Biology subjects

Klinman, J. P.

Publications and source records attributed to Klinman, J. P..

5 recordsLinked to original sources

bioRxiv2026

Mapping Light-Induced Conformational Dynamics of Pigeon Cryptochrome 4 by HDX-MS: Structural Transitions from Spin Pair Formation to Activated Conformational States

The navigational prowess of migratory birds is thought to arise from light-dependent radical-pair chemistry in cryptochrome 4 (CRY4), yet the slow structural transitions that couple photochemistry to signaling remain elusive. Here, we combine temperature-controlled steady-state UV-visible spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to elucidate the photochemical and conformational dynamics of pigeon CRY4 (ClCRY4). Steady-state measurements at 5-25 {degrees}C reveal that lower temperatures slow FAD photoreduction and prolong the FAD neutral semiquinone signaling state. This occurs without a solvent kinetic isotope effect, implicating a conformational change rather than proton transfer as the rate determining step in FAD neutral semiquinone formation. Simultaneous HDX-MS under blue-light exposure identifies protection near the FAD-binding site and C-terminal region. To enhance sensitivity, we developed a pump-probe HDX-MS approach at 10 {degrees}C. This reveals eight peptides (within the phosphate-binding loop, protrusion motif, electron-transfer-chain loops and C-terminal tail) that exhibit rapid ([≤]10 s) and sustained light-induced protection, delineating early conformational rearrangements as a prerequisite for FAD neutral semiquinone accumulation. The findings of slower onset HDX protection as well as a bimodal pattern of deuterium uptake in the phosphate-binding loop further identify a local redistribution of conformational substates on the time scale of the accumulation of the signaling species. Site specific mutagenesis within the CTT supports the findings, which lead to a model in which blue light triggers rapid clamping down of protein near the two regions of spin pair separation, followed by a rate limiting closure of a surface loop. The resolution of time-dependent structural transitions that follow photoactivation of CRY4 resolves the interface between quantum radical-pair formation and classical conformational changes, while providing an enhanced structural framework for the molecular events that underlie avian magnetoreception.

biophysics↗

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↗

bioRxiv2025

Correlated Motion-Based Residue Network Analysis Reveals the Distal Thermal Activation in Soybean Lipoxygenase

Enzyme catalysis has been shown to depend on distal pathways that channel thermal energy from solvent to the active site. In soybean lipoxygenase (SLO), experiments identified a cone-shaped network connecting loop residue Gln322 to Leu546 but not Leu754 in the active site. Here, microsecond molecular dynamics and a correlated motion-based protocol provide an atomistic analysis of such long-range communication. The developed approach enables systematic screening of communication between active site-specific residues that directly contact bound substrate and surface-exposed residues on the protein-solvent interface. In doing so, it provides a deeper molecular insight into experimentally mapped networks by resolving communication trends across diverse conformational ensembles. The simulations recover the experimentally demonstrated thermal initiation loop and the Leu546-directed cone in SLO, exclude the negative-control Ser596, and explain the preference for Leu546 over Leu754 through shorter, more correlated helical pathways. Mutational analysis further reveals the impact of single-site mutations on the network preference between Leu546 and Leu754. These results unify experiments and computation, corroborating an anisotropic channeling of thermal energy in SLO and establishing a general framework for computing distal intra-protein pathways that may enable the thermal activation of enzyme function.

biophysics↗

bioRxiv2025

Identification of Scaffold Specific Energy Transfer Networks in the Enthalpic Activation of Orotidine 5'-Monophosphate Decarboxylase

AbstractOrotidine 5-monophosphate decarboxylase (OMPDC) is one of the most efficient enzyme systems studied, enhancing the decarboxylation of OMP to uridine 5-monophosphate (UMP) by ca. 17 orders of magnitude, primarily by reducing the enthalpy of activation by ca. 28 kcal/mol. Despite a substantial reduction in activation enthalpy, OMPDC requires 15 kcal/mol of activation energy post- ES complex formation. This study investigates the physical basis of how thermal energy from solvent collisions is directed into the active site of enzyme to enable efficient thermal activation of the reaction. Comparative study of temperature-dependent hydrogen-deuterium exchange mass spectrometry (TDHDX) for WT and mutant forms of enzymes has recently been shown to uncover site specific protein networks for thermal energy transfer from solvent to enzyme active sites. In this study, we interrogate region-specific changes in the enthalpic barrier for local protein flexibility using a native OMPDC from Methanothermobacter thermautotrophicus (Mt-OMPDC) and a single site variant (Leu123Ala) that alters the activation enthalpy for catalytic turnover. The data obtained implicate four spatially resolved, thermally sensitive networks that originate at different protein/solvent interfaces and terminate at sites surrounding the substrate near the substrate phosphate-binding region (R203), the substrate- ribose binding region (K42), and a reaction enhancing loop5 (S127). These are proposed to act synergistically, transiently optimizing the position and electrostatics of the reactive carboxylate of the substrate to facilitate activated complex formation. The uncovered complexity of thermal activation networks in Mt-OMPDC distinguishes this enzyme from other members of the TIM barrel family previously investigated by TDHDX. The new findings extend the essential role of protein scaffold dynamics in orchestrating enzyme activity, with broad implications for the design of highly efficient biocatalysts.

biochemistry↗

bioRxiv2022

Temporal and Spatial Resolution of a Protein Quake that Activates Hydrogen Tunneling in Soybean Lipoxygenase

The enzyme soybean lipoxygenase (SLO) provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen deuterium exchange experiments to detect a radiating cone of aliphatic side chains that extends from the iron active site of SLO to the protein-solvent interface. Employing eight variants of SLO, nanosecond fluorescence Stokes shifts have been measured using a probe appended to the identified surface loop. We report a remarkable identity of the enthalpies of activation for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within the identified thermal network. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, these new data implicate a thermally initiated, cooperative protein quake as the source of the activation of SLO. These findings indicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis.

biophysics↗