Designed protein G core variants fold to native-like structures: Sequence selection by ORBIT tolerates variation in backbone specification

The solution structures of two computationally designed core variants of th e pi domain of streptococcal protein G (GP1) were solved by H-1 NMR methods to assess the robustness of amino acid sequence selection by the ORBIT pro tein design package under changes in protein backbone specification. One va riant has mutations at three of 10 core positions and corresponds to minima l perturbations of the native GP1 backbone. The other, with mutations at si x of 10 positions, was calculated for a backbone in which the separation be tween G beta1's alpha -helix and beta -sheet was increased by 15% relative to native G beta1. Exchange broadening of some resonances and the complete absence of others in spectra of the sixfold mutant bespeak conformational h eterogeneity in this protein. The NMR data were sufficiently abundant, howe ver, to generate structures of similar, moderately high quality for both va riants. Both proteins adopt backbone structures similar to their target fol ds. Moreover, the sequence selection algorithm successfully predicted all c ore chi (1) angles in both variants, five of six chi (2) angles in the thre efold mutant and four of seven chi (2) angles in the sixfold mutant. We con clude that ORBIT calculates sequences that fold specifically to a geometry close to the template, even when the template is moderately perturbed relat ive to a naturally occurring structure. There are apparently limits to the size of acceptable perturbations: In this study, the larger perturbation le d to undesired dynamic behavior.