SURFACE SALT BRIDGES STABILIZE THE GCN4 LEUCINE-ZIPPER

We present a study of the role of salt bridges in stabilizing a simpli fied tertiary structural motif, the coiled-coil. Changes in GCN4 seque nce have been engineered that introduce trial patterns of single and m ultiple salt bridges at solvent exposed sites. At the same sites, a se t of alanine mutants was generated to provide a reference for thermody namic analysis of the salt bridges. Introduction of three alanines sta bilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrang ement corresponding to a complex type of salt bridge involving three g roups stabilizes the dimer by 1.7 kcal/mol, an apparent elevation of t he melting temperature relative to wild type of about 22 degrees C. Wh ile identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cy cles. Introduction of alanines for side chains of lower helix propensi ty and complex salt bridges both stabilize the coiled-coil, so that co mbining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequence s.