Subunit association and structural analysis of platelet basic protein and related proteins investigated by 1H 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-thromboglobulin (81 residues), and neutrophil activating peptide-2 (70 residues). Chemical cross-linking and gel filtration data indicate that each homolog can form dimers and tetramers. Subunit association equilibria for dimer (KD) and tetramer (KT) formation have been derived for each species from 1H NMR (600 MHz) spectral analysis of slowly exchanging (NMR time scale) monomer- dimer-tetramer aggregation state populations. In general, raising the pH from about pH 3.5 to pH 6 increases KD by two to three orders in magnitude and decreases KT by some 50-fold. Ionic strength effects also suggest that intersubunit electrostatic interactions are critical to subunit association. Subunit stabilization can be ranked proportional to N-terminal 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 active state. CD and NMR data indicate conservation of alpha-helix and anti-parallel beta-sheet structure among PBP-related species and support the idea that the extended N terminus folds over and masks the neutrophil activation domain and is part of the intersubunit binding domain.
Yang
Y
Y
Department of Biochemistry, University of Minnesota Health Sciences Center, Minneapolis 55455.
Mayo
K H
KH
Daly
T J
TJ
Barry
J K
JK
La Rosa
G J
GJ
D000595:Amino Acid Sequence; D018925:Chemokines; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D001620:beta-Thromboglobulin
D018925:Chemokines; C061615:PPBP protein, human; D011506:Proteins; D001620:beta-Thromboglobulin