Formation of a CH-π Contact in the Core of Native Barstar during Folding Erix A. Mila ́ n-Garce ́ s, † Pallavi Thaore, † Jayant B. Udgaonkar,* ,† and Mrinalini Puranik* ,‡ † National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India ‡ Indian Institute of Science Education and Research, Pune 411008, India * S Supporting Information ABSTRACT: An important part of the protein folding process is the consolidation of the protein core through the formation of specific, directional contacts after the initial hydrophobic collapse. Here, we simultaneously monitor formation of core contacts and assembly of secondary structure through salt-induced folding by using resonance Raman spectroscopy. Unfolded barstar at pH 12 was refolded by gradual addition of sodium sulfate salt. Altered spectral characteristics of the Trp53 residue suggest that the core of the protein attains a CH-π interaction at a low concentration of the salt, with an increase in the packing density. Further increase in salt concentration produces a reduction in the solvent accessibility of the core. These data provide evidence that the core of the protein becomes rigid upon the addition of 0.6 M sodium sulfate. This is the first time that the formation of a CH-π interaction has been directly monitored during the folding of a protein. ■ INTRODUCTION The understanding of the role of specific interactions during the folding of a protein from a random chain unfolded conformation to the functionally relevant tertiary structure is a major problem in biology. Hydrophobic, hydrogen bonding, packing, and other interactions have been found to play important roles during protein folding. 1,2 Nothing, however, is known about the formation of a specific CH-π interaction during a protein folding reaction. 3,4 CH-π interactions have a high probability of occurrence inside a protein, 5 as suggested by survey studies of the structures of several proteins, and it has been suggested that they are important in determining the stability and function of proteins. 5-12 To monitor the formation of CH-π interactions during folding has been a challenge. Not only is a suitable probe and a sensitive experimental technique which can directly measure these interactions required, but a suitable protein model in which the CH-π interaction can be detected experimentally is also needed. The protein barstar, an inhibitor of the ribonuclease barnase, has been systematically used during the past 20 years as a model protein to study protein folding and unfolding reactions. 13-27 Barstar contains three tryptophans (Trp38, Trp44, and Trp53) residues, with Trp53 present inside the core of the protein, completely sequestered away from the solvent. The crystal structure of barstar also shows that the side chain of Trp53 is inside a hydrophobic environment surrounded by several aliphatic residues. 28 Its position in the protein has made Trp53 a useful probe to get insights into the structural changes inside the core as well as on the changes in the solvent accessibility of the core during the folding and unfolding reactions of barstar. In a previous study from our group, UVRR spectroscopy was used to characterize the local environment of Trp53 inside barstar. In that study, it was shown that the Raman bands of Trp53 provide experimental evidence of the CH-π interaction as well as steric interactions with Phe56 and Ile5, respectively. The fact that UVRR spectroscopy is sensitive to the CH-π and steric interactions between Trp53 and neighboring amino acid residues suggests that this technique can be used to directly monitor the formation of specific interactions during structural changes of the protein. It has been difficult to directly monitor the development of a specific interaction during the folding of any protein, without resorting to chemical labeling and mutagenesis, and both these procedures can potentially perturb the interaction being studied. On the other hand, UVRR spectroscopy offers itself as a nonperturbative probe to monitor the formation of specific interactions. The ability of the resonance Raman spectroscopy to monitor the folding and unfolding reactions of proteins is well- known. 29-40 This, together with the sensitivity of the UVRR spectrum of Trp to capture information about relevant interactions in proteins, makes UVRR spectroscopy an excellent methodology to study folding using barstar as a model. 41-49 In this study, the salt-induced equilibrium refolding reaction of a single Trp (Trp53) containing mutant form of barstar was monitored using UVRR spectroscopy. Excitation with a wavelength at the red edge of the Bb absorption band of Trp allows the changes in the Raman bands of Trp53 to be monitored. Received: December 2, 2014 Published: January 13, 2015 Article pubs.acs.org/JPCB © 2015 American Chemical Society 2928 DOI: 10.1021/jp512036p J. Phys. Chem. B 2015, 119, 2928-2932