SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Translational Diffusion of Fluorescent Proteins by Molecular Fourier Imaging Correlation Spectroscopy
Michael C Fink 1, Kenneth V. Adair 1, Marina G. Guenza 1 and Andrew H Marcus 1*
1 University of Oregon
* To whom correspondence should be addressed. E-mail: ahmarcus{at}uoregon.edu.
Submitted on March 23, 2006
Revised on May 9, 2006
Accepted on 1 August 2006
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Abstract |
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The ability to non-invasively observe translational diffusion of proteins and protein complexes is important to many biophysical problems. We report high signal-to-noise (S/N 250) measurements of the translational diffusion in viscous solution of the fluorescent protein, DsRed. This is carried out using a new technique: molecular Fourier imaging correlation spectroscopy (M-FICS). M-FICS is an interferometric method that detects a collective Fourier component of the fluctuating density of a small population of fluorescent molecules, and provides information about the distribution of molecular diffusivities. A theoretical analysis is presented that expresses the detected signal fluctuations in terms of the relevant time correlation functions for molecular translational diffusion. Furthermore, the role played by optical orientational degrees of freedom is established. We report Fickian self-diffusion of the DsRed tetramer at short time scales. The long-time deviation of our data from Fickian behavior is used to determine the variance of the distribution of the protein self-diffusion coefficient. We compare our results to the expected outcomes for i) a bidisperse distribution of protein species, and ii) dynamic disorder of the host solvent.
Key Words:
DsRed, fluctuation spectroscopy, low-signal-detection, molecular distributions, protein diffusion
