Circular dichroism and 1H NMR studies of Co2+- and Ni2+-substituted concanavalin A and the lentil and pea lectins.
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.
Bertini
I
I
Department of Chemistry, University of Florence, Italy.
Viezzoli
M S
MS
Luchinat
C
C
Stafford
E
E
Cardin
A D
AD
Behnke
W D
WD
Bhattacharyya
L
L
Brewer
C F
CF
D019325:3-O-Methylglucose; D002118:Calcium; D002942:Circular Dichroism; D003035:Cobalt; D003208:Concanavalin A; D037102:Lectins; D009682:Magnetic Resonance Spectroscopy; D008757:Methylglucosides; D009532:Nickel; D037121:Plant Lectins; D013696:Temperature
D037102:Lectins; D008757:Methylglucosides; D037121:Plant Lectins; C033619:lentil lectin; C036292:pea lectin; D003208:Concanavalin A; D019325:3-O-Methylglucose; D003035:Cobalt; D009532:Nickel; D002118:Calcium