A Fluorescence-Based Temperature-Jump Apparatus for Illustrating Protein Dynamics on the Millisecond Time Scale

Area of Science:

• Biophysics
• Protein dynamics
• Spectroscopy

Background:

• Understanding protein dynamics is crucial for elucidating biological functions.
• Existing techniques often lack the resolution to capture large-domain motions on biologically relevant timescales.
• Millisecond timescale dynamics bridge the gap between fast local motions and steady-state conformations.

Purpose of the Study:

• To develop and validate a fluorescence-based temperature jump (T-jump) module for studying protein dynamics.
• To investigate large-domain motions of proteins on the millisecond timescale.
• To characterize protein responses to thermal stimuli within physiological temperature ranges.

Main Methods:

• A fluorescence-based T-jump module utilizing a 1467 nm diode laser and "optical Riemann sum" for rapid heating (5.0 °C in ~2 ms).
• Monitoring temperature evolution via time-resolved fluorescence intensity changes of dissolved tryptophan.
• Utilizing microspot objective for low sample volume (1 μL) and a homemade thermostatic pad for temperature control.

Main Results:

• Successfully demonstrated millisecond-resolution T-jump measurements within physiological temperatures (35.0-39.9 °C).
• Characterized the dynamics of bovine serum albumin (BSA) with an apparent activation energy of 276 ± 23 kJ mol⁻¹.
• Human serum albumin (HSA) did not exhibit a discernible dynamic component under the tested conditions.

Conclusions:

• The developed millisecond-resolution T-jump technique effectively illustrates large-domain protein dynamics.
• This method bridges the gap between fast (ns-μs) and steady-state protein dynamics characterization.
• The technique is valuable for studying protein conformational changes and thermal responses in biophysical research.