DESIGN, STRUCTURE AND STABILITY OF A HYPERTHERMOPHILIC PROTEIN VARIANT

Here we report the use of an objective computer algorithm in the desig n of a hyperstable variant of the Streptococcal protein G beta 1 domai n (G beta 1). The designed seven-fold mutant, G beta 1-c3b4. has a mel ting temperature in excess of 100 degrees C and an enhancement in ther modynamic stability of 4.3 kcal mol(-1) at 50 degrees C over the wild- type protein. G beta 1-c3b4 maintains the G beta 1 fold, as determined by nuclear magnetic resonance spectroscopy, and also retains a signif icant level of binding to human IgG in qualitative comparisons with wi ld type. The basis of the stability enhancement appears to have multip le components including optimized core packing, increased burial of hy drophobic surface area, more favorable helix dipole interactions, and improvement of secondary structure propensity. The design algorithm is able to model such complex contributions simultaneously using empiric al physical/chemical potential functions and a combinatorial optimizat ion algorithm based on the dead-end elimination theorem. Because the d esign methodology is based on general principles, there is the potenti al of applying the methodology to the stabilization of other unrelated protein folds.