Translational diffusion of fluorescent proteins by molecular fourier imaging correlation spectroscopy.
The ability to noninvasively observe translational diffusion of proteins and protein complexes is important to many biophysical problems. We report high signal/noise (>or=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 timescales. 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 1), a bi-disperse distribution of protein species, and 2), dynamic disorder of the host solvent.
Fink
Michael C
MC
Department of Chemistry, Institute of Theoretical Sciences, University of Oregon, Eugene, USA.
Adair
Kenneth V
KV
Guenza
Marina G
MG
Marcus
Andrew H
AH
D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D021381:Protein Transport / Q000502:physiology*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D005456:Fluorescent Dyes