Soft–Soft Interactions in the Protein–Protein Recognition Process: The K 1 Channel-Charybdotoxin Case Felipe Aparicio, [a] Nelly Gonz alez-Rivas, [b] Joel Ireta, [c] Arturo Rojo, [a] Laura I. Escobar, [d] Andr es Cedillo, [c] and Marcelo Galv an* [c] Molecular recognition between peptide blockers and ionic channels is a complex process that involves many effects. To determine if the short-range charge transfer effects play a significant role in this interaction, a chemical reactivity analysis of charybdotoxin (ChTX) and six of its mutants was carried out using global and local reactivity indices. The results show that global softness correlates with the affinity of ChTX, and its mutants to the channel indicating that soft–soft interactions play a role in the recognition process between ChTX and a potassium channel. The analysis of the local reactivity indicates that the toxin as a whole can be seen as a complex polydentate ligand with several places to coordinate with the external vestibule of the pore of the potassium channels. The successful treatment of point mutations supports the idea of using this tool in the study of chemical reactivity in proteins, in a similar way as substituent effects in organic chemistry. V C 2012 Wiley Periodicals, Inc. DOI: 10.1002/qua.24278 Introduction The protein–protein recognition process is complex and involves many effects. The most common are the steric and electrostatic interactions, hydrogen bond formation, and hydrophobicity. [1] In some cases, the information about key residues involved in the recognition process is available from site directed mutagenesis experiments. In a more restricted number of cases, structural data of the interacting proteins are at hand. For some of the later cases, it may be even possible to pursue computational studies at an atomistic level including the evaluation of the electronic structure of such proteins. In such context, this work establishes a structure–function rela- tionship between short-range charge transfer effects and kinetic constants for the dissociation process in the interaction of a peptide type toxin and a potassium channel. To reach this goal, the electronic structure of charybdotoxin (ChTX) [2] and six of its mutants was obtained; and the reactivity analysis was performed using local and global density functional theory (DFT) concepts that have been widely used to rationalize the chemical behavior of small molecules. [3] In small molecules, the analysis is performed by changing atoms or functional groups, however, the experimental information available for ChTX is obtained by site directed mutagenesis, that is, the exchange of an amino acid for a different one at a particular position in the peptide chain. Consequently, it is desirable to identify global and local changes in the electronic structure of the peptide induced by such mutations and correlate them to changes in the reactivity of the protein. The present work tests if such analysis is feasible because that could open a new pro- cedure for the chemical reactivity analysis in proteins. Scorpion toxins inhibit ion conduction through potassium channels by occluding the pore at the extracellular vestibule. The stoichiometry of the process indicates that one toxin mol- ecule binds to a single channel. [4] These toxins comprise pep- tides of 35–40 amino acids in length, are rigid structures held by three disulfide bridges, [2] and according to their amino acid sequence homology, they have been classified in 18 sub- families. [5] Electrophysiological or binding experiments have shown that the affinity of these peptides toward a variety of potassium channels varies from picomolar to micromolar range. [6] Before the determination of the first high-resolution crystal structure of the pore of a potassium channel, [7] mutagenesis studies involving the toxin and the channel were the experi- mental approaches applied to get a qualitative description of the external surface of the vestibule of a K þ channel pore. [8–11] Several residues in the toxins were found to be crucial for bind- ing to a potassium channel. [8,12] One conserved lysine residue located in the b-strand of all of these toxins interacts directly with a tyrosine residue in the selectivity filter of the ‘Shaker ’-K þ channel. [11] Based on the observation that several channel-spe- cific toxins, with unrelated folds and different origins, possess a common dyad component of a lysine and an aromatic residue, [a] F. Aparicio, A. Rojo Departamento de Ciencias Naturales, Divisi  on de Ciencias Naturales e Ingenierı´a, Universidad Aut  onoma Metropolitana Cuajimalpa, Av. Pedro Antonio de los Santos 84, San Miguel Chapultepec, M exico D.F. 11850, M exico [b] N. Gonz  alez-Rivas Centro Conjunto de Investigaci  on en Quı´mica Sustentable UAEM-UNAM, Carretera Toluca-Atlacomulco Km 14.5, Unidad San Cayetano, Toluca, Estado de M exico, C. P. 50200, M exico [c] J. Ireta, A. Cedillo, M. Galv  an Departamento de Quı´mica, Divisi  on de Ciencias B asicas en Ingenierı´a, Universidad Aut  onoma Metropolitana-Iztapalapa, A.P. 55-534, M exico D.F. 09340, M exico [d] L. I. Escobar Departamento de Fisiologı´a, Facultad de Medicina, Universidad Nacional Aut  onoma de M exico, M exico D.F., M exico Fax: (þ52 55) 58 04 64 15 E-mail: mgalvan@xanum.uam.mx Contract grant sponsor: CONACYT; contract grant numbers: 155698, 41348-F, 41365, 60799 and 105532. V C 2012 Wiley Periodicals, Inc. 3618 International Journal of Quantum Chemistry 2012, 112, 3618–3623 WWW.CHEMISTRYVIEWS.ORG FULL PAPER WWW.Q-CHEM.ORG