No abstract

Dynorphin A (1-17) (dynorphin) acts preferentially and with high affinity at the kappa-opioid receptor, for which it is the natural, endogenous ligand. Interest in designing new ligands to interact at the kappa-opioid receptor is based in part on the desire to circumvent some of the problems associated with mu-opioid ligands such as morphine. The high-resolution structure of dynorphin in an environment which closely resembles its environment in vivo could be considered as an important lead for new drugs. The interactions that occur between dynorphin and a model membrane are potentially important, as peptide hormone activity is thought to be mediated by interactions with the cell membrane. Therefore, we have determined the high-resolution structures of dynorphin in a model membrane. Results from our laboratory have shown the existence of an alpha-helical region in dynorphin from residues Gly3 through Arg9 when bound to perdeuterated dodecylphosphocholine (DPC) micelles. In this report we show that dynorphin is bound to DPC micelles and describe a family of dynorphin structures that is alpha-helical from residues Gly3 through Pro10 and that contains a beta-turn from residues Trp14 through Gln17. A model of interaction with the micelle is also reported and is discussed in the context of hormone action in vivo. The structures were determined with 1D and 2D nuclear magnetic resonance spectroscopy, distance geometry in dihedral angle space, and restrained molecular dynamics simulations.

The compound c[Cys5,11]dynorphin A-(1-11)-NH2, 1, is a cyclic dynorphin A analog that shows similar selectivity and potency at the kappa-opioid receptor when compared to the native form of the peptide in central nervous system assays. Previous molecular mechanics calculations have shown that the ring portion of the isoform that is trans about the Arg9-Pro10 omega bond contains either a beta-turn from residues Arg6 to Arg9 or an alpha-helical conformation. Our results from solution state NMR indicate that the compound exhibits cis-trans isomerism about the Arg9-Pro10 omega bond in both aqueous solution and when bound to dodecylphosphocholine micelles. Restrained molecular dynamics calculations show that the cis isoform of the peptide contains a type III beta-turn from residues Arg7 to Pro10. Similar calculations on the trans isoform show it to contain a beta-turn from residues Cys5 and Arg8. In this report we describe the generation of three-dimensional models from NMR data for the ring portions of both the cis and trans isoforms of 1 bound to dodecylphosphocholine micelles. Comparison with other dynorphin A structural information indicates that both the cis and trans isoforms of the peptide may be active as kappa-opioid agonists.

No abstract

No abstract

The acceptor stem domain of tRNA Ala contains the major recognition elements for aminoacylation by Escherichia coli alanine-tRNA synthetase (AlaRS). Previous studies established that a simple base pair transversion (G1:C72 f C1:G72) completely abolishes in vitro aminoacylation with alanine of duplex Ala substrates. Subsequent atomic group "mutagenesis" experiments, wherein over 30 base pair variants were introduced at this site, revealed that the presence of a major groove carbonyl oxygen at position 72 functions as a negative determinant. Although, these previous biochemical studies strongly suggested the critical nature of a blocking element at this site, they could not rule out the possibility that conformational changes were responsible for the observed effects on aminoacylation. The mechanism of discrimination at the first base pair is further examined here. In particular, to establish whether conformational change contributes to the striking difference in aminoacylation activity, NMR spectroscopy and molecular dynamics (MD) simulations were carried out. The solution NMR assignments indicate that the global conformations of the wild-type microhelix versus the C1:G72-containing variant are very similar. This is consistent with the predicted structures based on MD simulations; on the basis of average structures and dynamical analysis of trajectories, there are no statistically significant differences between the variant and the wild-type microhelices with respect to either helical structure or A73 stacking interactions. Thus, AlaRS is indeed sensitive to specific atomic groups in the inactive C1:G72 variant rather than to conformational changes associated with this base pair transversion.

Fluorescence spectroscopy has been used to examine the interaction between the opioid peptide dynorphin A(1‐17) (dynorphin) and dodecylphosphocholine (DPC) micelles. Fluorescence emission spectra as a function of added lipid indicate insertion of the Trp14 side chain into the hydrophobic portion of the micelle, supporting NMR results from this laboratory. A model of interaction with micelles consistent with the fluorescence results and earlier NMR results is proposed. The critical micelle concentration in the presence of peptide was also determined, and is discussed in the context of relevance to both NMR spectroscopy and peptide‐lipid interactions. © Munksgaard 1997.