Biology subjects

Offenbacher, A. R.

Publications and source records attributed to Offenbacher, A. R..

4 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↗

bioRxiv2022

13C ENDOR Spectroscopy-Guided MD Computations Reveals the Structure of the Enzyme-Substrate Complex of an Active, N-linked Glycosylated Lipoxygenase

Lipoxygenases (LOXs) are enzymes responsible for producing important cell signaling mediators and have been extensively studied for their potential clinical relevance as well as to advance our understanding of enzyme catalysis. The common inability to capture and characterize LOX-substrate complexes by Xray co-crystallography requires the development of alternative structural methods. We previously reported the integration of 13C/1H electron nuclear double resonance (ENDOR) spectroscopy and molecular dynamics (MD) to visualize the complex structure of the paradigmatic LOX from soybean, SLO, with substrate linoleic acid (LA). However, this required substitution of the catalytic mononuclear, nonheme iron by the structurally faithful, yet inactive Mn2+ ion as a spin-probe. Unlike canonical Fe-LOXs from plants and animals, LOXs from pathogenic fungi contain active mononuclear manganese metallocentres. Here, we report the ground-state active-site structure of the native, fully glycosylated fungal LOX from M. oryzae, MoLOX complexed with LA obtained through the 13C/1H ENDOR-guided MD approach. The Mn-oxygen-to-LA donor carbon distance (DAD) for MoLOX-LA, 3.4 {+/-} 0.3 [A], matches the distance in the single representative X-ray co-structure of an animal 8R-LOX with its natural fatty acid substrate, and slightly elongated from that of the SLO-LA complex, 3.1 {+/-} 0.2 [A], despite its carboxylate-out substrate binding orientation versus carboxylate-in for SLO. The results provide unique insight into the evolutionary divergence of the ground-state DAD in the LOX family, which influences the activation barrier for hydrogen tunneling, and give a structural basis for guiding development of MoLOX inhibitors. The work highlights the robustness of ENDOR-guided MD approach to describe LOX-substrate structures that elude conventional X-ray techniques.

biochemistry↗

bioRxiv2022

Chorography and conformational dynamism of the Soluble Human Fibrinogen in solution

Fibrinogen is a soluble, multi-subunit and multi-domain dimeric protein, which, upon its proteolytic cleavage by thrombin, is converted to insoluble fibrin initiating polymerization that substantially contributes to clot growth. The consentaneous structural view of the soluble form of fibrinogen is relatively straight and rigid-like. However, fibrinogen contains numerous, transiently-accessible "cryptic" epitopes for hemostatic and immunologic proteins, suggesting that fibrinogen exhibits conformational flexibility, which may play functional roles in its temporal and spatial interactions. Hitherto, there has been limited information on the solution structure and internal flexibility of soluble fibrinogen. Here, utilizing an integrative, biophysical approach involving temperature-dependent hydrogen-deuterium exchange mass spectrometry, small angle X-ray scattering, and negative stain electron microscopy, we present a holistic, conformationally dynamic model of human fibrinogen in solution. Our data reveal a high degree of internal flexibility, accommodated by four major and distinct flexible sites along the central axis of the protein. We propose that the fibrinogen structure in solution consists of a complex, conformational landscape with multiple local minima, rather than a single, rigid topology. This is further supported by the location of numerous point mutations that are linked to dysfibrinogenemia, and post-translational modifications, residing near fibrinogen flexions. This work provides a molecular basis for the structural "dynamism" of fibrinogen that is expected to influence the broad swath of functionally diverse macromolecular interactions and fine-tune the structural and mechanical properties of blood clots.

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↗