distinct effects of guanidine thiocyanate on the structure of superfolder gfp不同的硫氰酸胍对superfolder gfp的结构的影响.pdfVIP

Distinct Effects of Guanidine Thiocyanate on the Structure of Superfolder GFP 1 1 1 2 Olesya V. Stepanenko , Olga V. Stepanenko , Irina M. Kuznetsova , Daria M. Shcherbakova , 2 1 Vladislav V. Verkhusha , Konstantin K. Turoverov * 1 Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology of the Russian Academy of Sciences, St. Petersburg, Russia, 2 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, New York, United States of America Abstract Having a high folding efficiency and a low tendency to aggregate, the superfolder GFP (sfGFP) offers a unique opportunity to study the folding of proteins that have a b-barrel topology. Here, we studied the unfolding–refolding of sfGFP that was induced by guanidine thiocyanate (GTC), which is a stronger denaturing agent than GdnHCl or urea. Structural perturbations of sfGFP were studied by spectroscopic methods (absorbance, fluorescence, and circular dichroism), by acrylamide quenching of tryptophan and green chromophore fluorescence, and by size-exclusion chromatography. Low concentrations of GTC (up to 0.1 M) induce subtle changes in the sfGFP structure. The pronounced changes in the visible absorption spectrum of sfGFP which are accompanied by a dramatic decrease in tryptophan and green chromophore fluorescence was recorded in the range 0–0.7 M GNC. These alterations of sfGFP characteristics that erroneously can be mixed up with appearance of intermediate state in fact have pure spectroscopic but not structural nature. Higher concentrations