title: The Non-native Helical Intermediate State May Accumulate at Low pH in the Folding and Aggregation Landscape of the Intestinal Fatty Acid Binding Protein.
abstract: There has been widespread interest in studying early intermediate states and their roles in protein folding. The interest in intermediate states has been further emphasized in the recent literature because of their implications for protein aggregation. Unfortunately, direct kinetic characterization of intermediates has been difficult because of the limited time resolutions offered by the kinetic techniques and the heterogeneity of the folding and aggregation landscape. Even in equilibrium experiments, the characterization of intermediate states could be difficult because (a) their populations in equilibrium could be low and/or (b) they lack any specific biochemical or biophysical signatures for their identification. In this paper, we have used fluorescence correlation spectroscopy to study the nature of a low-pH intermediate state of the intestinal fatty acid binding protein, a small protein with predominantly β-sheet structure. Our results have shown that the pH 3 intermediate diffuses faster than the folded protein and has strong helix forming propensity. These behaviors support Lim's hypothesis according to which even an entirely β-sheet protein would form helical bundles at the early stage. Using dynamic light scattering and thioflavin T binding measurements, we have observed that the pH 3 intermediate is prone to aggregation. We believe that early helix formation is the result of a local effect, which originates from the interaction of the neighboring amino acids around the hydrophobic core residues. This early intermediate reorganizes subsequently, and this structural reorganization is initiated by the destabilizing interactions induced by the distant residues, unfavorable entropic costs, and steric constraints of the hydrophobic side chains. Mutational analyses show further that the increase in the hydrophobicity in the hydrophobic core region increases the population of the α-helical intermediate, enhancing the aggregation propensity of the protein, while an identical change, distant from the hydrophobic core, does not show any effect. This study re-emphasizes an overlap between the folding and aggregation landscape of a protein, where the fine-tuning between the local and global effects may be important for the protein to fold efficiently or to aggregate.
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology , Kolkata 700032, India.
authors.forename: Suparna
authors.initials: S
authors.lastname: Sarkar-Banerjee
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology , Kolkata 700032, India.
authors.forename: Sourav
authors.initials: S
authors.lastname: Chowdhury
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology , 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 , Kolkata 700032, India.
authors.forename: Debashis
authors.initials: D
authors.lastname: Dutta
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology , Kolkata 700032, India.
authors.forename: Anisa
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology , Kolkata 700032, India.
authors.forename: Krishnananda
authors.initials: K
authors.lastname: Chattopadhyay
mesh_terms: D000595:Amino Acid Sequence; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D066329:Protein Aggregates; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D017510:Protein Folding; D058767:Protein Unfolding / Q000187:drug effects; D014508:Urea / Q000494:pharmacology
chemical_list: D050556:Fatty Acid-Binding Proteins; D066329:Protein Aggregates; D014508:Urea