Network reorganization of a cold-adapted β-glucosidase couples conformational flexibility to catalytic efficiency under low-temperature conditions

Dual Engineering of the Hydrophobic Core and Functional Loop Reshapes the Conformational Energy Landscape for Significantly Enhanced Xylanase Activity and Thermostability.

The industrial application of enzyme catalysts is often constrained by the trade-off between thermostability and catalytic activity. Here, a region-focused engineering strategy was applied to a thermophilic GH10 xylanase to simultaneously improve both properties. The strategy integrates qProtein-guided hydrophobic cluster design for scaffold stabilization and dynamic loop analysis for active-site optimization. The resulting triple mutant A206S-N209D-F130L exhibited substantially improved thermostability, with a 5.79 °C increase in melting temperature and an 18.8-fold extension of the half-life at 60 °C. Its optimum temperature increased from 60 to 70 °C, accompanied by a 129.4% enhancement in catalytic activity at 70 °C relatively to the wild type. Molecular dynamics simulations indicated that these mutations reshape the conformational energy landscape by stabilizing hydrophobic packing and modulating loop dynamics. This study provides a generalizable framework for simultaneously improving enzyme stability and catalytic performance.

Analysis of the Atypical Temperature Dependence and Conformational Changes During Turnover of a Lactobacillus Chlorogenic Acid Esterase

It is suggested that contrasting thermodynamic models for atypical temperature dependence in enzymes can give rise to similar looking fits, even though they have different underlying physical meaning.

Multidimensional Computational Engineering of Pectate Lyase for Enhanced Thermostability and Moderately Improved Catalytic Efficiency.

This study provides a practical strategy for engineering thermostable pectate lyases with improved catalytic performance by developing a multidimensional consensus computational framework integrating sequence conservation, structural dynamics, and thermodynamic prediction to identify functional mutation hotspots in PcPel1834.

Thermodynamic Profiling of Glucose Translocation in POPC embedded GLUT-4 protein models

A comparative biophysical analysis of glucose translocation using both the native Cryo-EM structure and the AI-predicted AlphaFold model is conducted, establishing that rigid AI-generated architectures lack the essential internal free volume and conformational plasticity required to sustain accurate thermodynamic profiling of dynamic membrane transporters.

Thermostability modification and simulated application of acidic catalase

Chemical Pathway-Dependent Structural Reorganization at a Hinge Microenvironment in the AcrA Adaptor Protein.

Adaptor protein AcrA plays a central role in the assembly and function of tripartite multidrug efflux pumps in Gram-negative bacteria, yet how its structural organization responds to coupled chemical perturbations rather than solely to equilibrium conditions remains unclear. Residues near His285 define a hinge microenvironment linking the lipoyl and β-barrel domains, suggesting a site for chemically sensitive structural modulation. Here, site-directed spin labeling combined with continuous-wave electron paramagnetic resonance spectroscopy was used to examine AcrA under an Mg2+-driven perturbation that simultaneously alters proton availability. Mg2+ addition produced spectral broadening at residue 62 that was fully reversed by spin dilution, indicating increased interspin proximity without changes in intrinsic side-chain dynamics. In contrast, direct acidification to a comparable bulk pH in the absence of Mg2+ did not reproduce this behavior. Structural mapping places residue 62 in proximity to the His285-centered hinge region, suggesting that coupled changes in protonation and metal coordination bias local interaction networks and modulate interdomain organization. These findings demonstrate that equivalent bulk conditions can mask distinct molecular states and identify chemical pathways as an important determinant of AcrA structural dynamics.