Disulfide bonds and thermal stability in T4 lysozyme.

Abstract

Disulfide bonds are thought to serve a stabilizing role in extracellular globular proteins, but little is known about the modes of stabilization or their mechanisms. Thermodynamic data presented here demonstrate that an engineered 3-97 disulfide bond previously shown to stabilize T4 lysozyme in vitro against irreversible thermal inactivation also stabilizes the molecule against reversible thermal unfolding. In this paper, we explore the relationship between the disulfide's thermodynamic contribution to protein folding and its role in providing resistance to irreversible thermal inactivation. In T4 lysozyme (C54V/C97S), a non-crosslinked mutant lacking the two cysteines found in the wild type, sensitivity toward irreversible thermal inactivation increases dramatically at temperatures above the melting temperature of the molecule. In addition, most of the lost activity can be restored by denaturation/renaturation with guanidine hydrochloride. In contrast, the crosslinked mutant T4 lysozyme (13C-97C/C54V) inactivates relatively slowly, even above its melting temperature, and the lost activity is not restored by denaturation/ renaturation. These observations suggest that the predominant inactivation pathways for non-crosslinked T4 lysozymes are conformation related, while those for the crosslinked variant are insensitive to the conformational route and thus are susceptible only to slower processes of a chemical nature. We also show that multiple mutants, constructed to contain the 3-97 disulfide plus a temperature-sensitive lesion, are more stable than the wild type to irreversible inactivation even though they are less stable to reversible thermal unfolding. These rmdings together suggest that the 3-97 disulfide provides stability to irreversible inactivation primarily via a pathway that is independent of its thermodynamic contribution. The 3-97 disulfide may stabilize T4 lysozyme by restricting the unfolded state to a class of more compact structures with less exposed hydrophobic surface, compared to the unfolded states of non-crosslinked T4 lysozymes. The results have implications both for the use of the stabilizing potential of disulfide bonds in protein engineering and for their roles in protein function and evolution.Many extracellular globular proteins contain disulfide bonds, which are thought to provide additional stability against environmental insults (1, 2). This hypothesis is based on observations that the disruption of disulfide crosslinks is often accompanied by a decrease in thermodynamic stability-that is, stability to reversible denaturation (2-5). In turn, thermodynamic stability is important because unfolded proteins are generally more susceptible to at least some of the irreversible processes (e.g., proteolysis, aggregation, adsorption, precipitation) by which protein structure and activity can be lost in vitro (6-8) and in vivo (9-12). Previously we have shown that the introduction, by directed mutagenesis, of a 3-97 disulfide bond into the normally disulfide-free enzyme T4 lysozyme sta...

Disulfide bonds and the stability of globular proteins

“…In addition, AH, for these variants is greatly reduced, indicating that the presence of these crosslinks destabilizes the protein (Wetzel et al, 1988). Three other novel disulfides increase T, by 5-10 "C (Matsumura et al, 1989a,b) at low pH, and combinations of these crosslinks are nearly additive in their ability to increase T,,, (Matsumura et al, 1989b), although the tendency of these crosslinks to increase resistance to denaturation at neutral pH (Wetzel et al, 1988) or under low-temperature denaturing conditions (Anderson et al, 1990) is diminished. However, each of these "stabilizing" crosslinks raises AC, and lowers AH, (Matsumura et al, 1989a).…”

“…Of these, the most extensively studied is T4 lysozyme, which has no disulfides in its native form. Five different novel disulfide bonds have been introduced into this protein (Perry & Wetzel, 1984;Wetzel et al, 1988;Matsumura et al, 1989a,b). Two have been found to lower T,,, by 20 "C at neutral pH.…”

Engineering multiple properties of a protein by combinatorial mutagenesis.

“…Recombinant DNA technology has allowed the construction of proteins of altered stability in vitro (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), catalytic efficiency (17)(18)(19), substrate specificity (20)(21)(22)(23), and resistance to in vivo thermal inactivation (24) through the use of single or multiple amino acid substitutions. This effort has been greatly helped by the fact that the effects of amino acid substitutions on such properties of proteins tend to be additive as mutations accumulate, provided that the substituting residues do not interact functionally or by direct contact (25).…”

Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase

“…When the enzyme was heated at 75 "C for 30 min at neutral pH, however, these cysteines were oxidized to cystine without affecting the catalytic activity of TBADH. As the results of many studies have suggested that disulfide bonds confer rigidity and stability on certain proteins (Nakamura et al, 1978;Wetzel et al, 1988;Matsumura et al, 1989;Dohlman et al, 1990;Mely & Gerard, 1990;Kanaya et al, 1991), several investigators have attempted to introduce disulfides to enhance the thermal stability of proteins, using such methods as site-directed mutagenesis (Wetzel et al, 1988;Matsumura et al, 1989;Luckey et al, 1991) or grafting cysteine-containing peptides (Magonet et al, 1992), for example. To probe the function of the cystine 283-295 disulfide in TBADH, we used site-directed mutagenesis to replace cys 283 with serine.…”