Resolving dynamics and function of transient states in single enzyme molecules

Area of Science:

• Biophysics
• Structural Biology
• Enzymology

Background:

• T4 Lysozyme (T4L) is a model enzyme extensively studied using crystallography.
• Existing structural data has not fully captured the dynamic conformational states relevant to T4L function.

Purpose of the Study:

• To investigate the kinetic and dynamic interplay of T4 Lysozyme's conformational states.
• To identify transient conformational states beyond those observed in crystal structures.

Main Methods:

• Utilized a hybrid fluorescence spectroscopic toolkit combining single-molecule and ensemble multiparameter fluorescence detection.
• Employed Electron Paramagnetic Resonance (EPR) spectroscopy, mutagenesis, and Förster Resonance Energy Transfer (FRET)-positioning and screening.
• Integrated other biochemical and biophysical techniques for comprehensive analysis.

Main Results:

• Characterized three short-lived conformational states of T4L on the nanosecond-to-millisecond timescale.
• Identified that T4L in solution primarily exists in known open and closed states, exchanging at 4 µs.
• Discovered a novel, minor conformational state, sampled at 230 µs, not previously observed in over 500 crystal structures.

Conclusions:

• The novel minor state may play an active role in the product release step of T4L catalysis.
• The developed fluorescence spectroscopic toolkit advances dynamic structural biology by revealing transient states.
• Transient conformational states are abundant in biological systems and critical for enzymatic reactions.