.
Interaction with electromagnetic radiation
(radio, micro, infrared, visible, ultraviolet,
Elucidation of protein structure.

disulfide bridge: a covalent bond formed when the
reduced SH groups of two cysteine residues react
with one another to make an oxidized SS linkage.
electrostatic interaction: noncovalent interaction
between atoms or groups of atoms due to attraction of
opposite charges.
hydrogen bond: a noncovalent interaction between
the donor atom, which is bound to a positively
polarized hydrogen atom, and the acceptor atom,
which is negatively polarized. Though not covalent, the
hydrogen bond holds the donor and acceptor atom
close together.
reducing environment: a chemical environment in
which the reduced states of chemical groups are
favored. In a reducing environment, free SH groups
are favored over SS bridges. The interior of most
cells is a highly reducing environment.
salt bridge: a hydrogen bond in which both donor
and acceptor atoms are fully charged. The
bonding energy of a salt bridge is significantly
higher than that of a hydrogen bond in which
only one participating atom is fully charged or in
which both are partially charged.
van der Waals interaction: a weak attractive
force between two atoms or groups of atoms,
arising from the fluctuations in electron
distribution around the nuclei. Van der Waals
forces are stronger between less electronegative
atoms such as those found in hydrophobic

native proteins are only marginally stable
entities under physiological conditions.
The free energy required to denature them is
0.4 kJ mol-1 of amino acid residues, so that
100-residue proteins are typically stable by
only around 40 kJ mol-1
Electrostatic Forces
Charged side chains in protein can interact favorably with an opposing
charge of another side chain according to Coulombs 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.

Methods are widely used to calculate the surface electrostatic potentials
of proteins using a program called GRASP (for Graphical Representation
and Analysis of Surface Properties) written by Anthony Nicholls, Kim
Sharp, and Barry Honig.

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 in proteins
Small charged metal ions can act as positive
charge in an ion pair.

Ionic Interactions Are Strong but Do Not
Greatly Stabilize Proteins
The association of two ionic protein groups of
opposite charge is known as an ion pair or salt
The energy of a typical ion pair, say the
carboxyl group of Glu and the ammonium
group of Lys, whose charge centers are
separated by 4.0 in a medium of dielectric
constant 4, is 86 kJ mol1 (one electronic
charge 16.0 1019 C).

van der Waals forces

van der Waals forces are also known as
London forces.
They are weak interactions caused by
momentary changes in electron density in a
They are the only attractive forces present in
nonpolar compounds.

Even though CH
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.

DipoleDipole Interactions Are Weak but
Significantly Stabilize Protein Structures
The non-covalent associations between
electrically neutral molecules, collectively
known as van der Waals forces, arise from
electrostatic interactions among permanent
and/or induced dipoles.
These forces are responsible for numerous
interactions of varying strengths between
nonbonded neighboring atoms.
Interactions among permanent dipoles are
important structural determinants in proteins
because many of their groups, such as the
carbonyl and amide groups of the peptide
backbone, have permanent dipole moments.
These interactions are generally much weaker
than the chargecharge interactions of ion
In helices, however, the negative ends of the
dipolar amide and carbonyl groups of the
polypeptide backbone all point in the same
direction , so that their interactions and bond
dipoles are additive (these groups, of course, also
form hydrogen bonds, but here we are concerned
with their residual electric fields).
The helix therefore has a significant dipole
moment that is positive toward the N-terminus
and negative toward the C-terminus.
Consequently, in the low dielectric constant core
of a protein, dipoledipole interactions
significantly influence protein folding.
Although nonpolar molecules are nearly electrically
neutral, at any instant they have a small dipole
moment resulting from the rapid fluctuating motions
of their electrons.
This transient dipole moment polarizes the electrons in
a neighboring group, thereby giving rise to a dipole
moment such that, near their van der Waals contact
distances, the groups are attracted to one another (a
quantum mechanical effect that cannot be explained in
terms of only classical physics).
These so-called London dispersion forces are
extremely weak
London forces are only significant for
contacting groups because their association
energy is proportional to r-6.
Nevertheless, the great numbers of
interatomic contacts in the closely packed
interiors of proteins make London forces a
major influence in determining their
London forces also provide much of the
binding energy in the sterically
complementary interactions between proteins
and the molecules that they specifically bind.

Hydrogen Bonds Only Weakly
Stabilize Proteins
H bonding has a major influence on the
structures of proteins. However, an unfolded
protein makes most of its H bonds with the water
molecules of the aqueous solvent (water, it will
be recalled, is a strong H bonding donor and
Consequently, it might be expected that H bonds
do not stabilize (and perhaps even slightly
destabilize) the structure of a native protein
relative to its unfolded state.
Despite their low stability, a proteins hydrogen
bonds provide a structural basis for its native
folding pattern: If a protein folded in a way that
prevented some of its internal H bonds from
forming, their free energy would be lost and such
conformations would be less stable than those
that are fully H bonded.
In fact, the formation of helices and sheets
efficiently satisfies the polypeptide backbones H
bonding requirements.
Most Hydrogen Bonds in Proteins Are
Most of the H bonds in a protein are local, that is, they
involve donors and acceptors that are close together in
sequence and hence can readily find their H bonding
1. On average, 68% of the H bonds in proteins are
between backbone atoms. Of these, 1/3 form n - n 4 H
bonds (as in ideal helices),1/3 form n -n 3 H bonds
(as in reverse turns and ideal 3
helices), and 1/3 are
between paired strands in sheets. In fact, only 5% of
the H bonds between backbone atoms are not wholly
within a helix, sheet, or turn.
2. Hydrogen bonds between side chains and backbones
are clustered at helix-capping positions. In an helix,
the first four NH groups and the last four groups
cannot form H bonds within the helix (which accounts
for half the potential H bonds involving backbone
atoms in an helix of 12 residues, the average length of
These potential H bonds are often made with nearby
side chains. In particular,1/2 of the N-terminal NH
groups of helices form H bonds with polar side chains
that are 1 to 3 residues distant, and 1/3 of their C-
terminal groups form H bonds with polar side chains
that are 2 to 5 residues distant.
3. Over half the H bonds between side chains
are between charged residues (i.e., they form salt
bridges) and are therefore located on protein
surfaces between and within surface loops .
However,85% of the remaining side chainside
chain H bonds are between side chains that are 1
to 5 residues apart.
Hence with the exception of those in salt bridges,
side chainside chain H bonds also tend to be

Hydrophobic forces are a major influence in causing proteins to fold
into their native conformations.
The amino acid side chain hydropathies (indexes of combined
hydrophobic and hydrophilic tendencies) are, in fact, good
predictors of which portions of a polypeptide chain are inside a
protein, out of contact with the aqueous solvent, and which
portions are outside, in contact with the aqueous solvent.
In proteins, the effects of hydrophobic forces are often termed
hydrophobic bonding, presumably to indicate the specific nature of
protein folding under the influence of the hydrophobic effect.
You should keep in mind, however, that hydrophobic bonding does
not generate the directionally specific interactions usually
associated with the term bond.

Intramolecular forces

covers those macromolecular structures
with dimensions between small molecules of
chemistry & the larger features of cell
structure visible in the light microscope

The methods by which the structures are determined
deserve study in structural biology
Dominant techniques are spectroscopies (mass, IR, UV and
NMR) - finds the covalent framework of macromolecules
Structural studies focus on CONFORMATION - the three-
dimensional structures consistent with free rotation about
single bonds; their description, enumeration and
In a protein, 3-D structures are infinite in number, which
interconvert under physiological conditions.
Dominant techniques must determine shapes:
based on image-formation (microscopy)
Diffraction (X-ray crystallography)

Electromagnetic spectrum
showing wavelengths used to study protein
X-rays have very short wavelengths: 0.15
x 10
m or less

At the opposite end of the frequency
spectrum, radio waves have longer
wavelengths ~ 10 m

The frequency scale is significant because
each domain is exploited in biochemistry
to examine different atomic properties or
motions present in proteins.

Radio waves & microwaves cause changes
to the magnetic properties of atoms -
detected by nuclear magnetic resonance
(NMR) & electron spin resonance (ESR).

In the microwave and infrared regions of
the spectrum irradiation causes bond
movements and in particular motions
about bonds such as stretching,
bending or rotation.

The ultraviolet (UV) and visible regions of the
electromagnetic spectrum are of higher energy and probe
changes in electronic structure through transitions
occurring to electrons in the outer shells of atoms.
Fluorescence and absorbance methods are widely used
in protein biochemistry and are based on these

X-rays are used to probe changes to the inner electron
shells of atoms. These techniques require high energies to
knock inner electrons from their shells and this is
reflected in the frequency of such transitions (~ 10

Frequency range & atomic parameters central
to physical techniques used to study protein