Hydrophobic Interactions at the Ccap Position of the C-capping Motif of a-Helices Dmitri N. Ermolenko 1,2 , Susan T. Thomas 1 , Rajeev Aurora 3 Angela M. Gronenborn 4 and George I. Makhatadze 1 * 1 Department of Biochemistry and Molecular Biology H171 Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033-2390, USA 2 A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, 117071 Moscow Russia 3 Computational Biology Group Pharmacia Corporation, 700 Chesterfield Parkway North BB5A St Louis, MO 63017 USA 4 Laboratory of Chemical Physics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 USA We investigated the possible role of residues at the Ccap position in an a-helix on protein stability. A set of 431 protein a-helices containing a C 0 -Gly from the Protein Data Bank (PDB) was analyzed, and the normal- ized frequencies for finding particular residues at the Ccap position, the average fraction of buried surface area, and the hydrogen bonding patterns of the Ccap residue side-chain were calculated. We found that on average the Ccap position is 70% buried and noted a significant corre- lation ðR ¼ 0:8Þ between the relative burial of this residue and its hydro- phobicity as defined by the Gibbs energy of transfer from octanol or cyclohexane to water. Ccap residues with polar side-chains are commonly involved in hydrogen bonding. The hydrogen bonding pattern is such that, the longer side-chains of Glu, Gln, Arg, Lys, His form hydrogen bonds with residues distal (. ^ 4) in sequence, while the shorter side- chains of Asp, Asn, Ser, Thr exhibit hydrogen bonds with residues close in sequence (, ^ 4), mainly involving backbone atoms. Experimentally we determined the thermodynamic propensities of residues at the Ccap position using the protein ubiquitin as a model system. We observed a large variation in the stability of the ubiquitin variants depending on the nature of the Ccap residue. Furthermore, the measured changes in stability of the ubiquitin variants correlate with the hydrophobicity of the Ccap residue. The experimental results, together with the statistical analysis of protein structures from the PDB, indicate that the key hydro- phobic capping interactions between a helical residue (C3 or C4) and a residue outside the helix (C 00 , C3 0 or C4 0 ) are frequently enhanced by the hydrophobic interactions with Ccap residues. q 2002 Elsevier Science Ltd. All rights reserved Keywords: helix termination; C-capping motifs; statistical analysis; hydrophobic interactions; differential scanning calorimetry *Corresponding author Introduction The a-helix is one of the basic motifs in protein secondary structure. It is characterized by consecu- tive main-chain hydrogen bonds between amide hydrogen and carbonyl oxygen of the previous helical turn. However, the first four amide hydro- gen bond donors and last four carbonyl oxygen hydrogen bond acceptors do not have hydrogen- bonding partners inside the helix. This leads to fraying of the helix ends. Statistical 1 and stereochemical 2 analysis of residues at the ends of the a-helix revealed the existence of specific capping interactions that compensate for the unsatisfied hydrogen bonds. The residues at the end of the helices appear to form additional inter- actions that include side-chain to main-chain hydrogen bonding at the N terminus and hydro- phobic interactions at the C terminus. 3–5 The importance of different capping interactions for the stability in a particular helical segment has been tested both in peptide 6–13 and protein 14 – 20 model systems. Initial theoretical observations were further extended and in 1998 Aurora & Rose 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved Present address: S. T. Thomas, Center for Biomolecular Analysis, Department of Biochemistry, Health Science Center at San Antonio, University of Texas, San Antonio, TX 78229, USA. E-mail address of the corresponding author: makhatadze@psu.edu Abbreviations used: PDB, Protein Data Bank; DSC, differential scanning calorimetry; SEHC, small exterior hydrophobic clusters. doi:10.1016/S0022-2836(02)00734-9 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 322, 123–135