|
Sequence/structure relationships have been explored by site-directed mutagenesis
using a structurally known adenylate kinase. In particular the effects of helix
capping and nonpolar core expansion on thermodynamic stability have been
analyzed. Six point mutations were produced and characterized by SDS/PAGE,
native PAGE, isoelectric focussing, electrophoretic titration, enzyme kinetics,
and X-ray structure analysis. Heat-denaturation experiments yielded melting
temperatures Tm and melting enthalpy changes delta Hm. The heat capacity change
delta Cp of the wild-type enzyme was determined by guanidine hydrochloride
denaturation in conjunction with Tm and delta Hm. Using the wild-type delta Cp
value, Gibbs free energy changes delta G at room temperature were calculated for
all mutants. Four mutants were designed for helix capping stabilization, but
only one of them showed such an effect. Because of electrostatic interference
with the induced-fit motion, one mutant decreased the catalytic activity
strongly. Two mutants expanded nonpolar cores causing destabilization. The
mutant with the lower stability could be crystallized and subjected to an X-ray
analysis at 223-pm resolution which showed the structural changes. The enzyme
was stabilized by adding a -Pro-His-His tail to the C-terminal alpha-helix for
nickel-chelate chromatography. This addition constitutes a helix cap. Taken
together, the results demonstrate that stabilization by helix capping is
difficult to achieve because the small positive effect is drowned by adverse
mutational disruption. Further addition of atoms to nonpolar cores destabilized
the protein, although the involved geometry changes were very small,
demonstrating the importance of efficient packing.
|
