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Non-native helical intermediate state may accumulate at low pH in the folding/aggregation landscape of the intestinal fatty acid binding protein Suparna Sarkar-Banerjee, Sourav Chowdhury, Simanta Sarani Paul, Debashis Dutta, Anisa Ghosh, and Krishnananda Chattopadhyay Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00390 • Publication Date (Web): 25 Jul 2016 Downloaded from http://pubs.acs.org on July 27, 2016
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Biochemistry
Non-native helical intermediate state may accumulate at low pH in the folding/aggregation landscape of the intestinal fatty acid binding protein Suparna Sarkar-Banerjee$,#, Sourav Chowdhury#, Simanta Sarani Paul#, Debashis Dutta, Anisa Ghosh, and Krishnananda Chattopadhyay* $
Present Address: Department of Integrative Biology and Pharmacology, McGovern Medical School at UTHealth, Houston, Texas 77030, USA. #
contributed equally,
*Corresponding author: Krishnananda Chattopadhyay, Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, Kolkata 700032, INDIA email:
[email protected] Funding information: The present study has been funded by CSIR network project grant, UNSEEN.
Keywords: Fluorescence correlation spectroscopy, single molecule fluorescence, intermediate states, protein folding
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Biochemistry
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Abstract: There have been widespread interests to study the early intermediate states and their roles in protein folding. The interests in the intermediate states have been further emphasized in recent literatures because of their implications in protein aggregation. Unfortunately, direct kinetic characterization of intermediates has been difficult because of limited time resolutions offered by the kinetic techniques and heterogeneity of the folding/aggregation landscape. Even for the equilibrium experiments, the characterization of the intermediate states could be difficult. This is 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 (FCS) to study the nature of a low pH intermediate state of the intestinal fatty acid binding protein (IFABP), 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 aggregation prone. We believe that the early helix formation is the result of a local effect, which is originated by 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 constrains of the hydrophobic side chains. Mutational analyses show further that the increase in the hydrophobicity at the hydrophobic core region increases the population of the alpha helical intermediate enhancing the aggregation propensity of the protein, while 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.
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Biochemistry
Introduction: Folding/aggregation landscape of a protein could be complex and heterogeneous involving intermediate states, which are difficult to characterize. This problem is particularly severe for the early intermediate states, because of the dead time limitations of mixing devices used for the kinetics measurements. Nevertheless, a large number of computational and experimental studies have been carried out to study the early events of protein folding and the associated intermediate states. Unfortunately, a coherent understanding is still lacking. A general description of the nature of the early intermediate states was hypothesized by Lim (1, 2). According to Lim’s hypothesis, proteins of different secondary structures (including the β sheet proteins) have strong potentials to form helices. This hypothesis predicts that the folding of even a completely β sheet protein would involve the formation of helical bundles (whose stabilities may vary depending on the nature of the protein), and these bundles would reorganize later to form the native folded structure. The support for this hypothesis has come from several computational studies (3, 4). However, the experimental proof is limited, and the most notable example is β-lactoglobulin. Although the native folded state of this protein is predominantly β sheet, the folding of β-lactoglobulin is characterized by the formation of helical intermediates. These intermediates are sufficiently stable and populated enough to be studied by stopped flow CD and other biophysical methods (5, 6). There may be several reasons why the experimental characterizations of the early intermediates could be difficult. The early intermediates are often short lived, with population too low to be detected by the spectroscopic methods. The transient nature of the early non-native helices has been evident by the observation of the helical potential at a period of
