Structural interpretation of lanthanide binding to the basic pancreatic trypsin inhibitor by 1H NMR at 360 MHz.
The weak binding of lanthanides to the five carboxyl groups of the basic pancreatic trypsin inhibitor (hereafter termed "the inhibitor"), has been investigated in detail using high resolution 1H NMR at 360 MHz. Lanthanides bind to the C-terminus with an apparent binding constant of 30 M-1, and thus competitively inhibit the formation of a salt-bridge between the C-terminus and the N-terminus, Lanthanides bind also to the side chain carboxyl groups of Asp 3, Glu 7, Glu 49 and Asp 50, with binding constants of 10--30 M-1. With the use of lanthanides individual resonance assignments for Phe 4 and Phe 45 were obtained in the 1H NMR spectrum of the inhibitor, and for several spin systems previous  identifications were independently confirmed. The present experiments also provide a nice illustration for the use of shift reagents to improve the resolution in 1H NMR spectra of proteins. The exchange broadening for Tyr 35 and Phe 45 over the temperature range 4--72 degrees C could thus be observed for almost all the components of these aromatic spin systems and new details on the dynamic properties were obtained also for other aromatic residues.
Structural interpretation of lanthanide binding to the basic pancreatic trypsin inhibitor by 1H NMR at 360 MHz. The weak binding of lanthanides to the five carboxyl groups of the basic pancreatic trypsin inhibitor (hereafter termed "the inhibitor"), has been investigated in detail using high resolution 1H NMR at 360 MHz. Lanthanides bind to the C-terminus with an apparent binding constant of 30 M-1, and thus competitively inhibit the formation of a salt-bridge between the C-terminus and the N-terminus, Lanthanides bind also to the side chain carboxyl groups of Asp 3, Glu 7, Glu 49 and Asp 50, with binding constants of 10--30 M-1. With the use of lanthanides individual resonance assignments for Phe 4 and Phe 45 were obtained in the 1H NMR spectrum of the inhibitor, and for several spin systems previous  identifications were independently confirmed. The present experiments also provide a nice illustration for the use of shift reagents to improve the resolution in 1H NMR spectra of proteins. The exchange broadening for Tyr 35 and Phe 45 over the temperature range 4--72 degrees C could thus be observed for almost all the components of these aromatic spin systems and new details on the dynamic properties were obtained also for other aromatic residues.