A fluorescence correlation spectroscopy study of hindered–probe diffusion in complex media

Monday, October 24, 2011 — Poster Session I
Noon – 2:00 p.m.	Natcher Conference Center	NICHD	BIOPHY-13

* FARE Award Winner

Authors

• S Zustiak
• R Nossal
• D Sackett

Abstract

Here, we present the application of fluorescence correlation spectroscopy (FCS) for the study of hindered–probe diffusion in complex media. This study recapitulates the intracellular environment, where molecular diffusion is impaired predominantly by crowding and charge-mediated binding. Intracellular diffusion has long been of interest for describing basic cellular processes but is still poorly understood. In this work, we developed a model system in which crowding and binding were addressed independently in a controlled manner. Using FCS, we obtained the diffusivity of the charged protein, RNase, in solutions of dextrans of various charges (binding) and concentrations (crowding). We observed an overall five-fold decrease in RNase diffusivity at the highest dextran concentration, where binding accounted for 75% and crowding for 25% of the decrease. Further, 100 μM crowder, as compared to 1 μM binder, was needed to achieve equivalent reduction in RNase diffusivity. However, the data suggested that at a higher crowder concentration (similar to the cell), crowding could “mask” the effect of binding. This is one of the first studies to highlight the relative contribution of binding and crowding to hindered diffusion in complex media and thus can facilitate future understanding of molecular transport implicated in key cellular processes.

back to top