Surface salt bridges stabilize the GCN4 leucine zipper

Abstract

We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcall mol, an apparent elevation of the melting temperature relative to wild type of about 22 "C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences. Keywords: GCN4; leucine zipper; salt bridge; thermal stabilityInteractions that stabilize the native state of proteins include the hydrophobic effect, van der Waals interactions, hydrogen bonds and ionic effects, including dipole interactions and salt bridges (Creighton, 1993). The question of which of these are most important in protein stabilization has been debated since the review by Kauzmann (1959). One aspect of the problem concerns how to account for the additional stabilization of proteins from thermophiles, which can have very high thermal stabilities (see Hiller et al., 1997). Since the pioneering work of Matthews et al. (1974) on thermolysin, structures of thermophilic and mesophilic proteins have been compared in a search for clues to what accounts for the higher stability of the former (Korndorfer et al., 1995;Yip et al., 1995;Hatanaka et al., 1997;Robb & Maeder, 1998). In 1978, Perutz (1978 observed that the main discernible difference between a thermophilic and mesophilic version of ferrodoxin lay in the greater number of salt bridges on the surface of the thermophile. As more crystal structures of thermophilic proteins have become available, other mechanisms have been proposed to explain their stability (Vogt & Argos, 1997): improved internal packing, burial of a greater hydrophobic area (Chan et al., 1995; Delboni et al., 1995), and networks of complex salt bridges (Yip et al., 1995;Pappenberger et al., 1997 Yip et al., 1995;Robb & Maeder, 1998).Here, we consider the role of complex salt bridges in stabilizing a simplified model protein structure. The strength of a salt bridge can be estimated by different experimental methodologies: changes in the helicity of model peptides (Merutka & Stellwagen, 1990;Lyu et al., 1992;Scholtz et al., 1993), shifts in pK, of interacting side chains (Anderson et al., 1990;, or T, differences in model proteins Dao-Pin et al., 1991). Using the first method, Smi...

Design of helical, oligomeric HIV-1 fusion inhibitor peptides with potent activity against enfuvirtide-resistant virus

“…Alanine has been shown to promote helix formation in peptides and has one of the highest helix propensities, as determined from host-guest experiments (24,(26)(27)(28)(29)(30). Based on the alanine scan data, eight noncritical residues that were not part of an ion pair were substituted with alanine ( Fig.…”

“…The rules that govern the stability of the ␣-helix have been extensively studied, and a variety of methods have been used to estimate the helical propensity of amino acids (17)(18)(19)(20). Introducing ion pair interactions, or ''salt bridges,'' has also been suggested to increase helical structure in peptides and stabilize coiled coils (21)(22)(23)(24). Some of these noncovalent methods have been successful in enhancing antiviral activity of monomeric HR2 peptides (15,25).…”

Contribution of Surface Salt Bridges to Protein Stability,

“…Theoretical and some experimental results suggest that forming a solvent-exposed ion pair may not be favorable, because in the process two ions have to be desolvated and immobilized (9,12,14,(44)(45)(46)(47). The surface salt bridge , and 6A49A (0) at 1°C in (a) guanidinium chloride and (b) urea (to have the curves appear on the same plot, the initial and final points were arbitrarily called 0 and 100% unfolded despite the fact that proteins 14Q, 14T, 1Q14Q, and 1Q14T probably have an unfolded population even at 0% denaturant).…”

Putative Interhelix Ion Pairs Involved in the Stability of Myoglobin

“…They are salt bridges (hydrogen-bonded ion pairs) in which the hydrogen bond is a major contributor to stability, as judged both by the effects of protonating carboxylate groups and by attempting to screen the interaction with NaCl. A complex RER salt bridge significantly stabilizes the GCN4 dimeric coiled coil (34), and the RER residues are expected to be solvent exposed. Complex networks of ionized side chains are commonly found on the surfaces of thermophilic proteins, but evaluation of their stabilizing effects is still under discussion (35).…”