title: Molecular Crowding Affects the Conformational Fluctuations, Peroxidase Activity, and Folding Landscape of Yeast Cytochrome c.
abstract: To understand how a protein folds and behaves inside living cells, the effects of synthetic crowding media on protein folding, function, stability, and association have been studied in detail. Because the effect of excluded volume is more prominent in an extended state than in the native protein, a majority of these studies have been conducted in the unfolded state of different model proteins. Here, we have used fluorescence correlation spectroscopy (FCS) and other biophysical methods to investigate the effect of crowding agents Ficoll70 and Dextran70 on the nativelike state of cytochrome c from yeast. Yeast cytochrome c (y-cytc) contains a substantial expanded state in its native folded condition, which is present in equilibrium with a compact conformer in aqueous buffer. We have found that the crowding medium affects the native state equilibrium between compact and expanded states, shifting its population toward the compact conformer. As a result, the peroxidase activity of y-cytc decreases. Urea-induced protein stability measurements show that the compaction destabilizes the protein due to charge repulsions between similar charged clusters. Interestingly, the time constant of conformational fluctuations between the compact and expanded conformers has been found to increase in the crowded milieu, suggesting a crucial role of the solution microviscosity.
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology , 4, Raja S. C. Mullick Road, Kolkata 700032, India.
authors.forename: Simanta Sarani
authors.initials: SS
authors.lastname: Paul
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology , 4, Raja S. C. Mullick Road, Kolkata 700032, India.
authors.forename: Pallabi
authors.initials: P
authors.lastname: Sil
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology , 4, Raja S. C. Mullick Road, Kolkata 700032, India.
authors.forename: Ritobrita
authors.initials: R
authors.lastname: Chakraborty
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute of Biochemistry , Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Shubhasis
authors.initials: S
authors.lastname: Haldar
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology , 4, Raja S. C. Mullick Road, Kolkata 700032, India.
authors.forename: Krishnananda
authors.initials: K
authors.lastname: Chattopadhyay
mesh_terms: D045304:Cytochromes c / Q000737:chemistry*; D003911:Dextrans / Q000737:chemistry*; D005362:Ficoll / Q000737:chemistry*; D008958:Models, Molecular; D010544:Peroxidases / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D058767:Protein Unfolding; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence
chemical_list: D003911:Dextrans; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D005362:Ficoll; D045304:Cytochromes c; D010544:Peroxidases