Date

Wednesday 21 May 2025

Time

15:15 - 15:45

Location

BW030

Supervisor

Marcellus Ubbink

2nd reviewer

Rolf Boot

Jury

Alia Alia

Enzymes are dynamic entities whose precise conformations and subtle motions are essential for catalysis. Flexible proteins tolerate mutations via their conformational plasticity that absorbs structural changes while retaining activity. In contrast, rigid proteins maintain a fixed active site geometry that ensures high catalytic efficiency. Surprisingly, class A -lactamases combine rigidity with high evolvability, making them excellent models for studying protein dynamics and evolution. These rigid proteins possess almost identical structures, while only sharing 38-47% sequence similarity. By comparing two distant -Lactamases, we try to find to what degree the interactions between second shell residues (which are responsible for the structural finetuning and rigidity of the active site) are conserved in evolution. To investigate how second shell residues surrounding active site of Temoneira-1 (TEM-1) contribute to regulate active site dynamics, we generated four mutants. Mutants A217S, A217V, D233A, and G245A were successfully expressed and purified for nuclear magnetic resonance spectroscopy, and kinetic and thermal stability measurements. In general, the mutations destabilized the enzyme and abolished activity, except for A217S, which had a similar melting temperature as WT and partial activity. Chemical shift perturbations revealed widespread conformational changes, with the largest disruptions in TEM-1 A217V and D233A. Comparative analysis with BlaC indicated limited similarity between the tested mutations in the two -lactamases, underscoring the need for a broader mutational survey to fully elucidate how second shell residues modulate active site entropy and balance rigidity with flexibility in -Lactamases and if these mechanisms are conserved.