A NEW METHOD TO CHARACTERIZE HYDROPHOBIC ORGANIZATION OF PROTEINS - APPLICATION TO RATIONAL PROTEIN ENGINEERING OF BARNASE

We present a new algorithm for characterization of protein spatial str ucture basing on the molecular hydrophobicity potential approach. The method is illustrated by the analysis of three-dimensional structure o f barnase and barnase-barstar complex. Current approach enables identi fication of amino acid residues situated in unfavorable environment (t hese residues may be ''active'' for binding), and to map quantitativel y hydrophobic, hydrophilic and unfavorable hydrophobic-hydrophilic int ra-and inter-molecular contacts involving backbone and side-chain segm ents of amino acid residues. Calculation of individual contributions o f amino acid residues to such contacts permits identification of struc turally-important residues. The contact plots obtained with molecular hydrophobicity potential calculations, provide easy rules to choose si tes for mutations, which can increase a strength of intra- or inter-mo lecular hydrophobic interactions. The unfavorable hydrophobic-hydrophi lic contact can be mutated to favorable hydrophobic, and already exist ing weak hydrophobic contact can be strengthen by increasing hydrophob icity of residues in contact. Basing on the analysis of the contact pl ots, we suggest several mutations of barnase which are supposed to inc rease intramolecular hydrophobic interactions, and thus might lead to increased stability of the protein. Part of these mutations was studie d previously experimentally, and indeed stabilized barnase. The other of predicted mutations were not studied experimentally yet. Several ne w mutations of barnase and barstar are also proposed to enhance the hy drophobic interactions on their binding interface.