Enkephalin structure determination

Fig. 2.11. Three-dimensional structure of Leu-enkephalin crystal determined by the REDOR experiment [27].

Perhaps the most striking example of this upsurge of interest has been in the area of pain research—in its genesis, expression, and, of greatest importance, its intrinsic and extrinsic control. These studies were placed on a molecular footing by the isolation and structure determination of the enkephalins (Hughes et al., 1975b)—two related peptides found in the brain with potent opiate activity—and the concomitant observation that the amino acid sequence of one of these species (met-enkephalin) was present in the protein 3-lipotropin. This led to the discovery of other... 

In the solid, dynamics occurring within the kHz frequency scale can be examined by line-shape analysis of 2H or 13C (or 15N) NMR spectra by respective quadrupolar and CSA interactions, isotropic peaks16,59-62 or dipolar couplings based on dipolar chemical shift correlation experiments.63-65 In the former, tyrosine or phenylalanine dynamics of Leu-enkephalin are examined at frequencies of 103-104 Hz by 2H NMR of deuterated samples and at 1.3 x 102 Hz by 13C CPMAS, respectively.60-62 In the latter, dipolar interactions between the 1H-1H and 1H-13C (or 3H-15N) pairs are determined by a 2D-MAS SLF technique such as wide-line separation (WISE)63 and dipolar chemical shift separation (DIP-SHIFT)64,65 or Lee-Goldburg CP (LGCP) NMR,66 respectively. In the WISE experiment, the XH wide-line spectrum of the blend polymers consists of a rather featureless superposition of components with different dipolar widths which can be separated in the second frequency dimension and related to structural units according to their 13C chemical shifts.63... 

Sequence Determination of the Brain Peptide Leucine Enkephalin A group of peptides that influence nerve transmission in certain parts of the brain has been isolated from normal brain tissue. These peptides are known as opioids, because they bind to specific receptors that also bind opiate drugs, such as morphine and naloxone. Opioids thus mimic some of the properties of opiates. Some researchers consider these peptides to be the brain s own pain killers. Using the information below, determine the amino acid sequence of the opioid leucine enkephalin. Explain how your structure is consistent with each piece of information. 

It has been suggested that y-turns are present in the solution structures of several peptides, and furthermore implicated in their bioactive conformations 101 including brady-kinin, 111 substance P,1121 cyclic somatostatin analogues, 131 cyclolinopeptide, 141 and the 6-opioid receptor bound conformation of enkephalin. 151 Yet, despite the fact that y-tums are frequently hypothesized to represent important features of secondary structure 161 based upon computational,1171 IR absorption,1181 NMR spectroscopic,119 201 and X-ray diffraction crystallographic determinations,1211 verification of the role of this predicted secondary structural element remains a difficult, but nonetheless critical step. 

Conformational analyses of JOM-13 and [L-Ala3]DPDPE have proven to be critical for the determination of the bioactive conformation of enkephalin-like peptides at the delta receptor. H-NMR studies of JOM-13 in aqueous solution revealed that this tetrapeptide exists in two distinct conformations on the NMR time scale as evidenced by two sets of resonances [63]. Large differences in the observed chemical shifts and coupling constants for the D-Cys2 residue in the two conformers suggested that the major differences between the two NMR conformers reside in the disulfide portion of the molecule however, a paucity of conformationally informative nuclear Overhauser enhancement (NOE) interactions precluded the development of a detailed structural model from the NMR studies. In order to develop such a model a thorough conformational analysis of JOM-13 was undertaken, in which the NMR data were complemented by x-ray diffraction results and by molecular mechanics calculations [64]. The results indicate that the 11-... 

It is demonstrated that polymorphs of the crystalline biologically-active peptides, Leu- and Met-enkephalins, are also easily distinguished by NMR on the basis of displacements of chemical shifts [80, 81]. Recently, we showed that high resolution solid-state NMR approach is a very useful mean to delineate local conformational change from a-helix to j8-sheet associated with fibril formation of selectively C-labeled human calcitonin [82]. In addition, we also demonstrated that chemical shift data were very conveniently used as an initial constraints to construct three-dimensional structures of Leu-enkephalin, based on accurately determined interatomic distances by REDOR [6, 83]. This aspect of work was already described in Chapter 2. 

Next, in Section 8.3.2, we describe long-time full atomic MD simulations (longer than 150 ns) for a small peptide, met-enkephalin (M-Enk), in addition to a larger chymotrypsin inhibitor 2 (CI2), both in ectoine aqueous solutions with the same concentration and in pure water at room temperature. To determine the spatial distribution of each solvent component, the atom number densities of water molecules and ectoine molecules around both solutes were analyzed. We found that one dominant structure of M-Enk does not preferentially exclude the ectoine molecules from its surface as CI2 does. To understand the reason for this difference in ectoine exclusion, in addition to the effect of direct interaction between M-Enk and ectoine, the influence of hydration (i.e., property alteration of the hydration layer near the solute surface) on the development of ectoine preferential exclusion around each solute was examined at the molecular level. 

The structure of the pentapeptide leucine enkephalin shown as (a) a structural drawing and (b) as a molecular model. The shape of the molecular model was determined by X-ray crystallography. Hydrogens have been omitted for clarity. 

Experimental results have indicated that met-enkephalin in aqueous solution does not possess an unique structure [79]. In general, experimentally determined aqueous conformations are found to exhibit characteristics of extended random-coil polypeptide with no discernible secondary structure. When considering the effects of hydration, the competition for backbone hydrogen bonding (with water), which contributes to the bending of the unsolvated conformation, should result in a more extended structure. 

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