Showing papers by "Stephen W. Fesik published in 1990"

Heteronuclear three-dimensional NMR spectroscopy of isotopically labelled biological macromolecules.

Abstract: Due to the development of two-dimensional Fourier transformation techniques (for reviews see Bax, 1982; Ernst et al. 1987), NMR spectroscopy has become a powerful tool for determining the 3D structures of small proteins (MW ≤ 10 kDa); for reviews see Wuthrich, 1986; Clore & Gronenborn, 1987. For larger molecules, however, the amount of detailed structural information that can be obtained using homonuclear 2D NMR techniques is limited because of the vast number of overlapping signals. In order to extend the capabilities of NMR to the study of larger systems, new approaches are required.

Isotope-edited NMR of cyclosporin A bound to cyclophilin: evidence for a trans 9,10 amide bond

Abstract: The binding of a 13C-labeled cyclosporin A (CsA) analog to cyclophilin (peptidyl prolyl isomerase) was examined by means of isotope-edited nuclear magnetic resonance (NMR) techniques. A trans 9,10 peptide bond was adopted when CsA was bound to cyclophilin, in contrast to the cis 9,10 peptide bond found in the crystalline and solution conformations of CsA. Furthermore, nuclear Overhauser effects (NOEs) were observed between the zeta 3 and epsilon 3 protons of the methylleucine (MeLeu) residue at position 9 of CsA and tryptophan121 (Trp121) and phenylalanine (Phe) protons of cyclophilin, suggesting that the MeLeu9 residue of CsA interacts with cyclophilin. These results illustrate the power of isotope-edited NMR techniques for rapidly providing useful information about the conformations and active site environment of inhibitors bound to their target enzymes.

Role of protonated and neutral forms of macrolides in binding to ribosomes from gram-positive and gram-negative bacteria.

Abstract: Erythromycin binds to a single site on the bacterial 50S ribosomal subunit and perturbs protein synthesis. However, erythromycin contains desosamine and thus exists in both protonated (greater than 96%) and neutral (less than 4%) forms at physiological pH because of the pKa of the dimethylamino group. We therefore examined the relative roles of both forms in binding to ribosomes isolated from two species each of gram-positive and gram-negative bacteria. We developed a system to directly measure the forward (association) rate constant of formation of the macrolide-ribosome complex, and we have measured both the forward and reverse (dissociation) rate constants as a function of pH. Forward rate constants and binding affinity did not correlate with pH when the interaction of erythromycin with ribosomes from both gram-positive and gram-negative bacteria was examined, demonstrating that the protonated form of this macrolide binds to ribosomes. Conversely, the neutral form of macrolide cannot be the sole binding species and appears to bind with the same kinetics as the protonated form. Forward rate constants were 3- to 4-fold greater at physiological pH, and binding affinity calculated from rate constants was 5- to 10-fold greater than previously estimated. Similar results were obtained with azithromycin, a novel 15-membered macrolide that contains an additional tertiary amine in the macrolide ring. Ribosome- and macrolide-specific kinetic parameters were demonstrated at neutral pH and may be related to the potency of the two macrolides against gram-positive and gram-negative bacteria.