Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals

Area of Science:

• Biophysics
• Structural Biology
• Biochemistry

Background:

• Protein dynamics are crucial for biological function.
• Protein activity can be arrested below a dynamical transition temperature (150-250 K).
• Investigating single protein crystal dynamics is challenging.

Purpose of the Study:

• To introduce and validate a new technique, temperature-derivative fluorescence microspectrophotometry, for studying single protein crystal dynamics.
• To probe local dynamical transitions within biomolecules.
• To investigate the influence of solvent and confinement on protein dynamics.

Main Methods:

• Development of temperature-derivative fluorescence microspectrophotometry.
• Demonstration of technique potential using water/glycerol mixtures.
• Application to fluorescein-doped protein crystals (human butyrylcholinesterase).

Main Results:

• Successful observation of glass transitions in water/glycerol mixtures.
• Detection of a clear dynamical transition at 175 K in the active site of crystalline human butyrylcholinesterase.
• Evidence that crystalline protein dynamics depend on solvent composition and crystal confinement.

Conclusions:

• Temperature-derivative fluorescence microspectrophotometry is a sensitive tool for studying biological nanosamples.
• Protein dynamics are influenced by their surrounding environment within crystals.
• The technique has potential applications in kinetic crystallography and beyond.