Interpretation of fluorescence decays in proteins using continuous lifetime distributions

Area of Science:

• Biophysics
• Protein Dynamics
• Fluorescence Spectroscopy

Background:

• Tryptophan emission decay in proteins is complex due to environmental sensitivity.
• Traditional exponential analysis assumes fixed protein conformations.
• An alternative model considers rapid interconversion between numerous protein conformations.

Purpose of the Study:

• To analyze protein fluorescence decay using continuous lifetime distributions.
• To investigate the influence of protein conformations and dynamics on fluorescence decay.
• To evaluate different models for describing tryptophan fluorescence decay, including temperature dependence.

Main Methods:

• Multifrequency phase fluorometry was used to measure fluorescence decay.
• Analysis involved sum of exponentials, probability-density functions, and a single potential well model.
• Temperature dependence of fluorescence decay was investigated for various proteins and tryptophan derivatives.

Main Results:

• Continuous lifetime distributions provide insights into protein mobility and conformational interconversion rates.
• Ribonuclease T1 and neurotoxin variant 3 were well-described by bimodal distributions.
• Phospholipase A2 required more complex models, suggesting multiple potential wells at lower temperatures.

Conclusions:

• Continuous lifetime distributions offer a powerful alternative for analyzing complex protein fluorescence decay.
• Protein dynamics and conformational flexibility significantly impact excited-state decay.
• The choice of model, including potential well complexity, is crucial for accurately describing fluorescence decay across temperatures.