Intricate packing in the hydrophobic core of barstar through a CH–π interaction Erix A. Milán-Garcés, a Sayan Mondal, b Jayant B. Udgaonkar a and Mrinalini Puranik b * Identification of specific packing interactions within in the hydrophobic core of proteins is important for understanding the integ- rity of protein structure. Finding such interactions is challenging because few tools allow monitoring of a specific interaction in the presence of several non-specific forces that hold proteins together. It is important to understand how and when such interactions develop during protein folding. In this study, we have used the intrinsic tryptophan residue, Trp53, as an ultraviolet resonance Raman probe to elucidate the packing interactions in the hydrophobic core of the protein barstar. Barstar is extensively studied for its folding, unfolding and aggregation properties. The Trp53 residue is known to be completely buried in the hydrophobic core of the protein and is used extensively as an intrinsic probe to monitor the folding and unfolding reactions of barstar. A comparison of the resonance Raman cross sections of some bands of Trp53 with those observed for N-acetyl-tryptophanoamide in water suggests that Trp53 in barstar is indeed isolated from water. Intensity ratio of the Fermi doublet suggests that Trp53 is surrounded by several aliphatic amino acid residues in corroboration with the crystal structure of barstar. Importantly, we show that the side chain of Trp53 is involved in a unique CH–π interaction with CH groups of Phe56 as well as a steric interaction with the methyl group of Ile5. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: UV resonance Raman spectroscopy; tryptophan; CH–π interactions; packing interactions Introduction The stability and native structure of a protein are maintained by a complex network of interactions including hydrophobic interac- tions, hydrogen bonding, electrostatic and packing interactions. Although the hydrophobic effect is considered to contribute most to the stability and folding of proteins, [1] packing interac- tions also play an important role. [2–5] In an early survey of protein structures, it was found that proteins are packed with densities similar to those of crystals of small organic molecules. [6–8] The higher packing density in proteins in comparison to liquids suggests that van der Waals forces make an important contribution to the stability of proteins. Mutations that resulted in the creation of large cavities inside the core were found to destabilize the native state due to a decrease in strength of the van der Waals interac- tions. [9–13] The role of these weak interactions is also important in protein folding and in stabilizing intermediates on the folding pathway, e.g. in the structures of dry molten globule intermediates found in a few proteins. [14–18] In the dry molten globule, a structur- ally expanded form of the native protein, the packing density is loose as compared to the native state but van der Waals interac- tions are still strong enough to prevent penetration of water into the core. These studies suggest that the core packing interactions are the first to be lost during unfolding and are the last to form during folding reaction of the proteins. [14,19] Barstar is an inhibitor of the ribonuclease barnase, and its struc- ture and folding have been systematically explored. [19–29] Packing interactions in the barstar core have been characterized using dif- ferent experimental and theoretical studies. [22,25,29–32] Calorimetric measurements of the enthalpy and entropy changes of barstar denaturation suggested that the native state conformation is more loosely packed than the native state conformations of other proteins. [32] Molecular dynamics (MD) simulations too report a highly dynamic core. [30] Nuclear magnetic resonance (NMR) spectroscopy studies have shown that the side chain of Phe74 in the hydrophobic core of barstar is able to flip without con- straint. [29,30,33] In contrast, Phe56 is in a rigid environment, possibly due to its interaction with Trp53. [29] Wt barstar contains three tryptophan (Trp) residues, with one Trp (Trp53) completely buried in the core. A mutant form of barstar that contains only Trp53 has been extensively used for fluorescence spectroscopy-based studies. The fluorescence of Trp53 decays with single exponential characteristics with a life- time of 4.9 ns against the typical biexponential decay observed for free Trp in water. [25] Time-resolved fluorescence anisotropy decay measurements of Trp53 in this mutant protein showed a single rotational correlation time of 4.1 ns that corresponds to the global tumbling of the protein. Thus, Trp53 is in a rigid local environment that constrains the rotation of the side chain so that within the protein it exists in a single rotational isomer. The rigidity of Trp53 was also confirmed by a NMR study carried out by Li et al. [29] * Correspondence to: Mrinalini Puranik, Indian Institute of Science Education and Research, Mendeleev, Dr. Homi Bhabha Road, Pune 411 008, India. E-mail: mrinalini@iiserpune.ac.in; puranik.mrinalini@gmail.com a National Centre for Biological Sciences, Tata Institute of Fundamental Re- search, Bangalore 560065, India b Indian Institute of Science Education and Research, Pune, 411008, India J. Raman Spectrosc. 2014, 45, 814–821 Copyright © 2014 John Wiley & Sons, Ltd. Research article Received: 14 March 2014 Revised: 27 June 2014 Accepted: 3 July 2014 Published online in Wiley Online Library: 7 August 2014 (wileyonlinelibrary.com) DOI 10.1002/jrs.4558 814