Professional Education

  • Doctor of Philosophy, Stanford University, BIOC-PHD (2021)
  • Master of Science, Stanford University, MED-MS (2018)
  • BS, University of Washington, Chemistry (2014)
  • BS, University of Washington, Biochemistry (2014)

Stanford Advisors

All Publications

  • Parallel molecular mechanisms for enzyme temperature adaptation. Science (New York, N.Y.) Pinney, M. M., Mokhtari, D. A., Akiva, E., Yabukarski, F., Sanchez, D. M., Liang, R., Doukov, T., Martinez, T. J., Babbitt, P. C., Herschlag, D. 2021; 371 (6533)


    The mechanisms that underly the adaptation of enzyme activities and stabilities to temperature are fundamental to our understanding of molecular evolution and how enzymes work. Here, we investigate the molecular and evolutionary mechanisms of enzyme temperature adaption, combining deep mechanistic studies with comprehensive sequence analyses of thousands of enzymes. We show that temperature adaptation in ketosteroid isomerase (KSI) arises primarily from one residue change with limited, local epistasis, and we establish the underlying physical mechanisms. This residue change occurs in diverse KSI backgrounds, suggesting parallel adaptation to temperature. We identify residues associated with organismal growth temperature across 1005 diverse bacterial enzyme families, suggesting widespread parallel adaptation to temperature. We assess the residue properties, molecular interactions, and interaction networks that appear to underly temperature adaptation.

    View details for DOI 10.1126/science.aay2784

    View details for PubMedID 33674467

  • Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray-derived conformational ensembles. Proceedings of the National Academy of Sciences of the United States of America Yabukarski, F., Biel, J. T., Pinney, M. M., Doukov, T., Powers, A. S., Fraser, J. S., Herschlag, D. 2020; 117 (52): 33204–15


    How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme's reaction cycle. To examine positioning and motions and test catalytic proposals, we collected "room temperature" X-ray crystallography data for Pseudomonas putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI's reaction cycle. Active site positioning was on the 1- to 1.5-A scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI's general base reduced catalysis, suggesting optimized Angstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure-function relationships, providing the basis for a new era of "ensemble-function" interrogation of enzymes.

    View details for DOI 10.1073/pnas.2011350117

    View details for PubMedID 33376217

  • Serum electrolytes can promote hydroxyl radical-initiated biomolecular damage from inflammation. Free radical biology & medicine Komaki, Y. n., Simpson, A. M., Choe, J. K., Pinney, M. M., Herschlag, D. n., Chuang, Y. H., Mitch, W. A. 2019; 141: 475–82


    Chronic inflammatory disorders are associated with biomolecular damage attributed partly to reactions with Reactive Oxygen Species (ROS), particularly hydroxyl radicals (•OH). However, the impacts of serum electrolytes on ROS-associated damage has received little attention. We demonstrate that the conversion of •OH to carbonate and halogen radicals via reactions with serum-relevant carbonate and halide concentrations fundamentally alters the targeting of amino acids and loss of enzymatic activity in catalase, albumin and carbonic anhydrase, three important blood proteins. Chemical kinetic modeling indicated that carbonate and halogen radical concentrations should exceed •OH concentrations by 6 and 2 orders of magnitude, respectively. Steady-state γ-radiolysis experiments demonstrated that serum-level carbonates and halides increased tyrosine, tryptophan and enzymatic activity losses in catalase up to 6-fold. These outcomes were specific to carbonates and halides, not general ionic strength effects. Serum carbonates and halides increased the degradation of tyrosines and methionines in albumin, and increased the degradation of histidines while decreasing enzymatic activity loss in carbonic anhydrase. Serum electrolytes increased the degradation of tyrosines, tryptophans and enzymatic activity in the model enzyme, ketosteroid isomerase, predominantly due to carbonate radical reactions. Treatment of a mutant ketosteroid isomerase indicated that preferential targeting of the active site tyrosine accounted for half of the total tyrosine loss. The results suggest that carbonate and halogen radicals may be more significant than •OH as drivers for protein degradation in serum. Accounting for the selective targeting of biomolecules by these daughter radicals is important for developing a mechanistic understanding of the consequences of oxidative stress.

    View details for DOI 10.1016/j.freeradbiomed.2019.07.023

    View details for PubMedID 31349038

  • Structural Coupling Throughout the Active Site Hydrogen Bond Networks of Ketosteroid Isomerase and Photoactive Yellow Protein JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Pinney, M. M., Natarajan, A., Yabukarski, F., Sanchez, D. M., Liu, F., Liang, R., Doukov, T., Schwans, J. P., Martinez, T. J., Herschlag, D. 2018; 140 (31): 9827–43


    Hydrogen bonds are fundamental to biological systems and are regularly found in networks implicated in folding, molecular recognition, catalysis, and allostery. Given their ubiquity, we asked the fundamental questions of whether, and to what extent, hydrogen bonds within networks are structurally coupled. To address these questions, we turned to three protein systems, two variants of ketosteroid isomerase and one of photoactive yellow protein. We perturbed their hydrogen bond networks via a combination of site-directed mutagenesis and unnatural amino acid substitution, and we used 1H NMR and high-resolution X-ray crystallography to determine the effects of these perturbations on the lengths of the two oxyanion hole hydrogen bonds that are donated to negatively charged transition state analogs. Perturbations that lengthened or shortened one of the oxyanion hole hydrogen bonds had the opposite effect on the other. The oxyanion hole hydrogen bonds were also affected by distal hydrogen bonds in the network, with smaller perturbations for more remote hydrogen bonds. Across 19 measurements in three systems, the length change in one oxyanion hole hydrogen bond was propagated to the other, by a factor of -0.30 ± 0.03. This common effect suggests that hydrogen bond coupling is minimally influenced by the remaining protein scaffold. The observed coupling is reproduced by molecular mechanics and quantum mechanics/molecular mechanics (QM/MM) calculations for changes to a proximal oxyanion hole hydrogen bond. However, effects from distal hydrogen bonds are reproduced only by QM/MM, suggesting the importance of polarization in hydrogen bond coupling. These results deepen our understanding of hydrogen bonds and their networks, providing strong evidence for long-range coupling and for the extent of this coupling. We provide a broadly predictive quantitative relationship that can be applied to and can be further tested in new systems.

    View details for DOI 10.1021/jacs.8b01596

    View details for Web of Science ID 000441475800011

    View details for PubMedID 29990421

  • Hydrogen Bonds: Simple after All? BIOCHEMISTRY Herschlag, D., Pinney, M. M. 2018; 57 (24): 3338–52


    Hydrogen bonds play integral roles in biological structure, function, and conformational dynamics and are fundamental to life as it has evolved on Earth. However, our understanding of these fundamental and ubiquitous interactions has seemed fractured and incomplete, and it has been difficult to extract generalities and principles about hydrogen bonds despite thousands of papers published on this topic, perhaps in part because of the expanse of this subject and the density of studies. Fortunately, recent hydrogen bond proposals, discussions, and debates have stimulated new tests and models and have led to a remarkably simple picture of the structure of hydrogen bonds. This knowledge also provides clarity concerning hydrogen bond energetics, limiting and simplifying the factors that need be considered. Herein we recount the advances that have led to this simpler view of hydrogen bond structure, dynamics, and energetics. A quantitative predictive model for hydrogen bond length can now be broadly and deeply applied to evaluate current proposals and to uncover structural features of proteins, their conformational restraints, and their correlated motions. In contrast, a quantitative energetic description of molecular recognition and catalysis by proteins remains an important ongoing challenge, although our improved understanding of hydrogen bonds may aid in testing predictions from current and future models. We close by codifying our current state of understanding into five "Rules for Hydrogen Bonding" that may provide a foundation for understanding and teaching about these vital interactions and for building toward a deeper understanding of hydrogen bond energetics.

    View details for DOI 10.1021/acs.biochem.8b00217

    View details for Web of Science ID 000436026000004

    View details for PubMedID 29678112

  • Evaluation of the Catalytic Contribution from a Positioned General Base in Ketosteroid Isomerase. Journal of the American Chemical Society Lamba, V., Yabukarski, F., Pinney, M., Herschlag, D. 2016; 138 (31): 9902-9909


    Proton transfer reactions are ubiquitous in enzymes and utilize active site residues as general acids and bases. Crystal structures and site-directed mutagenesis are routinely used to identify these residues, but assessment of their catalytic contribution remains a major challenge. In principle, effective molarity measurements, in which exogenous acids/bases rescue the reaction in mutants lacking these residues, can estimate these catalytic contributions. However, these exogenous moieties can be restricted in reactivity by steric hindrance or enhanced by binding interactions with nearby residues, thereby resulting in over- or underestimation of the catalytic contribution, respectively. With these challenges in mind, we investigated the catalytic contribution of an aspartate general base in ketosteroid isomerase (KSI) by exogenous rescue. In addition to removing the general base, we systematically mutated nearby residues and probed each mutant with a series of carboxylate bases of similar pKa but varying size. Our results underscore the need for extensive and multifaceted variation to assess and minimize steric and positioning effects and determine effective molarities that estimate catalytic contributions. We obtained consensus effective molarities of ∼5 × 10(4) M for KSI from Comamonas testosteroni (tKSI) and ∼10(3) M for KSI from Pseudomonas putida (pKSI). An X-ray crystal structure of a tKSI general base mutant showed no additional structural rearrangements, and double mutant cycles revealed similar contributions from an oxyanion hole mutation in the wild-type and base-rescued reactions, providing no indication of mutational effects extending beyond the general base site. Thus, the high effective molarities suggest a large catalytic contribution associated with the general base. A significant portion of this effect presumably arises from positioning of the base, but its large magnitude suggests the involvement of additional catalytic mechanisms as well.

    View details for DOI 10.1021/jacs.6b04796

    View details for PubMedID 27410422