Cowan, A. E. | Search bioRxiv

bioRxiv2023

Mapping the biochemical landscape of rubisco

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

biochemistry↗

bioRxiv2023

Beyond analytic solution: analysis of FRAP experiments by spatial simulation of the forward problem

Fluorescence redistribution after photobleaching (FRAP) is a commonly used method to understand the dynamic behavior of molecules within cells. Analytic solutions have been developed for specific, well-defined models of dynamic behavior in idealized geometries, but these solutions are inaccurate in complex geometries or when complex binding and diffusion behaviors exist. We demonstrate the use of numerical reaction-diffusion simulation approaches using the easily accessible Virtual Cell (VCell) software, to establish methods for analyzing photobleaching data. We show how multiple simulations employing parameter scans and varying bleaching locations and sizes can help to bracket diffusion coefficients and kinetic rate constants. This approach is applied to problems in membrane surface diffusion, diffusion and binding in cytosolic volumes in complex cell geometries, and analysis of diffusion and binding in intracellular liquid droplets. Statement of SignificanceFluorescence Redistribution After Photobleaching (FRAP) is a widely used experimental method that can reveal important parameters for reaction/diffusion events within cells. However, analytic methods to analyze FRAP experiments are limited to specific geometries and conditions. We demonstrate how spatial numerical simulation methods using the freely available software Virtual Cell can be used to obtain parameter information from FRAP experiments in situations that are not amenable to analytic solutions and that are accessible to most bench biologists.

biophysics↗