Conformational Changes in Concanavalin A

234

Circular dichroism spectra have been obtained for albumin, alpha-chymotrypsinogen, collagen, concanavalin A, elastase, hemoglobin, histone f2b, alpha-lactalbumin, lactate dehydrogenase, beta-lactoglobulin, lysozyme, myoglobin, papain, ribonuclease A, and thermolysin in the presence of sodium dodecyl sulfate and dithiothreitol. While all spectra have the shape anticipated for a mixture of random coil and alpha helix, the intensities differ markedly ([theta]222 ranges from --1400 to --15 000 deg cm2/dmol). The variation in the circular dichroism can be quantitatively explained by a model which assumes that the arginyl, histidyl, and lysyl residues have an enhanced probability of propagating a helical segment in the presence of the detergent. The model also permits the computation of dimensional properties (unperturbed end-to-end distance and radius of gyration) for polypeptides of known amino acid sequence. Such computations have been performed for 67 proteins. The computed dimensions are compatible with experimental values and with the molecular weight dependence of the transport properties of the complexes. Furthermore, the model can account for the abnormal transport properties of the sodium dodecyl sulfate complexes formed by ribonuclease A, collagen fragments, and histones f2a1, f2a2, f2b, and f3. Even though some of the protein--sodium dodecyl sulfate complexes have helical contents as high as 50%, their overall conformation more closely approximates that of a random coil than a rod.

Section: Resultssupporting

confidence: 90%

Conformational properties of the complexes formed by proteins and sodium dodecyl sulfate

Mattice¹,

Riser²,

Clark³

1976

234

“…The CD spectra of Con A in the near-UV region in the absence and presence of the Glc and Gal derivatives of Man(3,6) at 23 °C are shown in Figure 3. As previously shown, binding of MeaMan increases the molar ellipticity values of the protein by about 17% (Bhattacharyya et al, 1991;Pflumm et al, 1971;Young et al, 1982). In addition, the band at 267 nm disappears, and there are small shifts in the bands at 270 and 273 nm.…”

Section: Resultssupporting

confidence: 56%

Studies of the Binding Specificity of Concanavalin A. Nature of the Extended Binding Site for Asparagine-Linked Carbohydrates

Mandal¹,

Bhattacharyya²,

Koenig³

et al. 1994

In the preceding paper [Mandal, D. K., Kishore, N., & Brewer, C. F. (1994) Biochemistry (preceding paper in this issue)] the trisaccharide 3,6-di-O-(alpha-D-mannopyranosyl)-D-mannose, which is present in all asparagine-linked carbohydrates, was shown by titration microcalorimetry to bind to the lectin concanavalin A (Con A) with nearly -6 kcal mol-1 greater enthalpy change (delta H) than methyl alpha-D-mannopyranoside (Me alpha Man). These results indicate that Con A possesses an extended binding site for the trisaccharide. In the present paper, we have investigated the binding of a series of synthetic analogs of the methyl alpha-anomer of the trisaccharide using hemagglutination inhibition, solvent proton magnetic relaxation dispersion (NMRD), near ultraviolet circular dichroism, and titration microcalorimetry measurements. Four of the analogs tested possess an alpha-glucosyl or alpha-galactosyl residue substituted at either the alpha(1-6) or alpha(1-3) position. Analysis of the data indicates that the alpha(1-6) residue of the parent trimannoside binds to the so-called monosaccharide site and the alpha(1-3) residue to a weaker secondary site. Binding at the secondary site involves unfavorable interactions of the 2-equatorial hydroxyl of the alpha(1-3) Glc derivative since this analog binds with 12-fold lower affinity and -3.4 kcal mol-1 lesser delta H than the trimannoside, whereas the alpha(1-3)-2-deoxyGlc analog possesses essentially the same affinity and delta H as the trimannoside.(ABSTRACT TRUNCATED AT 250 WORDS)

“…A useful compendium of the many diversified effects and uses of con A has recently been published (Chowdhury and Weiss, 1975). These properties of con A in vivo, the utility of the protein as a membrane probe, and the widespread use of con A as an analytical tool in the laboratory appear to be related to the ability of con A to form complexes with specific saccharides.Evidence for conformational changes in the equilibrium structure of con A has been presented (Becker et al, 1976;Pflumm et al, 1971) and kinetic observation of such a change could help clarify the effects of con A upon the membranes of cells. The mechanism of ligand binding and the values of the rate constants are important when considering the various biological effects of lectins and a comparison of the kinetic parameters of several lectins may reveal parallels between lectins possessing similar properties.…”

mentioning

confidence: 99%

“…Evidence for conformational changes in the equilibrium structure of con A has been presented (Becker et al, 1976;Pflumm et al, 1971) and kinetic observation of such a change could help clarify the effects of con A upon the membranes of cells. The mechanism of ligand binding and the values of the rate constants are important when considering the various biological effects of lectins and a comparison of the kinetic parameters of several lectins may reveal parallels between lectins possessing similar properties.…”

mentioning

confidence: 99%

Binding of 4-methylumbelliferyl α-D-mannopyranoside to dimeric concanavalin A: fluorescence temperature-jump relaxation study

Clegg¹,

Loontiens²,

Jovin³

1977

The kinetics of saccharide binding to the dimer form of concanavalin A (con A) has been studied at pH 5.5 with the fluorescence temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside, a fluorescent carbohydrate derivative which is quenched upon binding to con A, was used as the ligand. Three relaxation effects were seen. The major relaxation (r = 20-400 ms) was investigated at four different temperatures. The behaviour of this relaxation as a function of reactant concentrations is consistent with a simple one-step bimolecular association reaction. These conclusions result from the analysis of both the relaxation times and amplitudes, and from the comparison of the kinetically determined equilibrium parameters (Kass = 3.5 x 10(4) M-1 at 18.5 degrees C, delta H degrees = -(6-7) kcal/mol) to those obtained from a parallel series of equilibrium experiments (Loontiens, F.G., Clegg R.M., and Jovin, T.M. (1977), Biochemistry 16, preceding paper in this issue). The association and dissociation rate constants are in the range of (6-15) x 10(4) M-1 s-1 and (1.5 - 5.6) s-1, respectively, within a temperature range of 13.5-28.1 degrees C. The activation energies for the forward and reverse reactions are approximately 10 and approximately 15 kcal/mol, respectively. The two additional relaxations which are also present in the absence of saccharides result from changes in the protein fluorescence and are attributed to protein conformational changes which are not affected by the binding of saccharides. These effects were further studied using succinylated, acetylated, and demetallized con A. The faster relaxation (13 ms at 18.5 degrees C) was independent of the concentration of the protein and was not present in the derivatized con A samples. The two derivatized forms of con A show almost identical carbohydrate binding parameters as the underivatized protein. A limited series of stopped-flow experiments yielded results which were fully compatible with those from the relaxation measurements.