Li-Ping Liu

Cationic Hydrophobic Peptides with Antimicrobial Activity

ABSTRACT The MICs of cationic, hydrophobic peptides of the prototypic sequence KKAAAXAAAAAXAAWAAXAAAKKKK-amide (where X is one of the 20 commonly occurring amino acids) are in a low micromolar range for a panel of gram-negative and gram-positive bacteria, with no or low hemolytic activity against human and rabbit erythrocytes. The peptides are active only when the average segmental hydrophobicity of the 19-residue core is above an experimentally determined threshold value (where X is Phe, Trp, Leu, Ile, Met, Val, Cys, or Ala). Antimicrobial activity could be increased by using peptides that were truncated from the prototype length to 11 core residues, with X being Phe and with 6 Lys residues grouped at the N terminus. We propose a mechanism for the interaction between these peptides and bacterial membranes similar to the “carpet model,” wherein the Lys residues interact with the anionic phospholipid head groups in the bacterial membrane surface and the hydrophobic core portion of the peptide is then able to interact with the lipid bilayer, causing disruption of the bacterial membrane.

TM Finder: a prediction program for transmembrane protein segments using a combination of hydrophobicity and nonpolar phase helicity scales.

Based on the principle of dual prediction by segment hydrophobicity and nonpolar phase helicity, in concert with imposed threshold values of these two parameters, we developed the automated prediction program TM Finder that can successfully locate most transmembrane (TM) segments in proteins. The program uses the results of experiments on a series of host‐guest TM segment mimic peptides of prototypic sequence KK AAAXAAAAAXAAWAAXAAAKKKK‐amide (where X = each of the 20 commonly occurring amino acids) through which an HPLC‐derived hydropathy scale, a hydrophobicity threshold for spontaneous membrane insertion, and a nonpolar phase helical propensity scale were determined. Using these scales, the optimized prediction algorithm of TM Finder defines TM segments by first searching for competent core segments using the combination of hydrophobicity and helicity scales, and then performs a gap‐joining operation, which minimizes prediction bias caused by local hydrophilic residues and/or the choice of window size. In addition, the hydrophobicity threshold requirement enables TM Finder to distinguish reliably between membrane proteins and globular proteins, thereby adding an important dimension to the program. A full web version of the TM Finder program can be accessed at http://www.bioinformatics‐canada.org/TM/.

Uncoupling hydrophobicity and helicity in transmembrane segments. Alpha-helical propensities of the amino acids in non-polar environments.

Although the chains of amino acids in proteins that span the membrane are demonstrably helical and hydrophobic, little attention has been paid toward addressing the range of helical propensities of individual amino acids in the non-polar environment of membranes. Because it is inappropriate to apply soluble protein-based structure prediction algorithms to membrane proteins, we have usedde novo designed peptides (KKAAAXAAAAAXAAWAAXAAAKKKK-amide, where X indicates one of the 20 commonly occurring amino acids) that mimic a protein membrane-spanning domain to determine the α-helical proclivity of each residue in the isotropic non-polar environment of n-butanol. Peptide helicities measured by circular dichroism spectroscopy were found to range from θ222 = −17,000 ° (Pro) to −38,800 ° (Ile) inn-butanol. The relative helicity of each amino acid is shown to be well correlated with its occurrence frequency in natural transmembrane segments, indicating that the helical propensity of individual residues in concert with their hydrophobicity may be a key determinant of the conformations of protein segments in membranes.

The hydrophobicity threshold for peptide insertion into membranes

Abstract Peptides designed as model transmembrane segments are shown to insert spontaneously from water into micellar membranes only when their mean residue hydrophobicity is equivalent to or greater than a polyalanine segment. By using circular dichroism-derived peptide helicity as a probe of membrane insertion and correlating high-performance liquid chromatography-derived peptide hydrophobicity with helicity, we were able to determine quantitatively the hydrophobicity threshold. This analysis allows (1) assignment of hydropathy indices to the 20 commonly occurring amino acids and (2) assessment of the membrane insertion potential of a given transmembrane segment as “all-or-nothing,” depending upon whether its segmental hydrophobicity, calculated from the indices of its component amino acids, surpasses or fails to meet the minimum hydrophobicity requirement for integration into the membrane. The minimum hydrophobicity threshold was found to be satisfied by >96% of over 5000 transmembrane segments derived from a database of single- and multispanning intrinsic membrane proteins. When applied in vivo , the notion of “threshold hydrophobicity” would allow the selective incorporation of transmembrane segments into the lipid bilayer during the biosynthetic translocation process without the requirement of any additional expenditure of energy.

Helicity of hydrophobic peptides in polar vs. non-polar environments

The helical contents of a series of designed Ala-based host–guest hydrophobic peptides (where the guest residues include the 20 commonly occurring amino acids) were determined in both polar (aqueous solution) and non-polar (n-butanol) environments using circular dichroism spectroscopy. While the relative helicities of these peptides in water (Ppolar) agree well with the helical propensity predicted by Chou and Fasman (Annu. Rev. Biochem., 1978, 47, 251) for globular proteins, their helicities in n-butanol (Pnon-polar) correlate loosely with the relative hydrophobicities of the peptides as determined by HPLC retention times. To determine the relevance of helical propensity demonstrated in these model peptides to membrane proteins, we analyzed a database of 5444 transmembrane (TM) segments and 4196 non-TM helices. The amino acid frequency of the non-TM segments is correlated (71%) with Ppolar, while the amino acid frequency of the TM segments is correlated (85%) with Pnon-polar. Further, when mean residue helicity is computed for TM and non-TM helices of comparable length using Ppolar and Pnon-polar, we found that the TM segments have 64% overlap with the non-TM helices in terms of polar helicity, but only 17% overlap in terms of non-polar helicity. The results suggest that despite the necessity to fulfill the requirement of hydrophobicity by using bulky side chains and aromatic residues, the TM segment nevertheless must maintain a relatively high level of helicity in order to assume a stable helical conformation in the membrane.

δ-Regions in proteins: Helices mispredicted as transmembrane segments by the threshold hydrophobicity requirement

Previous studies on model peptides with the prototypical sequence, KKAAAXAAAAAXAAWAAXAAAKKK-amide have demonstrated the existence of a threshold hydrophobicity which dictates the process of spontaneous membrane insertion [1,2]. It was found that the hydrophobicity requirement can be combined with the helicity propensity of the amino acids in the membrane environment [3,4] to produce better predictions than the commonly used KD [5] or GES scales [6]. The notion of threshold hydrophobicity was supported by a survey of over 5000 transmembrane (TM) helices and over 4000 globular helices. It was found that over 96% of TM segments are above the threshold hydrophobicity while the majority of globular helices fail to satisfy the minimal hydrophobicity requirement. In this study, we examined those globular helices that have hydrophobicity above the threshold requirement and compared them with the regular TM segments in search of ways to improve the accuracy of TM prediction.

Interplay of hydrophobicity and electrostatics in peptide-bilayer interactions

In previous studies, we established a ‘threshold hydrophobicity’ that dictates the interaction of peptides with membranes [1,2]. Based on this ‘threshold hydrophobicity’ hypothesis, membrane-interactive sequences can be divided into two groups: (1) intrinsically hydrophobic; and (2) hydrophobic/hydrophilic. The first group, which possesses a generally non-polar primary sequence of segmental hydrophobicity above the ‘threshold value,’ can insert into membranes spontaneously from an aqueous phase. The second group, whose segmental hydrophobicity is below the ‘threshold hydrophobicity’ because it contains some relatively polar side chains, can nevertheless interact with membranes efficiently if facilitated by intermediate assistance through electrostatic binding to oppositely-charged lipid head groups [2]. In this work, we describe two peptides that have similar structural features but differ from each other in hydrophobicity [1]. Their interactions with negatively-charged phospholipid vesicles are evaluated quantitatively and the salt effects on these interactions are examined.