Abstract
Visible absorption, circular dichroism (CD) and magnetic circular dichroism spectra have been recorded for the Ca2+-Co2+ derivatives of the lentil (CCoLcH) and pea (CCoPSA) lectins (Co2+ at the S1 sites and Ca2+ at the S2 sites) and shown to be very similar for both proteins. The visible absorption and magnetic circular dichroism spectra indicate similar octahedral geometries for high spin Co2+ at S1 in both proteins, as found in the Ca2+-Co2+ complex of concanavalin A (CCoPL) (Richardson, C. E., and Behnke, W. D. (1976) J. Mol. Biol. 102, 441-451). The visible CD data, however, indicate differences in the environment around S1 of CCoLcH and CCoPSA compared to CCoPL. 1H NMR spectra at 90 MHz of the Co2+ and Ni2+ derivatives of the lectins show a number of isotropically shifted signals which arise from protons in the immediate vicinity of the S1 sites. Analysis of the spectra of the Co2+ derivatives in H2O and D2O has permitted resonance assignments of the side chain ring protons of the coordinated histidine at S1 in the lectins. Differences are observed in the H-D exchange rate of the histidine NH proton at S1 in concanavalin A compared to the lentil and pea lectins. NMR data of the Ni2+-substituted proteins, together with spectra of the Co2+ derivatives, also indicate that the side chains of a carboxylate ligand and of the histidine residue at S1 are positioned differently in concanavalin A than in the other two lectins. These results appear to account, in part, for the differences observed in the visible CD spectra of the Co2+-substituted proteins. In addition, binding of monosaccharides does not significantly perturb the spectra of the lectins. An unusual feature in the 1H NMR spectra of all three Co2+-substituted lectins is the presence of two exchangeable downfield shifted resonances which appear to be associated with the two protons of a slowly exchanging water molecule coordinated to the Ca2+ ion at S2. T1 measurements of CCoLcH have provided an estimation of the distances from the Co2+ ion to these two protons of 3.7 and 4.0 A.
Highlights
The visible absorptionand magnetic circular dichroism spectra for CCoLcH and CCoPSA are very similar for both lectins and similar to those of eo2+ complexes of ConA (18, 19)
'H NMR spectra of the Coz+and Ni2+derivatives of the three lectins show a number of isotropically shifted signals which arise from protons in the immediate vicinity of the metal ion at S1
These results may account, in part, for the differences observed in the visible CD spectra of the lectins and themetal ion exchange properties of the proteins (11).In addition, all three Co2+-substituted lectins possess two un
Summary
An unusual feature in the ‘HNMR spectra of all ConAby Mn2+and Ca2+has been extensively investigated. With HZ0 as solvent, a number ofwell resolved isotropically (paramagnetically) shifted signals are apparent in the spectra in the 100 to -40 ppm range, well outside the diamagnetic region of the spectrum These resonances are clearly observable at relatively low magnetic fields (90 MHz), whereas experiments performed at 200 MHz showmuch broader resonances over this region. The spectra of the three Co2+-substituted lectins in D,O show evidenceof two verybroad signals in the downfield region (Fig. 3) These signals are approximately centered at 87 and 51 ppm in CCoLcH, 81 and 42 ppm in CCoPSA, and 77 and 35 ppm in CCoPL. These two resonances in CCoPL appear to be narrower than the corresponding pair in the other two lectins. The two broad signals in the spectra of all three proteins appear shifted differently
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