Biology subjects

Barondeau, D. P.

Publications and source records attributed to Barondeau, D. P..

2 recordsLinked to original sources

bioRxiv2026

Site Specific Hydration Contributes to the Thermal Stability of Proteins and Protein-Complexes

Water is integral to protein structure and stability, yet ensemble measurements rarely identify the hydration interactions associated with stabilization. Here, variable-temperature electrospray ionization ion mobility mass spectrometry, conformation-resolved thermodynamic analysis, and molecular dynamics simulations are combined to examine solvent-dependent thermal stability of chymotrypsin inhibitor 2 (CI-2), a model two-state folder, and the barnase-barstar (Bn-B*) complex, which contains a highly polar binding interface. Replacing H2O with D2O raises the thermal transition temperature of both systems by approximately 3 {degrees}C, favors compact conformations, and reduces the relative thermodynamic accessibility of extended states. The conformationally resolved enthalpy-entropy compensation reveals D2O increases enthalpy for CI-2 but reduces enthalpy for Bn-B* as a result of opposing effects on the individual subunits of the complex. Molecular dynamics simulations reveal that lower solvent-accessible surface areas and fewer protein-water hydrogen bonds in D2O coexist with longer water residence times at specific structural sites. In CI-2, the strongest changes localize to the second {beta}-strand/C-terminal region; in Bn-B*, persistent hydration is concentrated at the charged protein-protein interface. Together, these results support a model in which D2O dependent stabilization is associated not only with the extent of hydration, but the site and lifetime of water interactions. More broadly, this work provides an experimental framework for resolving solvent-dependent conformational thermodynamics of protein unfolding and interactions, enabling direct comparison of free-energy landscapes across solution environments.

biochemistry↗

bioRxiv2020

Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model

Iron-sulfur (Fe-S) clusters have a key role in many biochemical processes and are essential for most life forms. Despite recent mechanistic advances in understanding the Fe-S cluster biosynthetic pathway, critical questions remain unresolved. Although human NFS1 and E. coli IscS share [~]60% sequence identity, NFS1 exhibits low activity and requires activation by the Friedreichs ataxia protein frataxin (FXN) for in vivo function. Surprisingly, structures of the human complex reveal three distinct quaternary structures with one form exhibiting the same subunit interactions as IscS. An architectural switch model has been proposed in which evolutionarily lost interactions between NFS1 subunits results in the formation of low-activity architectures; FXN binding compensates for these lost interactions and facilitates a subunit rearrangement to activate the complex. Here, we used a structure and evolution-guided approach to identify three conserved residues proposed to weaken interactions between NFS1 subunits and transplanted these amino acids into IscS. Compared to native IscS, the engineered variant had a 4000-fold weaker dimer interface and diminished activity that correlated with the absence of the second catalytic subunit. Remarkably, the addition of the FXN homolog to the engineered variant stimulated the decay of the Cys-quinonoid pyridoxal 5-phosphate intermediate, shifted IscS from the monomeric to dimeric form, and increased the cysteine desulfurase activity, reproducing results from the human system and supporting the architectural switch model. Overall, these studies indicate a weakening of the homodimeric interface was a key development during the evolution of the eukaryotic system and provide new insights into the role of FXN.

biochemistry↗