Probing the modulation of enzyme kinetics by multi-temperature, time-resolved serial crystallography

Area of Science:

• Structural Biology
• Biophysics
• Enzymology

Background:

• Most protein structures are determined at cryogenic temperatures, not physiological conditions.
• Temperature significantly impacts protein dynamics and function.
• Current time-resolved crystallography methods often miss crucial states visible only at physiological temperatures.

Purpose of the Study:

• To develop and apply a method for multi-temperature, time-resolved serial crystallography.
• To investigate the interplay between protein structure, activity, and temperature.
• To capture protein conformational states at physiological temperatures.

Main Methods:

• Development of a 5D-SSX technique for time-resolved serial crystallography.
• Experiments conducted across a temperature range from below 10°C to above 70°C.
• Analysis of temperature-dependent turnover kinetics and protein structure.

Main Results:

• Demonstrated the ability to perform time-resolved experiments at physiological temperatures with long time delays.
• Observed temperature-dependent modulation of turnover kinetics for β-lactamase CTX-M-14.
• Revealed temperature-dependent functional modulation for xylose isomerase.

Conclusions:

• The new method provides critical insights into protein function and enzyme catalysis at native temperatures.
• Protein structure and activity are significantly modulated by temperature.
• Physiological temperature studies are essential for a complete understanding of protein dynamics.