The beta-beta-alpha fold: explorations in sequence space.

The computational redesign of the second zinc finger of Zif268 to produce a 28 residue peptide (FSD-1) that assumes a betabetaalpha fold without metal binding was recently reported. In order to explore the tolerance of this metal-free fold towards sequence variability, six additional peptides resulting from the ORBIT computational protein design process were synthesized and characterized. The experimental stabilities of five of these peptides are strongly correlated with the energies calculated by ORBIT. However, when a peptide with a mutation in the beta-turn is examined, the calculated stability does not accurately predict the experimentally determined stability. The NMR solution structure of a peptide incorporating this mutation (FSD-EY) reveals that the register between the beta-strands is different from the model structure used to select and score the sequences. FSD-EY has a type I' turn instead of the target EbaaagbE turn (rubredoxin knuckle). Two additional peptides that have improved side-chain to backbone hydrogen bonding and turn propensity for the target turn were characterized. Both are of stability comparable to that of FSD-1. These results demonstrate the robustness of the ORBIT protein design methods and underscore the need for continued improvements in negative design.

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 the beta 1 domain of streptococcal protein G (G beta 1) were solved by (1)H NMR methods to assess the robustness of amino acid sequence selection by the ORBIT protein design package under changes in protein backbone specification. One variant has mutations at three of 10 core positions and corresponds to minimal perturbations of the native G beta 1 backbone. The other, with mutations at six of 10 positions, was calculated for a backbone in which the separation between G beta 1's alpha-helix and beta-sheet was increased by 15% relative to native G beta 1. Exchange broadening of some resonances and the complete absence of others in spectra of the sixfold mutant bespeak conformational heterogeneity in this protein. The NMR data were sufficiently abundant, however, to generate structures of similar, moderately high quality for both variants. Both proteins adopt backbone structures similar to their target folds. Moreover, the sequence selection algorithm successfully predicted all core chi(1) angles in both variants, five of six chi(2) angles in the threefold mutant and four of seven chi(2) angles in the sixfold mutant. We conclude that ORBIT calculates sequences that fold specifically to a geometry close to the template, even when the template is moderately perturbed relative to a naturally occurring structure. There are apparently limits to the size of acceptable perturbations: In this study, the larger perturbation led to undesired dynamic behavior.

De novo protein design: towards fully automated sequence selection.

Several groups have applied and experimentally tested systematic, quantitative methods to protein design with the goal of developing general design algorithms. We have sought to expand the range of computational protein design by developing quantitative design methods for residues of all parts of a protein: the buried core, the solvent exposed surface, and the boundary between core and surface. Our goal is an objective, quantitative design algorithm that is based on the physical properties that determine protein structure and stability and which is not limited to specific folds or motifs. We chose the betabetaalpha motif typified by the zinc finger DNA binding module to test our design methodology. Using previously published sequence scoring functions developed with a combined experimental and computational approach and the Dead-End Elimination theorem to search for the optimal sequence, we designed 20 out of 28 positions in the test motif. The resulting sequence has less than 40% homology to any known sequence and does not contain any metal binding sites or cysteine residues. The resulting peptide, pda8d, is highly soluble and monomeric and circular dichroism measurements showed it to be folded with a weakly cooperative thermal unfolding transition. The NMR solution structure of pda8d was solved and shows that it is well-defined with a backbone ensemble rms deviation of 0. 55 A. Pda8d folds into the desired betabetaalpha motif with well-defined elements of secondary structure and tertiary organization. Superposition of the pda8d backbone to the design target is excellent, with an atomic rms deviation of 1.04 A.