10.4 Kinetic and Thermodynamic Aspects of Protein–Nucleic Acid Interactions
10.4.1 The Delicate Balance of Sequence-Specificity
The most common way of achieving sequence-specific recognition is direct readoutof the DNA sequence
by hydrogen bonding between the protein and the DNA. This type of readout commonly includes the par-
ticipation of water molecules. A typical DNA–protein complex in the cell might have a binding energy of
a few kJ mol^1 , so it seems that the binding energy and sequence discrimination of most protein–DNA
complexes can be accounted for easily just by a few hydrogen-bonding contacts to the bases and the phos-
phate backbone. However, bases that are not contacted by the protein may also significantly affect the
stability of certain complexes. This results in indirect readoutwhereby the sequence is recognized
through its conformational effects.
The energy of indirect readout is likely to be around 5–20 kJ mol^1 and is thus important for fine-tuning
specificity. This has been demonstrated experimentally in studies of DNA-binding by the 434 repressor,
where it is found that the binding affinity is affected by base substitutions in the central portion of the palin-
dromic site, where there are no contacts with the repressor.^17 The bases there must influence the moulding
of the DNA to the protein.
The formation of specific protein–DNA complexes may represent a delicate balance between favourable
interactions on the one hand and energy penalties, such as those associated with distorting the DNA, on the
other hand. In specific complexes, the free energy contributions are clearly all favourable and derive from
base contacts, phosphate contacts, bulk solvent and counter-ion displacement through the optimal burying
of surface area, and by stabilizing electrostatic interactions. The net free energy change ( G) is offset by
the entropy penalties of conformational restriction and induced-folding and the energy penalty for deform-
ing the DNA, which involves both conformational restriction and altered base stacking. In a non-specific
complex, there are fewer direct interactions with the bases, and the buried surface area is often smaller
(Section 10.4.3). Consequently, a non-specific complex has smaller favourable contributions from these
effects, while the entropic penalties associated with conformational effects are also smaller. The relative
energy contributions are illustrated for specific and non-specific complexes of the restriction enzyme EcoRI
with DNA (Figure 10.9). In this example, specific binding (left) is a fine balance of favourable effects (down-
ward arrows) against unfavourable ones (upward arrows), to leave a small energy gain. In the energetic
decomposition of the non-specific binding event (right), the free energy change is comparatively smaller.
This decomposition is only illustrative because the changes are highly context-dependent.
Complexes in which the DNA is highly distorted tend to be entropy-driven, whilst those with a more
‘relaxed’ DNA conformation tend to be enthalpy-driven (Figure 10.9, right). There are several reasons for
this, but the principal cause is the entropy cost associated with protein folding upon binding of the DNA
in the relaxed case and the enthalpy cost of bending the DNA in the ‘distorted’ DNA case.
10.4.2 The Role of Water
There are broadly two effects to be considered for the role of water in protein–nucleic acid interactions:
(1) the thermodynamics of assembly, and (2) the detailed optimization of surface interactions. In specific
complexes, buried surface area is optimised, and the matching surfaces fit together well. The mobilization
of water from the hydrated surfaces of the protein and of the oligonucleotide grooves and phosphate back-
bones of the nucleic acid provides an entropically favourable contribution to complex formation (Figure 10.9,
right). These bulk solvent effects account in part for the noted changes in heat capacity associated with the
formation of complexes.^18
Crystallographic structures of nucleic acid–protein complexes frequently reveal ordered water molecules
that lie at the molecular interfaces (where their diffraction limit is better than about 3 Å). These water mol-
ecules mediate the macromolecular interactions and also fill the occasional gap arising from interfacial
imperfections. Water molecules participate in hydrogen-bonding networks that link side- and main-chain
398 Chapter 10