Protein self-association in solution: the bovine pancreatic trypsin inhibitor decamer.
We have used magnetic relaxation dispersion to study bovine pancreatic trypsin inhibitor (BPTI) self-association as a function of pH, salt type and concentration, and temperature. The magnetic relaxation dispersion method sensitively detects stable oligomers without being affected by other interactions. We find that BPTI decamers form cooperatively under a wide range of solution conditions with no sign of dimers or other small oligomers. Decamer formation is opposed by electrostatic repulsion among numerous cationic residues confined within a narrow channel. Accordingly, the decamer population increases with increasing pH, as cationic residues are deprotonated, and with increasing salt concentration. The salt effect cannot be described in terms of Debye screening, but involves the ion-specific sequestering of anions within the narrow channel. The lifetime of the BPTI decamer is 101 +/- 4 min at 27 degrees C. We propose that the BPTI decamer, with a heparin chain threading the decamer channel, plays a functional role in the mast cell. We also detect a higher oligomer that appears to be a subcritical nucleation cluster of 3-5 decamers. We argue that monomeric crystals form at high pH despite a high decamer population in solution, because the ion pairs that provide the critical decamer-decamer contacts are disrupted at high pH.
Gottschalk
Michael
M
Department of Biophysical Chemistry, Lund University, SE-22100 Lund, Sweden.
Venu
Kandadai
K
Halle
Bertil
B
D000818:Animals; D007611:Aprotinin / Q000737:chemistry*; D001665:Binding Sites; D002417:Cattle; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry; D012492:Salts / Q000737:chemistry*; D012996:Solutions; D013696:Temperature
D046911:Macromolecular Substances; D011506:Proteins; D012492:Salts; D012996:Solutions; D007611:Aprotinin