In this study we report on the 1H, 13C and 15N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide a complete and internally consistent set of 1H, 13C and 15N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring for both alanine and proline (i.e., the residues in the Y position) are found to depend strongly on the type of amino acid substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in determining peptide and protein chemical shifts.

A new set of hydrophilicity high-performance liquid chromatography (HPLC) parameters is presented. These parameters were derived from the retention times of 20 model synthetic peptides, Ac-Gly-X-X-(Leu)3-(Lys)2-amide, where X was substituted with the 20 amino acids found in proteins. Since hydrophilicity parameters have been used extensively in algorithms to predict which amino acid residues are antigenic, we have compared the profiles generated by our new set of hydrophilic HPLC parameters on the same scale as nine other sets of parameters. Generally, it was found that the HPLC parameters obtained in this study correlated best with antigenicity. In addition, it was shown that a combination of the three best parameters for predicting antigenicity further improved the predictions. These predicted surface sites or, in other words, the hydrophilic, accessible, or mobile regions were then correlated to the known antigenic sites from immunological studies and accessible sites determined by X-ray crystallographic data for several proteins.

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In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V 681 ) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80°C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V 681 was improved 90-and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V 681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.

In the present study, the 26-residue amphipathic ␣-helical antimicrobial peptide V13K L (Y. Chen et al., J. Biol. Chem. 2005, 280:12316-12329, 2005) was used as the framework to study the effects of peptide hydrophobicity on the mechanism of action of antimicrobial peptides. Hydrophobicity was systematically decreased or increased by replacing leucine residues with less hydrophobic alanine residues or replacing alanine residues with more hydrophobic leucine residues on the nonpolar face of the helix, respectively. Hydrophobicity of the nonpolar face of the amphipathic helix was demonstrated to correlate with peptide helicity (measured by circular dichroism spectroscopy) and self-associating ability (measured by reversedphase high-performance liquid chromatography temperature profiling) in aqueous environments. Higher hydrophobicity was correlated with stronger hemolytic activity. In contrast, there was an optimum hydrophobicity window in which high antimicrobial activity could be obtained. Decreased or increased hydrophobicity beyond this window dramatically decreased antimicrobial activity. The decreased antimicrobial activity at high peptide hydrophobicity can be explained by the strong peptide self-association which prevents the peptide from passing through the cell wall in prokaryotic cells, whereas increased peptide self-association had no effect on peptide access to eukaryotic membranes.Antibiotic resistance, due to the extensive clinical use of classical antibiotics (22, 32), has become a great concern in recent years, prompting an urgent need for a new class of antibiotics. Antimicrobial peptides have been proposed as potent candidates of a new class of antibiotics, with characteristics including an ability to kill target cells rapidly, an unusually broad spectrum of activity, activity against some of the more serious antibiotic-resistant pathogens in clinics, and the relative difficulty in selecting resistant mutants in vitro (13,35). Although the exact mode of action of antimicrobial peptides has not been established, it is generally accepted that the cytoplasmic membrane is the main target of antimicrobial peptides, whereby peptide accumulation in the membrane causes increased permeability and a loss of barrier function, resulting in the leakage of cytoplasmic components and cell death (13,28).Factors believed to be important for antimicrobial activity have been identified, including peptide hydrophobicity, the presence of positively charged residues, an amphipathic nature that segregates basic and hydrophobic residues, and secondary structure. Recently, Hodges and coworkers increased this list to include (i) the importance of a lack of structure in benign medium (nondenaturing conditions; see Materials and Methods) but an inducible structure in the presence of the hydrophobic environment of the membrane, (ii) the presence of a positively charged residue in the center of the nonpolar face of amphipathic cyclic ␤-sheet and ␣-helical peptides as a determinant for locating the peptides at the interface r...

The structure of a synthetic peptide comprising the 28 amino-terminal residues of actin has been examined by 1H-NMR and CD spectroscopy. The peptide is largely unstructured and flexible in solution but becomes increasingly structured at higher trifluoroethanol (TFE) concentrations. As judged by CD with the use of two additional peptides (actin 1-20 and actin 18-28), TFE induces formation of up to 48% helical content within residues 1-20, while residues 21-28 exhibit no helical propensity. Similar results were obtained by using NMR-derived distance information in restrained molecular dynamics calculations. The calculated structure of actin 1-28 peptide in 80% TFE is well defined for the first 23 residues with a backbone root mean square deviation of 0.5 A. Two helices are formed from residues 4-13 and 16-20, and a beta-turn is formed from residues 13-16. The N-terminal residues 1-3 exhibit increased flexibility and a helix-like conformation while the C-terminal residues 21-28 show no regular secondary structure. These results are compared with the predicted secondary structure and the structure of the corresponding sequence in the crystal structure of actin [Kabsch et al. (1990) Nature 347, 37-44]. The significance of the TFE-induced peptide structure is discussed.

The biological function of transmembrane proteins is closely related to their insertion, which has most often been studied through their lateral mobility. For >30 years, it has been thought that hardly any information on the size of the diffusing object can be extracted from such experiments. Indeed, the hydrodynamic model developed by Saffman and Delbrü ck predicts a weak, logarithmic dependence of the diffusion coefficient D with the radius R of the protein. Despite widespread use, its validity has never been thoroughly investigated. To check this model, we measured the diffusion coefficients of various peptides and transmembrane proteins, incorporated into giant unilamellar vesicles of 1-stearoyl-2-oleoylsn-glycero-3-phosphocholine (SOPC) or in model bilayers of tunable thickness. We show in this work that, for several integral proteins spanning a large range of sizes, the diffusion coefficient is strongly linked to the protein dimensions. A heuristic model results in a Stokes-like expression for D, (D ؔ 1͞R), which fits literature data as well as ours. Diffusion measurement is then a fast and fruitful method; it allows determining the oligomerization degree of proteins or studying lipid-protein and protein-protein interactions within bilayers.bilayers ͉ transmembrane proteins ͉ diffusion ͉ peptides ͉ sponge phase I n the hydrodynamic model of Saffman and Delbrück (1), transmembrane peptides and proteins are described as diffusing in a perfectly continuous medium, ignoring the finite size of the lipids. This model predicts that the diffusion coefficient D of a simple cylinder embedded in a thin sheet of fluid matching its height ( Fig. 1) is given byIn this expression, the adjustable parameters are by order of importance: the thickness h and viscosity m of the liquid membrane, the radius R of the diffusing cylinder, and the viscosity of the surrounding aqueous phase w . This result follows from solving the flow field in the membrane and in the surrounding fluid, assuming no-slip boundary conditions at the surface of the cylinder, which is considered as large compared with the bilayer components (i.e., R Ͼ h). Numerous biological studies, both in model systems (2-4) and living cells (5, 6), refer to this continuum approach (7). Because D depends only weakly on R, the characterization of protein or rafts radii is delicate (8); for example, increasing the radius from 10 to 100 Å changes the mobility by a mere 30% [for h ϭ 30 Å and m ϭ 10 poise (P; 1 P ϭ 0.1 Pa⅐s)].To check the applicability of the Saffman-Delbrück formula (Eq. 1), we have used fringe pattern photobleaching under the microscope (9) to measure precisely the self-diffusion of transmembrane peptides and proteins of well characterized dimensions. Results and DiscussionThe weight of the bilayer thickness, h, has never been investigated. Rather than using lipids of various lengths, we opted for a unique system where the bilayer thickness can be continuously tuned, leaving the bilayer viscosity constant.We use a phase of model bilayers made of nonionic ...

In our previous study, we utilized a 26‐residue amphipathic α‐helical antimicrobial peptide L‐V13K (Chen et al., Antimicrob Agents Chemother 2007, 51, 1398–1406) as the framework to study the effects of peptide hydrophobicity on the mechanism of its antimicrobial action. In this study, we explored the effects of net charge and the number of positively charged residues on the hydrophilic/polar face of L‐V13K on its biological activity (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity, helicity, and peptide self‐association). The net charge of V13K analogs at pH 7 varied between −5 and +10 and the number of positively charged residues varied from 1 to 10. The minimal inhibitory concentrations (MIC) against six strains of Pseudomonas aeruginosa as well as other gram‐negative and gram‐positive bacteria were determined along with the maximal peptide concentration that produces no hemolysis of human red blood cells (MHC). Our results show that the number of positively charged residues on the polar face and net charge are both important for both antimicrobial activity and hemolytic activity. The most dramatic observation is the sharp transition of hemolytic activity on increasing one positive charge on the polar face of V13K i.e., the change from +8 to +9 resulted in greater than 32‐fold increase in hemolytic activity (250 μg/ml to <7.8 μg/ml, respectively). © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 369–383, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com