Role of Protein Stabilizers on the Conformation of the Unfolded State of Cytochrome c and Its Early Folding Kinetics

Abstract

An insight into the conformation and dynamics of unfolded and early intermediate states of a protein is essential to understand the mechanism of its aggregation and to design potent inhibitor molecules. Fluorescence correlation spectroscopy has been used to study the effects of several model protein stabilizers on the conformation of the unfolded state and early folding dynamics of tetramethyl rhodamine-labeled cytochrome c from Saccharomyces cerevisiae at single molecular resolution. Special attention has been given to arginine, which is a widely used stabilizer for improving refolding yield of different proteins. The value of the hydrodynamic radius (r H ) obtained by analyzing the intensity fluctuations of the diffusing molecules has been found to increase in a two-state manner as the protein is unfolded by urea. The results further show that the presence of arginine and other protein stabilizers favors a relatively structured conformation of the unfolded states (r H of 29 Å ) over an extended one (r H of 40 Å ), which forms in their absence. Also, the time constant of a kinetic component ( R ) of about 30 s has been observed by analyzing the correlation functions, which represents formation of a collapsed state. This time constant varies with urea concentration representing an inverted Chevron plot that shows a roll-over behavior in the absence of arginine. To the best of our knowledge, this is one of the first applications of fluorescence correlation spectroscopy to study direct folding kinetics of a protein.

Interconnection of Salt-induced Hydrophobic Compaction and Secondary Structure Formation Depends on Solution Conditions

“…The presence of an equilibrium between an extended and a compact conformer in the unfolded state of cytochrome c has been shown before by fluorescence spectroscopy and small angle x-ray scattering experiments (14,23,24). The formation of the collapsed unfolded states for multiple other proteins has also been demonstrated (25-29).…”

“…We have used FCS and far UV CD to study salt-induced collapse and secondary structure formation of TMR-labeled cytochrome c (cytochrome c-TMR) at pH 2. Labeling with TMR does not affect the conformation and folding of cytochrome c (14,21). At low pH (for example, at pH 2), the protein becomes positively charged.…”

“…Cytochrome c from S. cerevisiae contains one free cysteine (Cys-102), which was labeled with TMR using published procedures (14). Labeling of BSA with Alexa Fluor 488-maleimide was carried out using an identical method.…”

“…Labeling of BSA with Alexa Fluor 488-maleimide was carried out using an identical method. Labeling of cytochrome c with TMR does not result in any significant effect on the structure, conformation, and folding of the protein (14). FCS experiments were carried out with a ConfoCor 3 LSM setup (Carl Zeiss, Evotec, Jena, Germany) using a 40ϫ water immersion objective.…”

Ligand Binding Assay Critical Reagents and Their Stability: Recommendations and Best Practices from the Global Bioanalysis Consortium Harmonization Team

“…There is a significant body of literature describing protein, peptide, antibody purification, and conjugations thereof and formulation to minimize degradation (for review, see 19) and maximize stability (20,21). Generally, these approaches are not applied to in-house reagents, although commercial reagents may include them.…”