SUBUNIT ASSOCIATION AND STRUCTURAL-ANALYSIS OF PLATELET BASIC-PROTEINAND RELATED PROTEINS INVESTIGATED BY H-1-NMR SPECTROSCOPY AND CIRCULAR-DICHROISM

Platelet basic protein (PBP) (94 residues) is naturally processed via N-terminal cleavage to yield connective tissue activating peptide-III (85 residues), beta-thrombo globulin (81 residues), and neutrophil act ivating peptide-2 (70 residues). Chemical cross-linking and gel filtra tion data indicate that each homolog can form dimers and tetramers. Su bunit association equilibria for dimer (K-D) and tetramer (K-T) format ion have been derived for each species from H-1 NMR (600 MHz) spectral analysis of slowly exchanging (NMR time scale) monomer- dimer-tetrame r aggregation state populations. In general, raising the pH from about pH 3.5 to pH 6 increases K-D by two to three orders in magnitude and decreases K-T by some 50-fold. Ionic strength effects also suggest tha t intersubunit electrostatic interactions are critical to subunit asso ciation. Subunit stabilization can be ranked proportional to N-termina l chain length: platelet basic protein > connective tissue activating peptide-III > beta-thromboglobulin > neutrophil activating peptide-2. Under more physiologic conditions, PBP family monomers are favored at normal cytokine protein concentrations and may form the biologically a ctive state. CD and NMR data indicate conservation of alpha-helix and anti-parallel beta-sheet structure among PBP-related species and suppo rt the idea that the extended N terminus folds over and masks the neut rophil activation domain and is part of the intersubunit binding domai n.