Fluorescence Correlation Spectroscopy Study on the Effects of the Shape and Size of a Protein on Its Diffusion Inside a Crowded Environment
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
View Archive Info| Field | Value | |
| Title |
Fluorescence Correlation Spectroscopy Study on the Effects of the Shape and Size of a Protein on Its Diffusion Inside a Crowded Environment
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| Creator |
Basak, Sujit
Chattopadhyay, Krishnananda |
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| Subject |
Structural Biology & Bioinformatics
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| Description |
Fluorescence correlation spectroscopy (FCS)
has been commonly used to study the diffusional and
conformational fluctuations of labeled molecules at singlemolecule 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.
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| Publisher |
American Chemical Society
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| Date |
2013
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| Type |
Article
PeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/2406/1/LANGMUIR__Volume__29____Issue__47____Pages__14709%2D14717.pdf
Basak, Sujit and Chattopadhyay, Krishnananda (2013) Fluorescence Correlation Spectroscopy Study on the Effects of the Shape and Size of a Protein on Its Diffusion Inside a Crowded Environment. Langmuir, 28 (47). pp. 14709-14717. |
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| Relation |
http://dx.doi.org/10.1021/la4031987
http://www.eprints.iicb.res.in/2406/ |
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