Denaturation of proteins at elevated temperatures is usually the result of unfolding followed by an irreversible process, for example aggregation. The unfolding processes that precede irreversible inactivation often have a partial (as opposed to global) character. Thus, two thermodynamically equally stabilizing mutations at different locations in a protein may prove rather different in terms of their effect on resistence towards irreversible denaturation [1,2].

The above is inferred from a series of studies on the stability of the thermolysin-like protease produced by B. stearothermophilus (TLP-ste). Site-directed mutagenesis studies (using WHAT IF [3] for mutant design) first suggested [1] and later confirmed [2] that thermal inactivation of TLP-ste is governed by partial unfolding processes that precede the irreversible process of autolysis [1]. The fact that the rate-limiting unfolding step has a partial character is confirmed by the following experimental observations: (1) stability-determining differences between TLP-ste and its more stable natural counterpart thermolysin were clustered in a limited region of the protein [4]; (2) designed stabilizing mutations were highly effective when introduced in this region, whereas mutations in other parts of the protein had marginal effects on stability [4,5]; (3) the effects of mutations far apart in the TLP-ste structure displayed remarkable non-additivity [2].

Recently, the TLP-ste work has culminated in the construction of a hyperstable variant carrying eight mutations in only a limited region of the protein [6]. Interestingly, this variant shares few structural characteristics with "extremozymes" isolated from hyperthermophilic microorganisms. Its only conspicuous structural characteristic is the binding of four calcium ions whose role is currently under investigation (see also [7]). An important principle derived from the TLP-work is that effective rational manipulation of a protein against irreversible inactivation may require the identification of "weak" regions involved in crucial partial unfolding processes. This principle has recently been confirmed by site-specific immobilization studies of TLP-ste [8] and is currently being applied to other enzymes in our laboratories.

[1] Eijsink, V.G.H. et al., Proteins, 14, 224-236, 1992.

[2] Vriend, G. et al., J. Biol. Chem., 273, 35074-35077, 1998.

[3] Vriend, G., J. Mol. Graph., 8, 52-56, 1990.

[4] Eijsink, V.G.H. et al., Nature Struct. Biol., 2, 374-379, 1995.

[5] Mansfeld, J. et al., J. Biol. Chem., 272, 11152-11156, 1997.

[6] Van den Burg, B. et al., Proc. Natl. Acad. Sci. USA, 95, 2056-2060, 1998.

[7] Veltman, O.R. et al., Biochemistry, 37, 5312-5319, 1998.

[8] Mansfeld, J. et al., Biochemistry, 38, 8240-8245, 1999.