Mapping the receptor-recognition site of human transforming growth factor-alpha.

The receptor-recognition site human transforming growth factor-alpha (TGF alpha), a 50-residue tricyclic peptide with three disulfide bonds, was mapped by a set of 46 peptide analogs consisting of linear, monocyclic, bicyclic, and tricyclic structures representing individual and overlapping subdomains of human TGF alpha. Linear overlapping fragments ranging from 7 to 18 residues and spanning the entire length of TGF alpha as well as monocyclic analogs with one disulfide linkage were found to be inactive in both receptor-binding and mitogenic assays. Bicyclic analogs with two disulfide linkage and representing either the amino or carboxyl two-thirds of TGF alpha showed low activity at 0.1-0.9 mM concentrations. Tricyclic analogs containing all three disulfide linkages but lacking either the amino or carboxyl terminal heptapeptide was, respectively, 3% and 0.1% as active as TGF alpha. These results show that determinants for the receptor binding cannot be represented by a short continuous fragment or a single subdomain, but are located on a discontinuous surface on a folded structure with disulfide restraints. Furthermore, these results when combined with our previous results which shows that the middle subdomain (second disulfide loop) is not involved in the receptor binding suggest that the receptor-binding residues are constituted of three fragments located at the first and third subdomains as well as the external carboxyl peptide.     

Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor alpha subunit: effects of cysteine/cystine modification and species-specific amino acid substitutions

The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha subunit forms a binding site for alpha-bungarotoxin (alpha-BTX) [e.g., see Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230]. Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR alpha 1 subunits were tested for their ability to bind 125I-alpha-BTX, and differences in alpha-BTX affinity were determined by using solution (IC50S) and solid-phase (KdS) assays. Panels of overlapping peptides corresponding to the complete alpha 1 subunit of mouse and human were also tested for alpha-BTX binding, but other sequence segments forming the alpha-BTX site were not consistently detectable. The Torpedo alpha 1(181-200) and the homologous frog and chicken peptides bound alpha-BTX with higher affinity (KdS approximately 1-2 microM, IC50s approximately 1-2 microM) than the human and calf peptides (Kds approximately 3-5 microM, IC50s approximately 15 microM). The mouse peptide bound alpha-BTX weakly when attached to a solid support (Kd approximately 8 microM) but was effective in competing for 125I-alpha-BTX in solution (IC50 approximately 1 microM). The cobra nAChR alpha 1-subunit peptide did not detectably bind alpha-BTX in either assay. Amino acid substitutions were correlated with alpha-BTX binding activity peptides from different species. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased alpha-BTX binding, whereas oxidation of the peptides had little effect. Modifications of the cysteine/cystine residues of the cobra peptide failed to induce alpha-BTX binding activity. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/alpha 1-subunit interface a vicinal disulfide bound was not required for alpha-BTX binding.     

AlphaC-conotoxin PrXA: a new family of nicotinic acetylcholine receptor antagonists

We have purified a novel paralytic peptide with 32 AA and a single disulfide bond from the venom of Conus parius, a fish-hunting species. The peptide has the following sequence: TYGIYDAKPOFSCAGLRGGCVLPONLROKFKE-NH2, where O is 4-trans-hydroxyproline. The peptide, designated alphaC-conotoxin PrXA (alphaC-PrXA), is the defining member of a new, structurally distinct family of Conus peptides. The peptide is a competitive nAChR antagonist; all previously characterized conotoxins that competitively antagonize nAChRs are structurally and genetically unrelated. (Most belong to the alpha- and alphaA-conotoxin families.) When administered to mice and fish in vivo, alphaC-PrXA caused paralysis and death. In electrophysiological assays, alphaC-PrXA potently antagonized mouse muscle nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.8 and 3.0 nM for the adult (alpha1beta1 epsilondelta subunits) and fetal (alpha1beta1 gammadelta subunits) muscle nAChR subtypes, respectively. When tested on a variety of ligand-gated and voltage-gated ion channels, alphaC-PrXA proved to be a highly specific inhibitor of the neuromuscular nAChR. The peptide competes with alpha-bungarotoxin for binding at the alpha/delta and alpha/gamma subunit interfaces of the nAChR, with higher affinity for the alpha/delta subunit interface. AlphaC-PrXA is strikingly different from the many conopeptides shown to be nicotinic antagonists; it is most similar in its general biochemical features to the snake toxins known as Waglerins.     

Characterisation of a novel toxin active on potassium apanaine channels, purified from Buthus occitanus tunetanus scorpion venom

A new peptidyl inhibitor of the small-conductance Ca(2+)-activated K+ channels (SKca) was purified to homogeneity from the venom of the Tunisian scorpion Buthus occitanus tunetanus. The molecular mass determined by SDS-PAGE, shows that it's a short peptide (3300 Da). The primary sequence of this toxin shows that it is a 31-residue polypeptide cross-linked by three disulfide bridges and structurally related to subfamily 5 of short scorpion toxins. This molecule shows similar pharmacological properties with this group of peptides inducing high toxicity in mice after intracerebro-ventricular injection, and competing with iodinated apamin for binding to its receptor site from rat brain synaptosomes (K0.5 = 4 nM).     

Subunits of purified calcium channels. Alpha 2 and delta are encoded by the same gene

Polyacrylamide gel electrophoresis of purified rabbit skeletal muscle L-type calcium channel before and after reduction of disulfide bonds confirmed that 27- and 24-kDa forms of the delta subunit are disulfide-linked to the 143-kDa alpha 2 subunit. The amino acid sequences of three peptides obtained by tryptic digestion of the delta subunits corresponded to amino acid sequences predicted from the 3' region of the mRNA encoding alpha 2. One of these peptides had the same sequence as the N terminus of the 24- and 27-kDa forms of the delta subunit and corresponded to residues 935-946 of the predicted alpha 2 primary sequence. Anti-peptide antibodies directed to regions on the N-terminal side of this site recognized the 143-kDa alpha 2 subunit in immunoblots of purified calcium channels under reducing conditions, whereas an antipeptide antibody directed toward a sequence on the C-terminal side of this site recognized 24- and 27-kDa forms of the delta subunit. A similar result was obtained after immunoblotting using purified transverse tubules or crude microsomal membrane preparations indicating that alpha 2 and delta occur as distinct disulfide-linked polypeptides in skeletal muscle membranes. Thus, the delta subunits are encoded by the same gene as the alpha 2 subunit and are integral components of the skeletal muscle calcium channel.     

Covalent structure of collagen: amino acid sequence of alpha 2-CB5 of chick skin collagen containing the animal collagenase cleavage site.

The amino acid sequence of the 112 residues from the amino terminus of alpha 2-CB5 from chick skin collagen was determined by automated sequential degradation of intact alpha 2-CB5 and several chymotryptic and tryptic peptides. This segment of the peptide includes the site of the action of animal collagenases. As compared to the sequence around the alpha 1 cleavage site, the alpha 2 sequence is notable for the remarkable constancy of the residues to the amino side and the relative abundance of hydrophobic residues to the carboxyl side of the cleavage site, suggesting that these features are important in the recognition by the enzyme. The sequence of this region of the alpha 2 chain is consistent with the Gly-X-Y triplet structure and the preference of certain residues for either the X or Y position in distribution. However, three of the six residues of leucine were found in the Y position rather than the X position. Leucine residues were found only once in the Y position in the alpha 1 (I) chain. This preference does not appear to hold in the alpha 2 chain.     

The position of the disulfide bonds in human plasma alpha 2 HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma alpha 2 HS-glycoprotein were determined. alpha 2 HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)--Cys-14 (A-chain), Cys-71--Cys-82, Cys-96--Cys-114, Cys-128--Cys-131, Cys-190--Cys-201 and Cys-212--Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of alpha 2 HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the S--S bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two S--S bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second S--S loops and the end of domains A and B, and the positions of the ordered structures.     

Isolation and characterization of gomesin, an 18-residue cysteine-rich defense peptide from the spider Acanthoscurria gomesiana hemocytes with sequence similarities to horseshoe crab antimicrobial peptides of the tachyplesin family

We have purified a small size antimicrobial peptide, named gomesin, from the hemocytes of the unchallenged tarantula spider Acanthoscurria gomesiana. Gomesin has a molecular mass of 2270.4 Da, with 18 amino acids, including a pyroglutamic acid as the N terminus, a C-terminal arginine alpha-amide, and four cysteine residues forming two disulfide bridges. This peptide shows marked sequence similarities to antimicrobial peptides from other arthropods such as tachyplesin and polyphemusin from horseshoe crabs and androctonin from scorpions. Interestingly, it also shows sequence similarities to protegrins, antimicrobial peptides from porcine leukocytes. Gomesin strongly affects bacterial growth, as well as the development of filamentous fungi and yeast. In addition, we showed that gomesin affects the viability of the parasite Leishmania amazonensis.     

Three-dimensional solution structure of omega-conotoxin TxVII, an L-type calcium channel blocker

We determined the three-dimensional structure of omega-conotoxin TxVII, a 26-residue peptide that is an L-type calcium channel blocker, by (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 411 distance constraints obtained from nuclear Overhauser effect connectivities, 20 torsion angle constraints, and 21 constraints associated with hydrogen bonds and disulfide bonds. The root-mean-square deviations about the averaged coordinates of the backbone atoms (N, C(alpha), C, and O) and all heavy atoms were 0.50 +/- 0.09 A and 0.99 +/- 0.13 A, respectively. The structure of omega-conotoxin TxVII is composed of a triple-stranded antiparallel beta-sheet and four turns. The three disulfide bonds in omega-conotoxin TxVII form the classical cystine knot motif of toxic or inhibitory polypeptides. The overall folding of omega-conotoxin TxVII is similar to those of the N-type calcium channel blockers, omega-conotoxin GVIA and MVIIA, despite the low amino acid sequence homology among them. omega-Conotoxin TxVII exposes many hydrophobic residues to a certain surface area. In contrast, omega-conotoxin GVIA and MVIIA expose basic residues in the same way as omega-conotoxin TxVII. The channel binding site of omega-conotoxin TxVII is different from those of omega-conotoxin GVIA and MVIIA, although the overall folding of these three peptides is similar. The gathered hydrophobic residues of omega-conotoxin TxVII probably interact with the hydrophobic cluster of the alpha(1) subunit of the L-type calcium channel, which consists of 13 residues located in segments 5 and 6 in domain III and in segment 6 in domain IV.     

The structure of a novel insect peptide explains its Ca2+ channel blocking and antifungal activities

Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional 1H nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungal peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.     

The primary structure of aphrodisin

Aphrodisin is a protein which is secreted in hamster vaginal discharge and acts via the vomeronasal organ of the accessory olfactory system to elicit copulatory behavior in male hamsters. The complete primary structure of aphrodisin was determined by sequence analysis of intact aphrodisin after unblocking the amino terminus with pyroglutamate aminopeptidase and from peptides generated by trypsin and Lys-C digests. Alignment of the peptides was obtained from sequence analysis of peptides from cyanogen bromide and hydroxylamine cleavages. The protein consists of 151 residues of Mr = 17,000. It has disulfide bonds linking cysteine residues at positions 38 and 42 and at 57 and 149. N-acetylglucosamine residues are linked to asparagines at positions 41 and 69. Based on its similarity to the major urinary proteins in rats and mice, aphrodisin is a putative member of the alpha 2u-globulin superfamily of extracellular proteins.     

Identification of three novel peptides isolated from the venom of the neotropical social wasp Polistes major major.

Three novel peptides designated as PMM1, PMM2, and PMM3 were isolated and characterized from the venom of the social wasp Polistes major major, one of the most common wasps in the Dominican Republic. By Edman degradation, and MALDI-TOF and ESI-QTOF mass spectrometry, the primary sequences of these peptides were established as follows: PMM1, H-Lys-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH (1357.77 Da); PMM2, H-Ile-Asn-Trp-Lys-Lys-Ile-Ala-Ser-Ile-Gly-Lys-Glu-Val-Leu-Lys-Ala-Leu-NH2 (1909.19 Da); and PMM3, H-Phe-Leu-Ser-Ala-Leu-Leu-Gly-Met-Leu-Lys-Asn-Leu-NH2 (1317.78 Da). The suggested sequences were confirmed by MS analysis of peptide fragments obtained by enzymatic digestion. The peptide PMM1 is a lysyl-arginyl-Thr(6)-bradykinine that belongs to the wasp kinins group. The sequence of the PMM2 peptide is unique; it resembles somewhat the tetradecapeptide amides of the mastoparan group; however, the chain is extended by three additional amino acid residues. The sequence of PMM3 dodecapeptide is homologous to the peptides of the wasp chemotactic group.     

Role of recurrent hydrophobic residues in catalysis of helix formation by T cell-presented peptides in the presence of lipid vesicles

We tested the hypothesis that the recurrence of hydrophobic amino acids in a polypeptide at positions falling in an axial, hydrophobic strip if the sequence were coiled as an alpha helix, can lead to helical nucleation on a hydrophobic surface. The hydrophobic surface could anchor such residues, whereas the peptide sequence grows in a helical configuration that is stabilized by hydrogen bonds among carbonyl and amido NH groups along the peptidyl backbone of the helix, and by other intercycle interactions among amino acid side chains. Such bound, helical structures might protect peptides from proteases and/or facilitate transport to a MHC-containing compartment and thus be reflected in the selection of T cell-presented segments. Helical structure in a series of HPLC-purified peptides was estimated from circular dichroism measurements in: 1) 0.01 M phosphate buffer, pH 7.0, 2) that buffer with 45% trifluoroethanol (TFE), and 3) that buffer with di-O-hexadecyl phosphatidylcholine vesicles. By decreasing the dielectric constant of the buffer, TFE enhances intrapeptide interactions generally, whereas the lipid vesicles only provide a surface for hydrophobic interactions. The peptides varied in their strip-of-helix hydrophobicity indices (SOHHI; the mean Kyte-Doolittle hydrophobicities of residues in an axial strip of an alpha helix) and in proline content. Structural order for peptides with helical circular dichroism spectra was estimated as percentage helicity from circular dichroism theta 222 nm values and peptide concentration. A prototypic alpha helical peptide with three cycles plus two amino acids and an axial hydrophobic strip of four leucyl residues (SOHHI = 3.8) was disordered in phosphate buffer, 58% helical in that buffer with 48% TFE, and 36% helical in that buffer with vesicles. Percentage helicity in the presence of vesicles of the subset of peptides without proline followed their SOHHI values. Peptides with multiple prolyl residues had circular dichroism spectra with strong signals, but since they did not have altered spectra in the presence of vesicles relative to phosphate buffer alone, the hydrophobic surface of the vesicle did not appear to stabilize those structures.     

Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry

The cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W have been recently described, and the primary structure of this gene product was deduced using a bioinformatic approach. In this study, capillary electrophoresis-on-the-chip and mass spectrometry were used to characterize native and overexpressed extracellular lipase protein from S. rimosus . The exact molecular mass of the wild-type and the overexpressed lipase, determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, were in excellent agreement (Deltam=0.11 Da and Deltam=0.26 Da, respectively) with a value of 24165.76 Da calculated from the structure deduced from the nucleotide sequence, considering the mature enzyme with all six cysteines forming disulfide bridges. The primary structure derived from the nucleotide sequence was completely verified using a combination of tryptic digestion and formic acid cleavage of the protein, followed by peptide mass fingerprinting. Selected peptides were further investigated by MALDI low-energy collision-induced dissociation hybrid tandem mass spectrometry, allowing the unambiguous determination of their predicted amino acid sequence. No post-translational modifications of mature S. rimosus lipase were detected. Comparison of the peptide mass fingerprints from the reduced and non-reduced overexpressed enzyme unequivocally revealed three intramolecular disulfide bonds with the following linkages: C27-C52, C93-C101 and C151-C198.     

Protein chemistry of the Neurospora crassa plasma membrane H+-ATPase

A highly effective procedure for fragmenting the Neurospora crassa plasma membrane H+-ATPase and purifying the resulting peptides is described. The enzyme is cleaved with trypsin to form a limit digest containing both hydrophobic and hydrophilic peptides, and the hydrophobic and hydrophilic peptides are then separated by extraction with an aqueous ammonium bicarbonate solution. The hydrophilic peptides are fractionated by Sephadex G-25 column chromatography into three pools, and the individual peptides in each pool are purified by high-performance liquid chromatography. The hydrophobic peptides are dissolved in neat trifluoroacetic acid (TFA), diluted with chloroform-methanol (1:1), and the hydrophobic peptide solution thus obtained is then fractionated by Sephadex LH-60 column chromatography in chloroform-methanol (1:1) containing 0.1% TFA. The recoveries in all of the above procedures are greater than 90%. The N-terminal amino acid sequences of three of the hydrophobic H+-ATPase peptides purified by this methodology have been determined, which establishes the position of these peptides in the 100,000 Da polypeptide chain by reference to the published gene sequence, and documents the sequencability of the hydrophobic peptides purified in this way. This methodology should facilitate the identification of a variety of amino acid residues important for the structure and function of the H+-ATPase molecule. Moreover, the overall strategy for working with the protein chemistry of the H+-ATPase should be applicable to other amphiphilic integral membrane proteins as well.     

The structure of type IX collagen

We present a detailed analysis both of tryptic peptides and amino-terminal sequences of the subunits of two collagenous fragments (HMW and LMW) previously isolated from pepsin extracts of chicken cartilage (Reese, C.A., and Mayne, R. (1981) Biochemistry 20, 5443-5448). This analysis and a comparison with the nucleotide sequence of the cDNApYN1738 (Ninomiya, Y., and Olsen, B.R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3014-3018) shows that HMW and LMW are pepsin-resistant fragments of a unique collagen composed of molecules with three different polypeptide chains (alpha-chains). This collagen has been assigned the type number IX, and the alpha-chain encoded by pYN1738 has been given the designation alpha 1 (IX). Type IX collagen contains three triple-helical domains and at least two sets of interchain disulfide bridges. At the amino and carboxyl ends are noncollagenous domains which do not appear to be homologous to amino and carboxyl propeptides of interstitial collagens.     

Solution structures and integrin binding activities of an RGD peptide with two isomers

The Arg-Gly-Asp (RGD) sequence serves as the primary integrin recognition site in extracellular matrix proteins, and peptides containing this sequence can mimic the activities of the matrix proteins. Depending on the context of the RGD sequence, an RGD-containing peptide may bind to all of the RGD-directed integrins, to a few, or to only a single one. We have previously isolated from a phage-displayed peptide library a cyclic peptide that binds avidly to the alpha(v)beta3 and alpha(v)beta5 integrins but does not bind to other closely related integrins. This peptide, ACDCRGDCFCG, exists in two natural configurations depending on internal disulfide bonding. The peptide with the 1-4; 2-3 disulfide bond arrangement accounts for most of the alpha(v) integrin binding activity, whereas the 1-3; 2-4 peptide is about 10-fold less potent. Solution structure analysis by nuclear magnetic resonance reveals an entirely different presentation of the RGD motif in the two isomers of RGD-4C. These results provide new insight into the ligand recognition specificity of integrins.     

Optimization of the production of a honeybee odorant-binding protein by Pichia pastoris

A honeybee putative general odorant-binding protein ASP2 has been expressed in the methylotrophic yeast Pichia pastoris. It was secreted into the buffered minimal medium using either the alpha-factor preprosequence with and without the Glu-Ala-Glu-Ala spacer peptide of Saccharomyces cerevisiae or its native signal peptide. Whereas ASP2 secreted using the alpha-factor preprosequence with the spacer peptide showed N-terminal heterogeneity, the recombinant protein using the two other secretion peptides was correctly processed. Mass spectrometry showed that the protein secreted using the natural peptide sequence had a mass of 13,695.1 Da, in perfect agreement with the measured molecular mass of the native protein. These data showed a native-like processing and the three disulfide bridges formation confirmed by sulfhydryl titration analysis. After dialysis, the recombinant protein was purified by one-step anion-exchange chromatography in a highly pure form. The final expression yield after 7-day fermentation was approximately 150 mg/liter. To our knowledge, this is the first report of the use of a natural insect leader sequence for secretion with correct processing in P. pastoris. The overproduction of recombinant ASP2 should allow ligand binding and mutational analysis to understand the relationships between structure and biological function of the protein.     

Molecular determinants of the reversible membrane anchorage of the G-protein transducin.

Transducin is a heterotrimer formed by a fatty acylated alpha-subunit and a farnesylated betagamma-subunit. The role of these two covalent modifications and of adjacent hydrophobic and charged amino acid residues in reversible anchoring at disk model membranes is investigated at different pH values, salt concentrations, and lipid packing densities using the monolayer expansion technique and CD spectroscopy. The heterotrimer only binds if the acetylated alpha-subunit is transformed into its surface-active form by divalent cations. In the presence of salts the alpha(GDP)-subunit, the betagamma-complex, and the heterotrimer bind to POPC monolayers at 30 mN/m, estimated to mimic the lateral packing density of disk membranes, with apparent binding constants of Kapp = (1.1 +/- 0.3) x 10(6) M-1 (reflecting the penetration of the fatty acyl chain together with approximately three adjacent hydrophobic amino acid residues), Kapp = (3.5 +/- 0.5) x 10(6) M-1 (reflecting the penetration of the farnesyl chain), and Kapp = (1.6 +/- 0.3) x 10(6) M-1 (reflecting a major contribution of the alpha(GDP)-subunit with only a minor contribution from the betagamma-complex). The apparent binding constant of the alpha(GTP)-subunit is distinctly smaller than that of the alpha(GDP)-subunit. Binding to negatively charged POPC/POPG (75/25 mole/mole) monolayers is reinforced by 2-3 cationic residues for the betagamma-complex. The alpha-subunit shows no electrostatic attraction and the heterotrimer shows even a slight electrostatic repulsion which becomes the dominating force in the absence of salts.     

Correlation of three-dimensional structures with the antibacterial activity of a group of peptides designed based on a nontoxic bacterial membrane anchor

To understand the functional differences between a nontoxic membrane anchor corresponding to the N-terminal sequence of the Escherichia coli enzyme IIA(Glc) and a toxic antimicrobial peptide aurein 1.2 of similar sequence, a series of peptides was designed to bridge the gap between them. An alteration of a single residue of the membrane anchor converted it into an antibacterial peptide. Circular dichroism spectra indicate that all peptides are disordered in water but helical in micelles. Structures of the peptides were determined in membrane-mimetic micelles by solution NMR spectroscopy. The quality of the distance-based structures was improved by including backbone angle restraints derived from a set of chemical shifts ((1)H(alpha), (15)N, (13)C(alpha), and (13)C(beta)) from natural abundance two-dimensional heteronuclear correlated spectroscopy. Different from the membrane anchor, antibacterial peptides possess a broader and longer hydrophobic surface, allowing a deeper penetration into the membrane, as supported by intermolecular nuclear Overhauser effect cross-peaks between the peptide and short chain dioctanoyl phosphatidylglycerol. An attempt was made to correlate the NMR structures of these peptides with their antibacterial activity. The activity of this group of peptides does not correlate exactly with helicity, amphipathicity, charge, the number of charges, the size of the hydrophobic surface, or hydrophobic transfer free energy. However, a correlation is established between the peptide activity and membrane perturbation potential, which is defined by interfacial hydrophobic patches and basic residues in the case of cationic peptides. Indeed, (31)P solid state NMR spectroscopy of lipid bilayers showed that the extent of lipid vesicle disruption by these peptides is proportional to their membrane perturbation potential.