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.     

Main structural and functional features of the basic cytosolic bovine 21 kDa protein delineated through hydrophobic cluster analysis and molecular modelling.

A 21 kDa protein purified from bovine brain cytosol was previously described as a hydrophobic ligand binding protein; however, its accurate biological function remained still uncertain. In order to get further information about its potential biological role, an extended prediction of its secondary and three dimensional structures was undertaken. We describe here a process which permitted us to discover a structural homology between the 21 kDa protein and the N-domain of yeast phosphoglycerate kinase (PGK). This process is based on comparing the 21 kDa protein with all the proteins presenting a slight homology, by using the Hydrophobic Cluster Analysis (HCA) method. According to the observed similarity between the N-domain of yeast PGK and the 21 kDa protein, we built a model which was shown to possess a potential binding site for nucleotides. Moreover, the model obtained presents three-dimensional (3D) structure similarity with adenylate kinase. These results suggest two main hypotheses: (i) the 21 kDa protein may belong to the kinase family; (ii) the binding of a nucleotide could imply a modification of the 3D structure of the 21 kDa protein that can promote the transfer of hydrophobic ligands to the plasma membrane. Meanwhile, verification of these hypotheses has been in part performed experimentally: the 21 kDa protein binds MgATP as well as, to a lesser extent, phosphoglycerate.     

MANSEK and SUNHCA. Two interactive programs for the hydrophobic cluster analysis of protein sequences

Hydrophobic cluster analysis (HCA) is an efficient method foranalysing and comparing the amino acid sequences of proteins.It relies on two–dimensional representations of the sequencespresently generated by simple plot programs working on microcomputers.Two interactive programs, MANSEK. and SUNHCA, are describedhere that operate from Vax and Sun workstations respectively.These programs allow the display of several protein sequencesin the form of two–dimensional helical plots suitablefor HCA. Several tedious, repetitive and timeconsuming stepsof HCA have been suppressed by implementing several featuressuch as interactive on–screen manipulations (zoom, translations)of the plots and HCA score calculations on segments chosen bythe user. Plots on paper can be obtained through hard copiesor plotting subroutines.