Evolutionary Constraints Specifying Protein Folding and Function

Proteins are complex macromolecules that carry out biological functions while under constant mutational load and selective pressure during evolution. Consequently, evolution has generated protein families by exploring the set of sequences able to carry out a particular biological activity, maintaining sequence motifs critical for function while varying the rest of the protein. Statistical coupling analysis of a protein family examines an alignment of such sequences and detects the evolutionarily preserved interresidue interactions critical for the proteins' selective fitness. This set of information has provided a sufficiently detailed description of evolutionary design constraints to allow the design of novel WW domain sequences that fold and function like natural proteins. This work expands the initial investigations, and probes the minimal information content necessary to specify the WW domain fold, as well as the effect of increasing the coupling constraints in the design process. This work also evaluates the ability of coupling information to design larger and more complex protein folds and to specify their biological functions. Experimental expression and characterization of WW domains designed with varying levels of coupling information indicates that incorporating even small amounts of coupling information has a notable impact on these proteins' ability to fold. Moreover, different coupling-based design approaches produce results robust to details of how coupling information is incorporated. Similar experiments with designed PDZ domains and in vivo characterization of designed G-protein coupled receptors show that, to the extent studied, this design approach is successful with these larger and more complex proteins as well. This indicates that the typically sparse matrices of coupling values observed for a protein family capture the core evolutionary constraints on the proteins in sufficient detail to generate even complex proteins with natural-like folds and functions.