Navigating function in sequence space using resurrected ancestral proteins and fourier transform infrared spectroscopy

A general challenge in protein science and protein engineering is that proteins belonging to the same protein family but differ in functional properties (such as enzymatic rate and thermodynamic stability) often differ in a substantial number of positions. In many cases it is likely that a combination of a number of substitutions causes the observed functional changes, but there are too many combinations (a combinatorial explosion) of possible substitutions to experimentally verify. Currently, there is no method to address such challenges in structure-function studies. Our approach allows the degree of sequence similarity between these predicted ancestors to be quite small, greatly narrowing down the residues involved in causing this functional change and allowing them to be unambiguously identified by traditional site-directed mutagenesis. This approach promises to provide a generally applicable strategy for identifying structure-function relationships in protein families based on resurrected ancestral proteins. With this project we were able to identify that AlphaFold structural predictions are not as accurate as originally thought. Also, along the evolutionary path, there is a phenomenon that occurs that we attribute to molecular meandering. The characterization of site-directed mutants of proteins is a major tool to uncover protein structure-function relationships. In general, a clear interpretation of the properties of the mutant is only possible if the mutation does not cause substantial structural perturbations. However, currently it is often difficult to determine if a point mutation leaves the structure of a protein essentially intact or if it causes substantial structural perturbations. Here we aim to develop an FTIR-based method that can quickly, reliably, and sensitively answer that question. Also, proton transfer is fundamental to many processes in biology, ranging from receptor activation and bioenergetics to bio-catalysis. However, few methods in structural biology are able to determine residue-resolved protonation states, particularly for functional intermediates. We focus on the unambiguous determination of histidine side chain protonation state because histidine is a residue found at the active site of many enzymes, where it often functions by altering its protonation state. With this research, we also developed a new method using FTIR to identify secondary structure perturbations.