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Six designed mutants of T4 lysozyme were created in an attempt to create
putative salt bridges on the surface of the protein. The first three of the
mutants, T115E (Thr 115 to Glu), Q123E, and N144E, were designed to introduce a
new charged side chain close to one or more existing charged groups of the
opposite sign on the surface of the protein. In each of these cases the putative
electrostatic interactions introduced by the mutation include possible salt
bridges between residues within consecutive turns of an alpha-helix. Effects of
the mutations ranged from no change in stability to a 1.5 degrees C (0.5
kcal/mol) increase in melting temperature. In two cases, secondary (double)
mutants were constructed as controls in which the charge partner was removed
from the primary mutant structure. These controls proteins indicate that the
contributions to stability from each of the engineered salt bridges is very
small (about 0.1-0.25 kcal/mol in 0.15 M KCl). The structures of the three
primary mutants were determined by X-ray crystallography and shown to be
essentially the same as the wild-type structure except at the site of the
mutation. Although the introduced charges in the T115E and Q123E structures are
within 3-5 A of their intended partner, the introduced side chains and their
intended partners were observed to be quite mobile. It has been shown that the
salt bridge between His 31 and Asp 70 in T4 lysozyme stabilizes the protein by
3-5 kcal/mol [Anderson, D. E., Becktel, W. J., & Dahlquist, F. W. (1990)
Biochemistry 29, 2403-2408]. To test the effectiveness of His...Asp interactions
in general, three additional double mutants, K60H/L13D, K83H/A112D, and
S90H/Q122D, were created in order to introduce histidine-aspartate charge pairs
on the surface of the protein. Each of these mutants destabilizes the protein by
1-3 kcal/mol in 0.15 M KCl at pH values from 2 to 6.5. The X-ray
crystallographic structure of the mutant K83H/A112D has been determined and
shows that there are backbone conformational changes of 0.3-0.6 A extending over
several residues. The introduction of the histidine and aspartate presumably
introduces strain into the folded protein that destabilizes this variant. It is
concluded that pairs of oppositely charged residues that are on the surface of a
protein and have freedom to adopt different conformations do not tend to come
together to form structurally localized salt bridges. Rather, such residues tend
to remain mobile, interact weakly if at all, and do not contribute significantly
to protein stability. It is argued that the entropic cost of localizing a pair
of solvent-exposed charged groups on the surface of a protein largely offsets
the interaction energy expected from the formation of a defined salt bridge.
There are examples of strong salt bridges in proteins, but such interactions
require that the folding of the protein provides the requisite driving energy to
hold the interacting partners in the correct rigid alignment.
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