Dimerization of A82846B, vancomycin and ristocetin: influence on antibiotic complexation with cell wall model peptides.

The thermodynamics of glycopeptide antibiotic dimerization have been studied by means of sedimentation equilibrium, using A82846B, vancomycin, ristocetin and complexes formed with several cell wall model peptides. These results indicate that vancomycin dimerization can be strongly promoted in two ways: i) stabilization of the antibiotic conformation in which the carbonyl group of residue three is on the back face of the molecule and ii) preferential interaction of the dimer with the lysine residue of N,N'-diacetyl-lysyl-D-alanyl-D-alanine. This effect was not found in ristocetin. A82846B forms stable dimers at very low antibiotic concentration. Two conformational forms have been found for complexed A82846B by 1H NMR. However, calorimetric binding experiments have shown that all its binding sites are thermodynamically equivalent. The affinity of the A82846B dimer for the tripeptide has been estimated to be about 3kJ x mol-1 higher than that of the vancomycin monomer and about -2.6kJ x mol-1 lower than that of dimeric vancomycin. The possible role of dimerization in the biological activity of glycopeptide antibiotics is discussed further on the basis of present thermodynamic data.

Temperature-induced changes in protein structures studied by Fourier transform infrared spectroscopy and global analysis.

Fourier transform infrared (FTIR) spectroscopy has been used to study temperature-induced structural changes which occur in albumin, immunoglobulin G, fibrinogen, lysozyme, alpha-lactalbumin, and ribonuclease S when dissolved in 2H2O. In order to analyze the data, a new method was developed in which the data were analyzed globally with the aid of a spectral model. Seven or eight bands were sufficient to fit the full data set of spectra ranging from 1420 to 1760 cm-1 with a root mean square error of 1-2% of the maximum. Subsequently, the estimated band amplitude curves which showed a sigmoidal progression with increasing temperature were (globally) fitted with a two-state thermodynamic model. In this way, information on structural changes as well as on the thermal stability of the proteins was obtained. In all proteins investigated, enhanced 1H-2H exchange occurred at temperatures well below the unfolding of the secondary structure. This was interpreted as a change in tertiary structure leading to enhanced solvent accessibility. In all the proteins investigated, except for ribonuclease S, an intermolecular beta-sheet band indicative of aggregation appeared concomitant with the denaturation of the secondary structure. The results are compared with data from other techniques and discussed in terms of local unfolding and folding intermediates.

Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.

Cryoprotectant toxicity is a fundamental obstacle to the full potential of artificial cryoprotection, yet it remains in general a poorly understood phenomenon. Unfortunately, most relevant biochemical studies to date have not met the basic criteria required for demonstrating mechanisms of toxicity. A model biochemical study of cryoprotectant toxicity was that of Baxter and Lathe, which demonstrated that alteration of a specific enzyme (fructose diphosphatase, or FDPase) was the cause of impaired glycolysis after treatment with and removal of dimethyl sulfoxide (D). FDPase alteration by D was reported to be preventable by the simultaneous presence of amides. This protection could be due to a "counteracting solute" effect similar to that employed by nature, but we find no meaningful correlation between the general protein stabilizing or destabilizing tendency of the cryoprotectant medium and its toxicity. Baxter and Lathe postulated that the effect of D arises from hydrogen bonding between D and the epsilon amino groups of surface lysine residues on FDPase, and it was found that molecules which resembled this group could block the alteration induced by D, presumably by competing with lysine residues for association with D. However, we find that the interaction between D and lysine in the presence of water is actually thermochemically repulsive, and that the presence of formamide does not affect the interaction between D and lysine, implying no useful complex formation between formamide and D. We were also unable to demonstrate that the blocking compounds consistently reduce toxicity when added to D rather than substituting for D, contrary to predictions based on complex formation between blocking compounds and D. In summary, it seems that present concepts of cryoprotectant toxicity are in need of serious revision.