Honors & Awards

  • Arnold O. Beckman Postdoctoral Fellowship, Arnold O. Beckman Foundation (2022-2024)
  • Hanna H. Gray Fellows Finalist, Howard Hughes Medical Institute (2022)
  • Dean’s Postdoctoral Fellowship, Stanford School of Medicine (2021)
  • Schmidt Science Fellows Finalist, Schmidt Science Foundation (2021)
  • Graduate Research Fellowship, National Science Foundation (2014)
  • Barry M. Goldwater Scholar, Barry M. Goldwater Foundation (2008)

Boards, Advisory Committees, Professional Organizations

  • Strategic Vision Team Member, Office of the Vice Provost of Graduate Education (2022 - Present)
  • Grant Coach, Stanford Grant Writing Academy (2022 - Present)

All Publications

  • Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context. eLife Radkov, A., Sapiro, A. L., Flores, S., Henderson, C., Saunders, H., Kim, R., Massa, S., Thompson, S., Mateusiak, C., Biboy, J., Zhao, Z., Starita, L. M., Hatleberg, W. L., Vollmer, W., Russell, A. B., Simorre, J. P., Anthony-Cahill, S., Brzovic, P., Hayes, B., Chou, S. 2022; 11


    Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

    View details for DOI 10.7554/eLife.79796

    View details for PubMedID 35762582

  • 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


    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


    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-+


    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

  • Altered expression of a quality control protease in E. coli reshapes the in vivo mutational landscape of a model enzyme ELIFE Thompson, S., Zhang, Y., Ingle, C., Reynolds, K. A., Kortemme, T. 2020; 9


    Protein mutational landscapes are shaped by the cellular environment, but key factors and their quantitative effects are often unknown. Here we show that Lon, a quality control protease naturally absent in common E. coli expression strains, drastically reshapes the mutational landscape of the metabolic enzyme dihydrofolate reductase (DHFR). Selection under conditions that resolve highly active mutants reveals that 23.3% of all single point mutations in DHFR are advantageous in the absence of Lon, but advantageous mutations are largely suppressed when Lon is reintroduced. Protein stability measurements demonstrate extensive activity-stability tradeoffs for the advantageous mutants and provide a mechanistic explanation for Lon's widespread impact. Our findings suggest possibilities for tuning mutational landscapes by modulating the cellular environment, with implications for protein design and combatting antibiotic resistance.

    View details for DOI 10.7554/eLife.53476

    View details for Web of Science ID 000555424200001

    View details for PubMedID 32701056

    View details for PubMedCentralID PMC7377907

  • Flex ddG: Rosetta Ensemble-Based Estimation of Changes in Protein-Protein Binding Affinity upon Mutation JOURNAL OF PHYSICAL CHEMISTRY B Barlow, K. A., Conchuir, S. O., Thompson, S., Suresh, P., Lucas, J. E., Heinonen, M., Kortemme, T. 2018; 122 (21): 5389-5399


    Computationally modeling changes in binding free energies upon mutation (interface ΔΔ G) allows large-scale prediction and perturbation of protein-protein interactions. Additionally, methods that consider and sample relevant conformational plasticity should be able to achieve higher prediction accuracy over methods that do not. To test this hypothesis, we developed a method within the Rosetta macromolecular modeling suite (flex ddG) that samples conformational diversity using "backrub" to generate an ensemble of models and then applies torsion minimization, side chain repacking, and averaging across this ensemble to estimate interface ΔΔ G values. We tested our method on a curated benchmark set of 1240 mutants, and found the method outperformed existing methods that sampled conformational space to a lesser degree. We observed considerable improvements with flex ddG over existing methods on the subset of small side chain to large side chain mutations, as well as for multiple simultaneous non-alanine mutations, stabilizing mutations, and mutations in antibody-antigen interfaces. Finally, we applied a generalized additive model (GAM) approach to the Rosetta energy function; the resulting nonlinear reweighting model improved the agreement with experimentally determined interface ΔΔ G values but also highlighted the necessity of future energy function improvements.

    View details for DOI 10.1021/acs.jpcb.7b11367

    View details for Web of Science ID 000434236900015

    View details for PubMedID 29401388

    View details for PubMedCentralID PMC5980710

  • Conformational Freedom of the LRP6 Ectodomain Is Regulated by N-glycosylation and the Binding of the Wnt Antagonist Dkk1 CELL REPORTS Matoba, K., Mihara, E., Tamura-Kawakami, K., Miyazaki, N., Maeda, S., Hirai, H., Thompson, S., Iwasaki, K., Takagi, J. 2017; 18 (1): 32-40


    LDL-receptor-related protein 6 (LRP6) is a single-pass membrane glycoprotein with a large modular ectodomain and forms a higher order signaling platform upon binding Wnt ligands on the cell surface. Although multiple crystal structures are available for fragments of the LRP6 ectodomain, we lack a consensus view on the overall molecular architecture of the full-length LRP6 and its dynamic aspects. Here, we used negative-stain electron microscopy to probe conformational states of the entire ectodomain of LRP6 in solution and found that the four-module ectodomain undergoes a large bending motion hinged at the junction between the second and the third modules. Importantly, the extent of inter-domain motion is modulated by evolutionarily conserved N-glycan chains proximal to the joint. We also found that the LRP6 ectodomain becomes highly compact upon complexation with the Wnt antagonist Dkk1, suggesting a potential role for the ectodomain conformational change in the regulation of receptor oligomerization and signaling.

    View details for DOI 10.1016/j.celrep.2016.12.017

    View details for Web of Science ID 000396465300004

    View details for PubMedID 28052259

  • Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting ELIFE Mayor, D., Barlow, K., Thompson, S., Barad, B. A., Bonny, A. R., Cario, C. L., Gaskins, G., Liu, Z., Deming, L., Axen, S. D., Caceres, E., Chen, W., Cuesta, A., Gate, R. E., Green, E. M., Hulce, K. R., Ji, W., Kenner, L. R., Mensa, B., Morinishi, L. S., Moss, S. M., Mravic, M., Muir, R. K., Niekamp, S., Nnadi, C. I., Palovcak, E., Poss, E. M., Ross, T. D., Salcedo, E. C., See, S. K., Subramaniam, M., Wong, A. W., Li, J., Thorn, K. S., Conchuir, S. O., Roscoe, B. P., Chow, E. D., DeRisi, J. L., Kortemme, T., Bolon, D. N., Fraser, J. S. 2016; 5


    Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.

    View details for DOI 10.7554/eLife.15802

    View details for Web of Science ID 000376605700001

    View details for PubMedID 27111525

    View details for PubMedCentralID PMC4862753

  • Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer BIOCHEMISTRY Ando, N., Li, H., Brignole, E. J., Thompson, S., McLaughlin, M. I., Page, J. E., Asturias, F. J., Stubbe, J., Drennan, C. L. 2016; 55 (2): 373-381


    Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (β) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α6 is assembled from dimers (α2) without a previously proposed tetramer intermediate (α4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α6 were further examined by SAXS in the presence of the β subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α6 is more stable than the ATP-induced α6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the β subunit to the active site of α.

    View details for DOI 10.1021/acs.biochem.5b01207

    View details for Web of Science ID 000368562100014

    View details for PubMedID 26727048

    View details for PubMedCentralID PMC4722859

  • Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells BIOCHEMISTRY Cohen, J. D., Thompson, S., Ting, A. Y. 2011; 50 (38): 8221-8225


    Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter's ligase ((W37V)LplA) or any tryptophan 37 point mutant. Here we report the results of a structure-guided, two-residue screening matrix to discover an LplA double mutant, (E20G/W37T)LplA, that ligates Pacific Blue as efficiently as (W37V)LplA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated.

    View details for DOI 10.1021/bi201037r

    View details for Web of Science ID 000295058700013

    View details for PubMedID 21859157

    View details for PubMedCentralID PMC3181222

  • A fluorophore ligase for site-specific protein labeling inside living cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Uttamapinant, C., White, K. A., Baruah, H., Thompson, S., Fernandez-Suarez, M., Puthenveetil, S., Ting, A. Y. 2010; 107 (24): 10914-10919


    Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.

    View details for DOI 10.1073/pnas.0914067107

    View details for Web of Science ID 000278807400027

    View details for PubMedID 20534555

    View details for PubMedCentralID PMC2890758

  • Yeast Display Evolution of a Kinetically Efficient 13-Amino Acid Substrate for Lipoic Acid Ligase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Puthenveetil, S., Liu, D. S., White, K. A., Thompson, S., Ting, A. Y. 2009; 131 (45): 16430-16438


    Escherichia coli lipoic acid ligase (LplA) catalyzes ATP-dependent covalent ligation of lipoic acid onto specific lysine side chains of three acceptor proteins involved in oxidative metabolism. Our lab has shown that LplA and engineered mutants can ligate useful small-molecule probes such as alkyl azides ( Nat. Biotechnol. 2007 , 25 , 1483 - 1487 ) and photo-cross-linkers ( Angew. Chem., Int. Ed. 2008 , 47 , 7018 - 7021 ) in place of lipoic acid, facilitating imaging and proteomic studies. Both to further our understanding of lipoic acid metabolism, and to improve LplA's utility as a biotechnological platform, we have engineered a novel 13-amino acid peptide substrate for LplA. LplA's natural protein substrates have a conserved beta-hairpin structure, a conformation that is difficult to recapitulate in a peptide, and thus we performed in vitro evolution to engineer the LplA peptide substrate, called "LplA Acceptor Peptide" (LAP). A approximately 10(7) library of LAP variants was displayed on the surface of yeast cells, labeled by LplA with either lipoic acid or bromoalkanoic acid, and the most efficiently labeled LAP clones were isolated by fluorescence activated cell sorting. Four rounds of evolution followed by additional rational mutagenesis produced a "LAP2" sequence with a k(cat)/K(m) of 0.99 muM(-1) min(-1), >70-fold better than our previous rationally designed 22-amino acid LAP1 sequence (Nat. Biotechnol. 2007, 25, 1483-1487), and only 8-fold worse than the k(cat)/K(m) values of natural lipoate and biotin acceptor proteins. The kinetic improvement over LAP1 allowed us to rapidly label cell surface peptide-fused receptors with quantum dots.

    View details for DOI 10.1021/ja904596f

    View details for Web of Science ID 000271723000036

    View details for PubMedID 19863063

    View details for PubMedCentralID PMC2799336

  • Photonic Hydrogels with Poly(ethylene glycol) Derivative Colloidal Spheres as Building Blocks MACROMOLECULES Cai, T., Wang, G., Thompson, S., Marquez, M., Hu, Z. 2008; 41 (24): 9508-9512

    View details for DOI 10.1021/ma802035d

    View details for Web of Science ID 000261767400003