A series of designed peptide 33-mers (betapep peptides) areknown to be bactericidal [Mayo, Haseman, Ilyina and Gray (1998)Biochim. Biophys. Acta 1425, 81-92]. Here dodecapeptides (SC-1-SC-8), which 'walk through' the sequence ofbetapep-25, were investigated for their ability to kill Gram-negativeand -positive bacteria and to neutralize endotoxin. SC-4 (KLFKRHLKWKI I-NH(2); the -NH(2) at the right of each sequenceindicates amidation of the C-terminal carboxylate group) is the mosteffective, more so than betapep-25, at killing Gram-negative bacteriawith nanomolar LD(50) values. Against Gram-positive bacteria,SC-4 also shows good activity with submicromolar LD(50)values. Leakage studies indicate rapid bacterial membrane permeability,with t(1/2) valuesof 10-15 min. SC-4 in the micromolar range also effectivelyneutralizes endotoxin and is not haemolytic below 10(-4)M. For all SC peptides, CD and NMR data indicate the presence of both 3(10)- and alpha-helix. For SC-4, nuclear-Overhauser-effect-based computational modelling yields an amphipathic helix with K1, K4,R5, and K8 arrayed on the same face (K is lysine, R is arginine). Activity differences among SC peptides and single-site variants of SC-4allow some structure-function relationships to be deduced. Relative to other known bactericidal peptides in the linear peptide,helix-forming category, SC-4 is the most potent broad-spectrumantibacterial identified to date. The present study contributes to thedevelopment of agents involved in combating the ever-recurring problemof drug-resistant micro-organisms.

Nociceptin, a 17 amino acid opioid-like peptide that has an inhibitory effect on synaptic transmission in the nervous system, is involved in learning, memory, attention, and emotion and is also implicated in the perception of pain and visual, auditory, and olfactory functions. In this study, we investigated the NMR solution structure of nociceptin in membrane-like environments (trifluoroethanol and SDS micelles) and found it to have a relatively stable helix conformation from residues 4 -17 with functionally important N-terminal residues being folded aperidoically on top of the helix. In functional assays for receptor binding and calcium flux, alanine-scanning variants of nociceptin indicated that functionally important residues generally followed helix periodicity, consistent with the NMR structural model. Structure-activity relationships allowed identification of pharmacophore sites that were used in small molecule data base searches, affording hits with demonstrated nociceptin receptor binding affinities.Nociceptin is a 17 amino acid opioid-like peptide that was identified as a natural ligand of the orphan opioid receptor ORL1 (also referred to as OP4) (1). Reinscheid et al. (2) called the same peptide orphanin FQ to signify that it is a ligand for ORL1 with an N-terminal phenylalanine and a C-terminal glutamine. Nociceptin signaling through ORL1 elicits many of the same responses induced by opioid signaling through the opioid receptors. Nociceptin causes inhibition of adenylyl cyclase (1, 2), activation of potassium channels (3-8), inhibition of calcium channels (9 -11), mobilization of intracellular calcium (10), and activation of mitogen-activated protein kinase (12)(13)(14). These effects indicate that, similar to opioids, nociceptin has an inhibitory effect on synaptic transmission in the nervous system, acting to reduce the secretion of neurotransmitters. Consistent with its cellular effects, nociceptin inhibits the release of glutamate (5, 15-17), ␥-Aminobutyric acid (5), acetylcholine (18, 19), tachykinin (20, 21), and noradrenaline (22) neurotransmitters. Based on their distribution in the brain and spinal cord, nociception and its receptor may be involved in a wide range of functions, including learning, memory, attention, and emotion. Nociceptin is also implicated in various sensory processes such as perception of pain, visual, auditory, and olfactory functions.Nociceptin is related to dynorphin A, a peptide 17-mer ligand of the -opioid receptor. Dynorphin A also binds ORL1 but with 100-fold lower affinity than nociceptin (23). Orphanin FQ2 is another biologically active peptide 17-mer processed from the same nociceptin precursor, prenociceptin (1, 2, 24). Fig. 1 shows the amino acid sequences of orphanin FQ2, nociceptin, and dynorphin A. Although nociceptin and dynorphin A are the most homologous of these three peptides (particularly at the N and C termini and being polycationic), there are clear differences in amino acid sequence that should affect conformation and receptor binding as noted abov...

␣-Chemokines are known heparin-binding proteins. Here, a heparin dodecasaccharide (H12) was purified and used in NMR studies to investigate binding to growth-related protein-␣ (Gro-␣) and to platelet factor-4-M2 (PF4-M2), an N-terminal chimera of PF4. Pulsed field gradient NMR was used to derive diffusion coefficients as the protein (monomer):H12 ratio was varied. In the absence of H12, both PF4-M2 and Gro-␣ give diffusion coefficients consistent with the presence of mostly dimers. As the PF4-M2:H12 ratio is increased from 1:6 to 2:1, the diffusion coefficient increases, indicating dissociation to the monomer state. On addition of H12 to either protein, 15 N/ 1 H heteronuclear single quantum coherence NMR data demonstrate loss of 1 H resonance dispersion and intensity, particularly at protein:H12 ratios of 2:1 to 4:1, indicating significant perturbation to native structures. For Gro-␣ in particular, 1 H resonance dispersion appears random coil-like. At these same ratios, circular dichroism (CD) data show general retention of secondary structure elements with a slight shift to additional helix formation. Random coil NMR resonance dispersion suggests a shift to a less compact, partially folded, and/or more flexible state. Further addition of H12 causes resonance intensity and dispersion to return making NMR spectra appear native-like. At low PF4-M2:H12 ratios, loss of resonance intensity for residues proximal to Arg-20 and Arg-22 in three-dimensional NMR HCCH-TOCSY spectra suggests that the Arg-20-Arg-22 loop either interacts most strongly with H12 and/or that binding at this site is heterogeneous. This domain was previously shown to be crucial to heparin binding. Of particular interest to the biology of PF4-heparin complex formation, heparin-induced thrombocytopenia antibody binding occurs at about the same PF4-M2:H12 ratio as does this transition to a partially folded PF4-M2 state, strongly suggesting that heparin-induced thrombocytopenia antibody recognizes a less folded, lower aggregate state of the protein.

Neutrophil-activating peptide 2 (NAP-2), which demonstrates a range of proinflammatory activities, is a 72-residue protein belonging to the alpha-chemokine family. Although NAP-2, like other alpha-chemokines, is known to self-associate into dimers and tetramers, it has been shown that the monomeric form is physiologically active. Here we investigate the solution structure of monomeric NAP-2 by multi-dimensional 1H-NMR and 15N-NMR spectroscopy and computational modelling. The NAP-2 monomer consists of an amphipathic, triple-stranded, anti-parallel beta-sheet on which is folded a C-terminal alpha-helix and an aperiodic N-terminal segment. The backbone fold is essentially the same as that found in other alpha-chemokines. 15N T1, T2 and nuclear Overhauser effects (NOEs) have been measured for backbone NH groups and used in a model free approach to calculate order parameters and conformational exchange terms that map out motions of the backbone. N-terminal residues 1 to 17 and the C-terminus are relatively highly flexible, whereas the beta-sheet domain forms the most motionally restricted part of the fold. Conformational exchange occurring on the millisecond time scale is noted at the top of the C-terminal helix and at proximal residues from beta-strands 1 and 2 and the connecting loop. Dissociation to the monomeric state is apparently responsible for increased internal mobility in NAP-2 compared with dimeric and tetrameric states in other alpha-chemokines.