Biology subjects

Wolff, A. M.

Publications and source records attributed to Wolff, A. M..

8 recordsLinked to original sources

bioRxiv2026

Conformational flexibility of soybean lipoxygenase is coupled to crystal solvent content in serial crystallography

X-ray crystallography is increasingly employed to study protein conformational ensembles under physiological conditions, but the effects of the crystal lattice on protein motions remains understudied. Here, we report the structure determination of soybean lipoxygenase-1 (SLO) from microcrystal slurries using serial femtosecond crystallography (SFX) at the Linac Coherent Light Source. During data analysis, we observed unexpected polymorphism in SLOs unit-cell parameters, arising from two compounding factors: indexing ambiguities caused by the pseudo-tetragonal symmetry of the SLO crystal lattice, and true non-isomorphism between individual crystal populations consistent with different solvent content. By combining unit-cell clustering with systematic reindexing, we resolved two distinct polymorphs and determined two independent structures from a single experiment. The two structures exhibit a small overall RMSD (0.34 [A]), yet a difference distance matrix reveals coordinated rearrangements that are not readily apparent from simple structural overlays. Furthermore, a difference of approximately 8.5% in crystal solvent content produces measurable differences in crystal contacts and conformational flexibility. The more hydrated (large-cell) polymorph exhibits greater inter-domain flexibility, as well as higher B-factors in key hydrophobic core residues. In the dehydrated (small-cell) polymorph, which shows less interdomain flexibility, these same residues adopt discrete alternative conformations resolvable in the electron density. Our results highlight that subtle changes in crystal packing can give rise to distinct conformational landscapes for crystallized proteins, with potential implications for the interpretation of protein intramolecular dynamics from crystallographic data. SynopsisUsing soybean lipoxygenase-1 as a model system, we determine two independent structures within a single experiment that differ in crystal solvent content. Despite a small overall RMSD, a detailed analysis reveals coordinated domain-level rearrangements and shows that conformational flexibility is sensitive to crystal packing, a consideration of broad relevance for the study of protein dynamics using X-ray crystallography.

biochemistry↗

bioRxiv2023

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

biochemistry↗

bioRxiv2022

Mapping Protein Dynamics at High-Resolution with Temperature-Jump X-ray Crystallography

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the sub-millisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature-jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.

biophysics↗

bioRxiv2022

Molecular-Dynamics Simulation Methods for Macromolecular Crystallography

To assess the potential benefits of molecular-dynamics (MD) simulations for macromolecular crystallography (MX), we performed room-temperature X-ray diffraction studies of the catalytic subunit of mouse protein kinase A (PKA-C). We then performed crystalline MD simulations of PKA-C, computed simulated electron densities from the water, protein, and ion components of the MD simulations, and carefully compared them to the initial crystal structure. The results led to the development of an MD-MX analysis procedure and several associated methods: 1) density comparison to evaluate consistency between the MD and the initial crystal structure model; 2) water building to generate alternative solvent models; and 3) protein remodeling to improve the crystal structure where interpretation of density is unclear. This procedure produced a revised structure of PKA with a new ordered water model and a modified protein structure. The revisions yield new insights into PKA mechanisms, including: a sensitivity of the His294 conformation to protonation state, with potential consequences for regulation of substrate binding; a remodeling of the Lys217 side chain along with a bound phosphate; an alternative conformation for Lys213 associated with binding to the regulatory subunit; and an alternative conformation for catalytic base Asp166 and nearby waters, suggesting a mechanism of progression of the phosphotransfer reaction via changes in Mg2+ coordination. Based on the benefits seen applying these methods to PKA, we recommend incorporating our MD-MX procedure into MX studies, to decide among ambiguous interpretations of electron density that occur, inevitably, as part of standard model refinement.

biophysics↗

bioRxiv2019

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample preparation and delivery methods required for each type of experiment effect the crystal structure of the enzyme.

biophysics↗

bioRxiv2018

Temperature-Jump Solution X-ray Scattering Reveals Distinct Motions in a Dynamic Enzyme

Correlated motions of proteins and their bound solvent molecules are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved methods hold promise for addressing this challenge but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature-jumps (T-jumps), through thermal excitation of the solvent, have been implemented to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform T-jump small- and wide-angle X-ray scattering (SAXS/WAXS) measurements on a dynamic enzyme, cyclophilin A (CypA), demonstrating that these experiments are able to capture functional intramolecular protein dynamics. We show that CypA displays rich dynamics following a T-jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes in the enzyme. Two relaxation processes are resolved, which can be characterized by Arrhenius behavior. We also used mutations that have distinct functional effects to disentangle the relationship of the observed relaxation processes. A fast process is related to surface loop motions important for substrate specificity, whereas a slower process is related to motions in the core of the protein that are critical for catalytic turnover. These results demonstrate the power of time-resolved X-ray scattering experiments for characterizing protein and solvent dynamics on the s-ms timescale. We expect the T-jump methodology presented here will be useful for understanding kinetic correlations between local conformational changes of proteins and their bound solvent molecules, which are poorly explained by the results of traditional, static measurements of molecular structure.

biophysics↗

bioRxiv2018

Co-occurring genetic alterations in the RAS pathway promote resistance to MET inhibitor treatment in non-small cell lung cancer with a MET exon 14 skipping mutation

PURPOSEWhile patients with advanced-stage non-small cell lung cancers (NSCLCs) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood.\n\nMETHODSTargeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC.\n\nRESULTSProminent co-occurring RAS-MAPK pathway gene alterations (e.g. in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared to EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by co-treatment with crizotinib and the MEK inhibitor trametinib.\n\nCONCLUSIONOur study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.

cancer biology↗

bioRxiv2017

Bringing Diffuse X-ray Scattering Into Focus

X-ray crystallography is experiencing a renaissance as a method for probing the protein conformational ensemble. The inherent limitations of Bragg analysis, however, which only reveals the mean structure, have given way to a surge in interest in diffuse scattering, which is caused by structure variations. Diffuse scattering is present in all macromolecular crystallography experiments. Recent studies are shedding light on the origins of diffuse scattering in protein crystallography, and provide clues for leveraging diffuse scattering to model protein motions with atomic detail.

biophysics↗