We use a single-step immobilization procedure to modify glass surface with a silane-reactive PEG/PEG-biotin mixture. A range of cpSRP43 proteins were site specifically-labeled with FRET dye pairs to probe specific interdomain conformational dynamics. The proteins were also labeled with biotin to enable immobilization via a Neutravidin-biotin linkage. Negligible non-specific adsorption was observed for these proteins on such surfaces verifying both the homogeneity of the surface and its ability to inhibit interaction of the surface with the proteins. We analyzed the dynamics of immobilized single protein molecules using time-resolved single molecule FRET. FRET histograms were compared to freely-diffusing molecules to confirm that the dynamics were intrinsic. A wide distribution of states were obtained, indicating a complex energy landscape underlying the protein's functions of binding multiple substrates and targeting LHCP to the thylakoid membrane in chloroplasts.

Immobilizing and characterizing the interdomain conformational dynamics of CpSRP43 proteins on PEG-modified surfaces

245th National Meeting (2013)

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