Hydrophobic cluster analysis: procedures to derive structural and functional information from 2-D-representation of protein sequences

Hydrophobic cluster analysis (HCA) 15] is a very efficient method to analyse and compare protein sequences. Despite its effectiveness, this method is not widely used because it relies in part on the experience and training of the user. In this article, detailed guidelines as to the use of HCA are presented and include discussions on: the definition of the hydrophobic clusters and their relationships with secondary and tertiary structures; the length of the clusters; the amino acid classification used for HCA; the HCA plot programs; and the working strategies. Various procedures for the analysis of a single sequence are presented: structural segmentation, structural domains and secondary structure evaluation. Like most sequence analysis methods, HCA is more efficient when several homologous sequences are compared. Procedures for the detection and alignment of distantly related proteins by HCA are described through several published examples along with 2 previously unreported cases: the beta-glucosidase from Ruminococcus albus is clearly related to the beta-glucosidases from Clostridum thermocellum and Hansenula anomala although they display a reverse organization of their constitutive domains; the alignment of the sequence of human GTPase activating protein with that of the Crk oncogene is presented. Finally, the pertinence of HCA in the identification of important residues for structure/function as well as in the preparation of homology modelling is discussed.