Conformation and dynamics of dimeric ureido-balhimycin

The conformation of the antibiotic ureido-balhimycin has been investigated by proton nmr and refined with computer simulations. The dimeric structure cj ureido-balhimycin was established by unambiguous identification of 6 nuclear Overhauser effects (NOEs) as intermonomeric, out of the total of 186 NOEs observed. Via distance geometry calculations the antiparallel orientation of the two monomers was demonstrated. Further refinement by molecular dynamics simulations provided the essential hydrogen bonds and aromatic interactions responsible for interfacial stabilization. The computational protocol illustrated here (distance geometry to define the coarse topology and molecular dynamics for refinement) should find general applicability in the study of homodimers. © 1995 John Wiley & Sons, Inc.     

Gas-phase IR spectra of substituted alkylamides

The source of the splittings observed in the amide A, I, II, and III absorption bands of N-methylacetamide (NMA) have been determined by high-resolution gas-phase FTIR. Analysis of isotopically labeled NMA, along with simpler amides (formamide, acetamide) show that the observed splitting arise from rotational transitions within the vibrational manifold. The ir spectra of a homologous series of monosubstituted alkylamides are also examined. The observed effects on the ir spectra from incorporating substituents from methyl to t-butyl on both sides of the amide linkage are discussed in terms of rotational transitions and coupled oscillations. During these studies we did not observe any evidence for the presence of cis conformations of the amide bond.     

Assembly and Turnover of Short Actin Filaments by the Formin INF2 and Profilin

INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2.     

Structure of two microcystins: Refinement with nuclear overhauser effects and ensemble calculations

Ensemble calculations employing restraints developed from ¹H-nmr were used to examine the conformational states of the two microcystins LR and LY. Despite the fast “flip-flop” dynamics about the N-methyl-dehydroalanine residue and adjacent residues, the main conformational characteristics of the cyclic heptapeptides consist of a compact ring formed by five of the seven amino acid residues with expulsion of a dipeptide portion out of the plane and the unnatural C20 β-amino acid (2S. 3S. 8S, 9S)-3-amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4. 6-dienoic acid pointing upward from the ring. This structure of microcystin LR shows high degrees of similarity with the energy-minimized structure of nodularin, a cyclic pentapeptide of identical inhibitory potency against protein phosphatases 1 and 2A. Comparison of these structures with those of the less toxic LY variant and with the structurally unrelated okadaic acid, known as potent inhibitor of the protein phosphatases 1 and 2A, allowed us to propose a rational binding mode of this structural diverse set of natural inhibitors. © 1995 John Wiley & Sons, Inc.     

14-membered cyclic opioids related to dermorphin and their partially retro-inverso modified analogues. I. Synthesis and biological activity.

As a continuation of our program to study structure-activity relationships of opiate peptides, we report the syntheses and biological activities of a series of 14-membered cyclic dermorphin analogues closely related to enkephalin analogue Tyr-c[D-A2bu-Gly-Phe-Leu] incorporating a phenylalanine at the third position in place of glycine. In addition to two parent dermorphin analogues Tyr-c[D-A2bu-Phe-Phe-(L and D)-Leu], four stereoisomeric retro-inverso modified analogues Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu] with a reversed amide bond between residues four and five, and Tyr-c[D-Glu-Phe-gPhe-(L and D)-rLeu] with two reversed amide bonds between residues four and five, and between residue five and the side chain of residue two have been synthesized. The results from the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays show that all analogues are superactive at either one or both opiate receptors and in general display higher activities as compared to the corresponding enkephalin analogues with a glycine at the third position. Results from the in vitro biological assays and conformational analysis using 1H-NMR spectroscopy (adjoining paper) will provide useful information to understand the role of the Phe3 aromatic side chain in dermorphin, and that of the Phe4 aromatic side chain in enkephalin, on opiate activity since these cyclic dermorphin analogues contain two Phe residues at both the third and fourth positions.