1 research outputs found
Fluorescence Correlation Spectroscopy Study on the Effects of the Shape and Size of a Protein on Its Diffusion Inside a Crowded Environment
Fluorescence correlation spectroscopy
(FCS) has been commonly used
to study the diffusional and conformational fluctuations of labeled
molecules at single-molecule resolution. Here, we explored the applications
of FCS inside a polyacrylamide gel to study the effects of molecular
weight and molecular shape in a crowded environment. To understand
the effect of molecular weight, we carried out FCS experiments with
four model systems of different molecular weights in the presence
of varying concentrations of acrylamide. The correlation curves were
fit adequately using a model containing two diffusing components:
one representing unhindered diffusion and one representing slow hindered
diffusion in the gel phase. A large number of measurements carried
out at different randomly chosen spots on a gel were used to determine
the most probable diffusion time values using Gaussian distribution
analysis. The variation of the diffusivity with the molecular weight
of the model systems could be represented well using the effective
medium model. This model assumes a combination of hydrodynamic and
steric effects on solute diffusivity. To study the effects of solute
shape, FCS experiments were carried inside a urea gradient gel to
probe the urea-induced unfolding transition of Alexa488Maleimide-labeled
bovine serum albumin. We showed that the scaling behavior, relating
the hydrodynamic radius and the number of amino acids, changes inside
an acrylamide gel for both folded and unfolded proteins. We showed
further that crowding induced by a polyacrylamide gel increases the
resolution of measuring the difference in hydrodynamic radii between
the unfolded and folded states