Measuring rotational diffusion of macromolecules by fluorescence correlation spectroscopy.

Area of Science:

• Biophysics
• Biochemistry
• Physical Chemistry

Background:

• Measuring rotational diffusion of large biomolecules is crucial for understanding their structure and dynamics.
• Conventional fluorescence anisotropy methods are limited by fluorescence decay times, restricting their application to molecules with short rotational diffusion times.
• There is a need for methods capable of measuring rotational diffusion for macromolecules with longer rotational diffusion times.

Purpose of the Study:

• To present a novel fluorescence correlation method for measuring rotational diffusion of large biomolecules in solution.
• To enable the study of macromolecules with rotational diffusion times significantly longer than typical fluorescence lifetimes.
• To provide a reliable method for determining hydrodynamic radii of proteins.

Main Methods:

• Utilizing fluorescence correlation on the nanosecond time scale.
• Employing a pulsed interleaved excitation scheme with crossed excitation polarization.
• Analyzing the time-dependent amplitude of the measured correlation curve.

Main Results:

• The developed method successfully measures rotational diffusion times from dozens to hundreds of nanoseconds.
• The technique is effective even when rotational diffusion times exceed fluorescence decay times.
• Precise hydrodynamic radii were obtained for a set of common globular proteins.

Conclusions:

• The novel fluorescence correlation method offers a powerful tool for studying the rotational dynamics of large biomolecules.
• This approach expands the scope of fluorescence-based techniques for macromolecular characterization.
• The method provides accurate sizing of proteins based on their rotational diffusion coefficients.