Permutation of modules or secondary structure units creates proteins with basal enzymatic properties

Abstract

The RNase activity of barnase mutants obtained by the permutation of modules or secondary structure units was investigated. Four of the 45 mutants had weak but distinct RNase activity, and they had unique optimum pHs and temperatures like natural enzymes. One of the active mutants had an ordered conformation, but the others did not. An active mutant having disordered conformation formed an ordered conformation in the presence of GMP, which is an inhibitor of this mutant. These results indicate that the amino acid sequences derived from barnase have sufficient plasticity to be rearranged into different proteins with basal enzymatic properties.z 1999 Federation of European Biochemical Societies.

Random multi-recombinant PCR for the construction of combinatorial protein libraries

“…In our previous paper (20,34), the foldability and RNase activity of 45 barnase mutants obtained by the permutation of modules or secondary structure units were investigated. This study suggests that amino acids not involved directly in catalysis in foldable proteins have been selected as scaffolds to stabilize the active site with appropriate conformations in the course of globular protein evolution.…”

“…This study suggests that amino acids not involved directly in catalysis in foldable proteins have been selected as scaffolds to stabilize the active site with appropriate conformations in the course of globular protein evolution. The mutant having a nascent active site, but not a stable scaffold, can be considered as globular protein in an 'evolutionarily intermediate state' (34). Theoretical studies have successfully simulated the trajectory in which evolution of stability around the active site leads to a sequence which folds globally into a conformation (35,36).…”

Towards the Creation of Novel Proteins by Block Shuffling

“…The RNase activity of the 46 barnase mutants was also investigated, and we found that eight secondary structure unit mutants and two module mutants had weak but distinct RNase activity [30,31]. RNase activity of several of the mutants was further investigated at different pHs, and we found that they each showed a bell-shaped curve with an optimum pH.…”

“…On the other hand, because S4523 does not have such a stable conformation, the overall structure of the mutant would be easily affected by conformational change of the active site. Amino acids that are not involved directly in the activites would have been selected as scaffolds to generate active sites with thermal stability in the early stages of globular protein evolution [31,33,34]. If this is the case, the barnase mutants that have an active site, but not a stable scaffold can be considered as globular proteins in evolutionarily intermediate states.…”

Estimating the Prevalence of Protein Sequences Adopting Functional Enzyme Folds

“…A commonly accepted view is that new folds are pieced together from small parts of existing folds. 32,33,39,40 But to the extent that a new fold is really new, its formation must require the joint solution of at least a considerable number of new local stabilization problems of the kind described above. How likely is it that sequences that carry the hydropathy signatures of other folds and provide joint solutions to the stabilization problems for those folds may be pieced together in such a way that they satisfy a new set of constraints, equally demanding but substantially different?…”