Electrostatic stabilization of a thermophilic cold shock protein

The cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus di ffers from its mesophilic homolog Bs-CspB from Bacillus subtilis by 15.8 kj mol(-1) in the Gibbs free energy of denaturation (DeltaG(D)). The two prot eins vary in sequence at 12 positions but only two of them, Arg3 and Leu66 of Bc-Csp, which replace Glu3 and Glu66 of Bs-CspB, are responsible for the additional stability of Bc-Csp. These two positions are near the ends of t he protein chain, but close to each other in the three-dimensional structur e. The Glu3Arg exchange alone changed the stability by more than 11 kj mol( -1). Here, we elucidated the molecular origins of tl e stability difference between the two proteins by a mutational analysis. Electrostatic contribut ions to stability were characterized by measuring the thermodynamic stabili ties of many variants as a function of salt concentration. Double and tripl e mutant analyses indicate that the stabilization by the Glu3Arg exchange o riginates from three sources. Improved hydrophobic interactions of the alip hatic moiety of Arg3 contribute about 4 kj mol(-1). Another 4 kj mol(-1) is gained from the relief of a pairwise electrostatic repulsion between Glu3 and Glu66, as in the mesophilic protein, and 3 kj mol(-1) originate from a general electrostatic stabilization by the positive charge of Arg3, which i s not caused by a pairwise interaction. Mutations of all potential partners for an ion pair within a radius of 10 Angstrom around Arg3 had only margin al effects on stability. The Glu3 Arg3 charge reversal thus optimizes ionic interactions at the protein surface by both local and global effects. Howe ver, it cannot convert the coulombic repulsion with another Glu residue int o a corresponding attraction. Avoidance of unfavorable coulombic repulsions is probably a much simpler route to thermostability than the creation of s tabilizing Surface ion pairs, which can form only at the expense of conform ational entropy. (C) 2001 Academic Press.