Electrostatic stabilization in four‐helix bundle proteins

Abstract

Charge substitutions generated by site‐directed mutagenesis at the termini of adjacent anti‐parallel α‐helices in a four‐helix bundle protein were used to determine a precise value for the contribution of indirect charge‐charge interactions to overall protein stability, and to simulate the electrostatic effects of α‐helix macrodipoles. Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti‐parallel helices in the four‐helix bundle cytochrome b₅₆₂ from Escherichia coli. Previously, theoretical calculations of helix macrodipole interactions using modeled four‐helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol. Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges. At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti‐parallel helix pair. Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude. The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect. Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti‐parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.