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PubMed · 8388289

Electrostatic stabilization in four-helix bundle proteins.

C R Robinson·S G Sligar
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Abstract

Charge substitutions generated by site-directed mutagenesis at the termini of adjacent anti-parallel alpha-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 alpha-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.

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ApoproteinsBase SequenceCircular DichroismCytochrome c GroupEscherichia coliModels, ChemicalModels, MolecularMolecular Sequence DataMutagenesis, Site-DirectedProtein ConformationProtein DenaturationProtein Structure, SecondaryRecombinant ProteinsThermodynamicsComparative StudyJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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C R Robinson, S G Sligar. 1993. Electrostatic stabilization in four-helix bundle proteins.. https://doi.org/10.1002/pro.5560020512

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