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All Publications


  • Fundamentals to function: Quantitative and scalable approaches for measuring protein stability. Cell systems Atsavapranee, B., Stark, C. D., Sunden, F., Thompson, S., Fordyce, P. M. 2021; 12 (6): 547-560

    Abstract

    Folding a linear chain of amino acids into a three-dimensional protein is a complex physical process that ultimately confers an impressive range of diverse functions. Although recent advances have driven significant progress in predicting three-dimensional protein structures from sequence, proteins are not static molecules. Rather, they exist as complex conformational ensembles defined by energy landscapes spanning the space of sequence and conditions. Quantitatively mapping the physical parameters that dictate these landscapes and protein stability is therefore critical to develop models that are capable of predicting how mutations alter function of proteins in disease and informing the design of proteins with desired functions. Here, we review the approaches that are used to quantify protein stability at a variety of scales, from returning multiple thermodynamic and kinetic measurements for a single protein sequence to yielding indirect insights into folding across a vast sequence space. The physical parameters derived from these approaches will provide a foundation for models that extend beyond the structural prediction to capture the complexity of conformational ensembles and, ultimately, their function.

    View details for DOI 10.1016/j.cels.2021.05.009

    View details for PubMedID 34139165

  • Structurally distributed surface sites tune allosteric regulation. eLife McCormick, J. W., Russo, M. A., Thompson, S., Blevins, A., Reynolds, K. A. 2021; 10

    Abstract

    Our ability to rationally optimize allosteric regulation is limited by incomplete knowledge of the mutations that tune allostery. Are these mutations few or abundant, structurally localized or distributed? To examine this, we conducted saturation mutagenesis of a synthetic allosteric switch in which Dihydrofolate reductase (DHFR) is regulated by a blue-light sensitive LOV2 domain. Using a high-throughput assay wherein DHFR catalytic activity is coupled to E. coli growth, we assessed the impact of 1548 viable DHFR single mutations on allostery. Despite most mutations being deleterious to activity, fewer than 5% of mutations had a statistically significant influence on allostery. Most allostery disrupting mutations were proximal to the LOV2 insertion site. In contrast, allostery enhancing mutations were structurally distributed and enriched on the protein surface. Combining several allostery enhancing mutations yielded near-additive improvements to dynamic range. Our results indicate a path towards optimizing allosteric function through variation at surface sites.

    View details for DOI 10.7554/eLife.68346

    View details for PubMedID 34132193

  • Negative-Stain Electron Microscopy Reveals Dramatic Structural Rearrangements in Ni-Fe-S-Dependent Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase STRUCTURE Cohen, S. E., Brignole, E. J., Wittenborn, E. C., Can, M., Thompson, S., Ragsdale, S. W., Drennan, C. L. 2021; 29 (1): 43-+

    Abstract

    The Ni-Fe-S-containing A-cluster of acetyl-coenzyme A (CoA) synthase (ACS) assembles acetyl-CoA from carbon monoxide (CO), a methyl group (CH3+), and CoA. To accomplish this feat, ACS must bind CoA and interact with two other proteins that contribute the CO and CH3+, respectively: CO dehydrogenase (CODH) and corrinoid Fe-S protein (CFeSP). Previous structural data show that, in the model acetogen Moorella thermoacetica, domain 1 of ACS binds to CODH such that a 70-Å-long internal channel is created that allows CO to travel from CODH to the A-cluster. The A-cluster is largely buried and is inaccessible to CFeSP for methylation. Here we use electron microscopy to capture multiple snapshots of ACS that reveal previously uncharacterized domain motion, forming extended and hyperextended structural states. In these structural states, the A-cluster is accessible for methylation by CFeSP.

    View details for DOI 10.1016/j.str.2020.08.011

    View details for Web of Science ID 000606462800006

    View details for PubMedID 32937101

    View details for PubMedCentralID PMC7796957