Characterization of the low-field proton magnetic resonance spectrum of plasminogen kringle 4 via selective Overhauser experiments in 1H2O.
The low-field 1H NMR spectrum of the kringle 4 domain of human plasminogen has been investigated at 300 and 600 MHz for the protein dissolved in 1H2O. The spectrum exhibits six well-resolved resonances, spanning the 9.8 approximately less than delta approximately less than 13 ppm chemical shift range, which arise from exchange-labile H atoms. The acid-base response of the six resonances was monitored in order to characterize the signals in terms of their pH titration profiles. The sensitivity of the low-field resonances to kringle binding the antifibrinolytic ligands N alpha-acetyl-L-lysine and p-benzylaminesulfonic acid was also investigated. The lowest field resonance, at 12.6 ppm, is a doublet of J approximately 7.9 Hz, a splitting that is unprecedented for His or Trp ring NH signals. Selective Overhauser experiments centered on the exchangeable proton transitions identify four of the other resonances as stemming from the His31, His33, Trp I, and Trp II side-chain NH groups, where the latter two are, as yet, not definitely assigned to the specific residues, Trp25 and Trp62. The relative narrowness of the His imidazole NH signals indicates that the two rings are sterically shielded from direct water accessibility. In particular, the His33 NH site appears to be the most protected. The Overhauser evidence conclusively shows that the two identified exchangeable His ring proton signals arise from imidazole NH3 sites rather than from the NH1 tautomers. Similarly, these experiments lead to an unambigous characterization of the corresponding Trp aromatic CH spin systems.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010446:Peptide Fragments; D010958:Plasminogen; D011487:Protein Conformation
D010446:Peptide Fragments; D010958:Plasminogen