The membrane-bound conformation of alpha-lactalbumin studied by NMR-monitored 1H exchange

The interaction of bovine alpha-lactalbumin (BLA) with negatively charged phospholipid bilayers was studied by NMR monitored 1H exchange to characterize the conformational transition that enables a water-soluble protein to associate with and partially insert into a membrane. BLA was allowed to exchange in deuterated buffer in the absence (reference) and the presence (membrane-bound) of acidic liposomes at pH 4.5, experimental conditions that allow efficient protein-membrane interaction. After adjusting the pH to 6.0, to dissociate the protein from the membrane, reference and membrane-released samples of BLA were analysed by (F1) band-selective homonuclear decoupled total correlation spectroscopy in the alphaH-NH region. The overall exchange behaviour of the membrane-bound state is molten globule-like, suggesting an overall destabilization of the polypeptide. Nevertheless, the backbone amide protons of residues R10, L12, C77, K94, K98, V99 and W104 show significant protection against solvent exchange in the membrane-bound protein. We propose a mechanism for the association of BLA with negatively charged membranes that includes initial protonation of acidic side-chains at the membrane interface, and formation of an interacting site with the membrane which involves helixes A and C. In the next step these helices would slide away from each other, adopting a parallel orientation to the membrane, and would rotate to maximize the interaction between their hydrophobic residues and the lipid bilayer.     

Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two-dimensional NMR.

In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein-ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO-D2O mixtures was investigated by NMR methods. As in water, H-D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid- and base-catalyzed processes and negligible water catalysis. However, the base-catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is approximately 100-fold reduced relative to that in D2O. The solvent-dependent decrease in base-catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKW seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent-quenching method, melittin was exchanged in D2O (pH 3.2, 12 degrees C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.     

NMR spectroscopy as a tool for the rapid assessment of the conformation of GST-fusion proteins

Glutathione-S-transferase (GST)-fusion proteins are used extensively for structural, biochemical, and functional analyses. Although the conformation of the target protein is of critical importance, confirmation of the folded state of the target is often not undertaken or is cumbersome because of the requirement to first remove the GST tag. Here, we demonstrate that it is possible to record conventional (15)N-HSQC NMR spectra of small GST-fusion proteins and that the observed signals arise almost exclusively from the target protein. This approach constitutes a rapid and straightforward means of assessing the conformation of a GST-fusion protein without having to cleave the GST and should prove valuable, both to biochemists seeking to check the conformation of their proteins prior to functional studies and to structural biologists screening protein constructs for suitability as targets for structural studies.     

The interactions of the HIV gp41 fusion peptides with zwitterionic membrane mimics determined by NMR spectroscopy

The wild-type (wt) N-terminal 23-residue fusion peptide (FP) of the human immunodeficiency virus (HIV) fusion protein gp41 and its V2E mutant have been studied by nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles as membrane mimics. A number of NMR techniques have been used. Pulsed field-gradient diffusion measurements in DPC and in 4:1 DPC/sodium dodecylsulfate mixed micelles showed that there is no major difference between the partition coefficients of the fusogenic wt peptide and the V2E mutant in these micelles, indicating that there is no correlation between the activity of the fusion peptides and their membrane affinities. The nuclear Overhauser enhancement (NOE) patterns and the chemical shift index for these two peptides indicated that both FP are in an α helical conformation between the Ile⁴ to Leu¹² or to Ala¹⁵ region. Simulated annealing showed that the helical region extends from Ile⁴ to Met¹⁹. The two FPs share similar conformational characteristics, indicating that the conformation of the FP is not an important factor determining its activity. The spin-label studies, utilizing spin labels 5- and 16-doxystearic acids in the DPC micelles, provided clear indication that the wt FP inserts its N-terminus into the micelles while the V2E mutant does not insert into the micelles. The conclusion from the spin-label results is corroborated by deuterium amide proton exchange experiments. The correlation between the oblique insertion of the FP and its fusogenic activity is in excellent agreement with results from our molecular dynamics simulation and from other previous studies.     

Determination of the degree of acetylation of chitin materials by 13C CP/MAS NMR spectroscopy

¹³C CP/MAS NMR spectroscopy has been shown to be a powerful tool to quantify the degree of acetylation of chitin and chitosan. In order to optimise the parameters which afford quantitative ¹³C cross-polarisation magic-angle spinning NMR spectra, a detailed relaxation study has been carried out on selected chitin and deacetylated chitin samples. A relaxation delay of 5 s and a contact time of 1 ms have been found to yield quantitative NMR spectra of samples with deacetylation degree values of 0.68 and 0.16. The measured spin-lattice relaxation times in the rotating frame, T_1ρH, are in the range 6.4–8.9 ms for chitin and 4.3–7.3 ms for deacetylated chitin, while T_CH values for both samples are very similar and range from 0.03 to 0.19 ms. Spin-counting experiments indicate that, within experimental error, all carbon is detected by NMR indicating that the samples studied contain no (or very few) paramagnetic centres.     

Bioactive conformation of a potent stromelysin inhibitor determined by X-nucleus filtered and multidimensional NMR spectroscopy

The biologically active conformation of a novel, very potent, nonpeptidic stromelysin inhibitor was determined by X-nucleus filtered and multidimensional NMR spectroscopy. This bound conformer was subsequently docked into the stromelysin catalytic domain (SCD) using intermolecular distance constraints derived from NOE data. The complex showed the S1′ pocket of stromelysin to be the major site of enzyme-inhibitor interaction with other portions of the inhibitor spanning the S2′ and S1 binding sites. Theoretical predictions of SCD-inhibitor binding from molecular modeling studies were consistent with the NMR data. Comparison of modeled enzyme-inhibitor complexes for stromelysin and collagenase revealed an alternate binding mode for the inhibitor in collagenase, suggesting a similar binding interaction might also be possible for stromelysin. The NMR results, however, revealed a single SCD-inhibitor binding mode and provided a structural template for the design of more potent stromelysin inhibitors.     

Observation of slow dynamic exchange processes in Ras protein crystals by 31P solid state NMR spectroscopy

The folding, structure and biological function of many proteins are inherently dynamic properties of the protein molecule. Often, the respective molecular processes are preserved upon protein crystallization, leading, in X-ray diffraction experiments, to a blurring of the electron density map and reducing the resolution of the derived structure. Nuclear magnetic resonance (NMR) is known to be an alternative method to study molecular structure and dynamics. We designed and built a probe for phosphorus solid state NMR that allows for the first time to study static properties as well as dynamic processes in single-crystals of a protein by NMR spectroscopy. The sensitivity achieved is sufficient to detect the NMR signal from individual phosphorus sites in a 0.3mm(3) size single-crystal of GTPase Ras bound to the nucleotide GppNHp, that is, the signal from approximately 10(15) phosphorus nuclei. The NMR spectra obtained are discussed in terms of the conformational variability of the active center of the Ras-nucleotide complex. We conclude that, in the crystal, the protein complex exists in three different conformations. Magic angle spinning (MAS) NMR spectra of a powder sample of Ras-GppNHp show a splitting of one of the phosphate resonances and thus confirm this conclusion. The MAS spectra provide, furthermore, evidence of a slow, temperature-dependent dynamic exchange process in the Ras protein crystal.     

Helix-helix interconversion rates of short 13C-labeled helical peptides as measured by dynamic NMR spectroscopy

The rates at which a peptide hexamer and a peptide octamer interconvert between left- and right-handed helical forms in CD2Cl2 solution have been characterized by 13C dynamic NMR (DNMR) spectroscopy. The peptide esters studied are Fmoc-(Aib)n-OtBu (n = 6 and 8), where Fmoc is 9-fluorenylmethyoxycarbonyl and Aib is the strongly helix-forming residue alpha-aminoisobutyric acid. Because the Aib residue is itself achiral, homooligomers of this residue form a 50/50 mixture of enantiomeric 3(10)-helices in solution. It has been demonstrated (R.-P. Hummel, C. Toniolo, and G. Jung, Angewandte Chemie International Edition, 1987, Vol. 26, pp. 1150-1152) that oligomers of Aib interconvert on the millisecond timescale. We have performed lineshape analysis of 13C-NMR spectra collected for our peptides enriched with 13C at a single residue. Rate constants for the octamer range from 6 s(-1) at 196 K to about 56,500 s(-1) at 320 K. At all temperatures, the hexamer interconverts about three times faster than the octamer. Eyring plots of the data reveal experimentally indistinguishable DeltaH++ values for the hexamer and octamer of 37.8 +/- 0.6 and 37.6 +/- 0.4 kJ mol(-1) respectively. The difference in the rates of interconversion is dictated by entropic factors. The hexamer and octamer exhibit negative DeltaS++ values of -29.0(-1) +/- 2.5 and -37.3 +/- 1.7 J K(-1) mol(-1), respectively. A mechanism for the helix-helix interconversion is proposed. and calculated DeltaG++ values are compared to the estimate for a decamer undergoing a helix-helix interconversion.     

Structural biology of transmembrane domains: efficient production and characterization of transmembrane peptides by NMR

Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.     

Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy.

Magainin 2 is a 23-residue peptide that forms an amphipathic alpha-helix in membrane environments. It functions as an antibiotic and is known to disrupt the electrochemical gradients across the cell membranes of many bacteria, fungi, and some tumor cells, although it does not lyse red blood cells. One- and two-dimensional solid-state 15N NMR spectra of specifically 15N-labeled magainin 2 in oriented bilayer samples show that the secondary structure of essentially the entire peptide is alpha-helix, immobilized by its interactions with the phospholipids, and oriented parallel to the membrane surface.     

Characterization of secondary amide peptide bond isomerization: thermodynamics and kinetics from 2D NMR spectroscopy

Secondary amide cis peptide bonds are of even lower abundance than the cis tertiary amide bonds of prolines, yet they are of biochemical importance. Using 2D NMR exchange spectroscopy (EXSY) we investigated the formation of cis peptide bonds in several oligopeptides: Ac-G-G-G-NH(2) , Ac-I-G-G-NH(2) , Ac-I-G-G-N-NH(2) and its cyclic form: I-G-G-N in dimethylsulfoxide (DMSO). From the NMR studies, using the amide protons as monitors, an occurrence of 0.13-0.23% of cis bonds was obtained at 296 K. The rate constants for the trans to cis conversion determined from 2D EXSY spectroscopy were 4-9 × 10(-3) s(-1) . Multiple minor conformations were detected for most peptide bonds. From their thermodynamic and kinetic properties the cis isomers are distinguished from minor trans isomers that appear because of an adjacent cis peptide bond. Solvent and sequence effects were investigated utilizing N-methylacetamide (NMA) and various peptides, which revealed a unique enthalpy profile in DMSO. The cyclization of a tetrapeptide resulted in greatly lowered cis populations and slower isomerization rates compared to its linear counterpart, further highlighting the impact of structural constraints.     

Conformational Analysis of the Thrombin Receptor Agonist Peptides SFLLR and SFLLR-NH2 by NMR: Evidence for a Cyclic Bioactive Conformation

The conformational properties of the pentapeptide Ser-Phe-Leu-Leu-Arg (P5), a human thrombin receptor-derived sequence forming part of a tethered ligand which activates the thrombin receptor, and its more active amide derivative Ser-Phe-Leu-Leu-Arg-NH₂ (P5-NH₂), have been studied by proton NMR spectroscopy in dimethylsulfoxide. Measurements of nuclear Overhauser effects, performed using two-dimensional rotating frame nuclear Overhauser (ROESY) and one-dimensional nuclear Overhauser enhancement (NOE) spectroscopy, revealed that P5 exists mainly in an extended conformation. However, proton–proton 1D-NOEs between Phe CH and Ser CH, Leu³ CH and Leu³ NH, and Leu⁴ CH and Leu⁴ NH, as well as between the Ser and Arg sidechains, also implicated a minor conformer for P5 having a curved backbone and a near-cyclic structure. In contrast to P5, measurements of NOEs and ROEs for P5-NH₂ revealed a more stabilized cyclic structure which may account for its higher biological potency. Thus strong interresidue sequential NH (i)–NH (i + 1) interactions, as well as C-terminal carboxamide to N-terminal side-chain interactions, i.e., Arg CONH₂ to Phe ring and Arg CONH₂ to Ser , observed at lower levels of the ROESY spectrum, supported a curved backbone structure for SFLLR-NH₂. Since the higher potaency P5-NH₂ analogue adopts predominantly a cyclic structure, a cyclic bioactive conformation for thrombin receptor agonist peptides is suggested.     

Conformational and biochemical analysis of the cyclic peptides which modulate serine protease activity.

Prostate-specific antigen (PSA), a member of the kallikrein sub-group of the trypsin serine protease family, is a widely used marker for prostate cancer. Several sequences with specific binding to PSA have been identified by using phage display peptide libraries. The GST-fusion proteins of the characterized sequences have been shown to increase the enzyme activity of PSA to a synthetic substrate. The corresponding three cyclic synthetic analogues CVFTSNYAFC (A-1), CVFAHNYNYLVC (B-2) and CVAYCIEHHCWTC (C-4) have similar PSA promoting activity. Despite differences in the amino acid sequences, all three peptides bind to the same region of PSA. The conformation of the peptides was investigated by proton NMR spectroscopy. In addition, alanine replacement was used to characterize the prerequisites for binding. It is proposed that interactions with PSA are based on the aromatic and hydrophobic features of the amino acid side chains. Furthermore, it is suggested that peptides form beta-turn structures forced by cysteine bridges directing important aromatic side chains to the same side of the turn-structure.     

The Ac-RGD-NH2 peptide as a probe of slow conformational exchange of short linear peptides in DMSO

According to general belief, the conformational information on short linear peptides in solution derived at ambient temperature from NMR spectrometry represents a population-weighted average over all members of an ensemble of rapidly interconverting conformations. Usually the search for discrete conformations is concentrated at low temperatures especially when sharp NMR resonances are detected at room temperature. Using the peptide Ac-RGD-NH(2) (Ac-Arg-Gly-Asp-NH(2), Ac: acetyl) as a model system and following a new approach, we have been able to demonstrate that short linear peptides can adopt discrete conformational states in DMSO-d(6) (DMSO: dimethylsulfoxide) which vary in a way critically dependent on the reconstitution conditions used before their dissolution in DMSO-d(6). The conformers are stabilized by intramolecular hydrogen bonds, which persist at high temperatures and undergo a very slow exchange with their extended structures in the NMR chemical shift time scale. The reported findings provide clear evidence for the occurrence of solvent-induced conformational exchange and point to DMSO as a valuable medium for folding studies of short linear peptides.     

Dynamic membrane interactions of antibacterial and antifungal biomolecules,and amyloid peptides,revealed by solid-state NMR spectroscopy

A variety of biomolecules acting on the cell membrane folds into a biologically active structure in the membrane environment. It is, therefore, important to determine the structures and dynamics of such biomolecules in a membrane environment. While several biophysical techniques are used to obtain low-resolution information, solid-state NMR spectroscopy is one of the most powerful means for determining the structure and dynamics of membrane bound biomolecules such as antibacterial biomolecules and amyloidogenic proteins; unlike X-ray crystallography and solution NMR spectroscopy, applications of solid-state NMR spectroscopy are not limited by non-crystalline, non-soluble nature or molecular size of membrane-associated biomolecules. This review article focuses on the applications of solid-state NMR techniques to study a few selected antibacterial and amyloid peptides. Solid-state NMR studies revealing the membrane inserted bent α-helical structure associated with the hemolytic activity of bee venom melittin and the chemical shift oscillation analysis used to determine the transmembrane structure (with α-helix and 3₁₀-helix in the N- and C-termini, respectively) of antibiotic peptide alamethicin are discussed in detail. Oligomerization of an amyloidogenic islet amyloid polypeptide (IAPP, or also known as amylin) resulting from its aggregation in a membrane environment, molecular interactions of the antifungal natural product amphotericin B with ergosterol in lipid bilayers, and the mechanism of lipid raft formation by sphingomyelin studied using solid state NMR methods are also discussed in this review article. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.     

New insight into the binding mode of peptides at urotensin‐II receptor by Trp‐constrained analogues of P5U and urantide

Urotensin II (U‐II) is a disulfide bridged peptide hormone identified as the ligand of a G‐protein‐coupled receptor. Human U‐II (H‐Glu‐Thr‐Pro‐Asp‐c[Cys‐Phe‐Trp‐Lys‐Tyr‐Cys]‐Val‐OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of human U‐II termed P5U (H‐Asp‐c[Pen‐Phe‐Trp‐Lys‐Tyr‐Cys]‐Val‐OH) and the compound termed urantide (H‐Asp‐c[Pen‐Phe‐d ‐Trp‐Orn‐Tyr‐Cys]‐Val‐OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized four analogues of P5U and urantide in which the Trp⁷ residue was replaced by the highly constrained l ‐Tpi and d ‐Tpi residues. The replacement of the Trp⁷ by Tpi led to active analogues. Solution NMR analysis allowed improving the knowledge on conformation–activity relationships previously reported on UT receptor ligands. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Debranched cassava starch crystallinity determination by Raman spectroscopy: Correlation of features in Raman spectra with X-ray diffraction and C CP/MAS NMR spectroscopy

Because starch crystallinity influences the physical, mechanical, and technological aspects of numerous starch-based products during production and storage, rapid techniques for its assessment are vital. Samples of different levels of crystallinity were obtained by debranching gelatinized cassava starch, followed by subjection to various hydrothermal treatments. The recrystallized products were further subjected to partial hydrolysis with a mixture of α-amylase and glucoamylase prior to freeze-drying. Crystallinities were determined using X-ray diffraction (XRD) and ¹³C CP/MAS NMR spectroscopy, and correlated with FT-Raman spectra features. XRD crystallinities ranged between 0 and 58%, and agreed with crystalline-phase fractions (R² = 0.99) derived from the respective ¹³C CP/MAS NMR spectra. A strong linear correlation was found between crystallinities and integrated areas of the skeletal mode Raman band at 480 cm⁻¹ (R² = 0.99). With appropriate calibration, FT-Raman spectroscopy is a promising tool for rapid determination of starch crystallinity.     

Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approaches.

Most synthesized peptides are nowadays produced using solid-phase procedures. Due to cleavage and purification conditions, they are mainly obtained in the presence of trifluoroacetic acid (TFA) and, for cationic peptides, as trifluoroacetate (TF-acetate) salts. However, TF-acetate interferes with physicochemical characterizations using infrared spectroscopy and might significantly affect the in vivo studies. Thus, TF-acetate exchange by another counter-ion is often required. Up to now, the classical procedure has consisted of freeze-drying the peptide several times in the presence of an excess of a stronger acid than TFA (pKa approximately 0): generally HCl (pKa = - 7). This approach means that working at pH < 1 can induce peptide degradation. We therefore tested three different approaches to exchange the tightly bound TF-acetate counter-ion from the dicationic octapeptide lanreotide: (i) reverse-phase HPLC, (ii) ion-exchange resin, and (iii) deprotonation/reprotonation cycle of the amino groups. The first two approaches allow the partial to almost complete exchange of the TF-acetate counter-ion by another ion from an acid weaker than TFA, such as acetic acid (pKa = 4.5), and the third requires a basic solution that permits the complete removal of TF-acetate counter-ion. The efficiency of these three procedures was tested and compared by using different analytical techniques such as 19F-NMR, 1H-NMR and attenuated total reflectance Fourier transformed infrared spectroscopy (ATR FT-IR). We also show that ATR-IR can be used to monitor the TFA removal. The counter-ion exchange procedures described in this study are easy to carry out, fast, harmless and reproducible. Moreover, two of them offer the very interesting possibility of exchanging the initial TF-acetate by any other counter-ion.     

NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins

Summary Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4°C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide¹H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional¹H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.[/p]     

Bilayer stabilizing peptides and the inhibition of viral infection: antimeasles activity of carbobenzoxy-Ser-Leu-amide

A number of carbobenzoxy-dipeptide-amides raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine (stabilizes the bilayer). The potency of the peptides in stabilizing the bilayer phase is Z-Tyr-Leu-NH₂= Z-Gly-Phe-NH₂>Z-Ser-Leu-NH₂>Z-Gly-Leu-NH₂>Z-Gly-Gly-NH₂. A linear correlation was found between the respective HPLC retention time parameterk for the peptide and the slope of the bilayer stabilization curve determined with model membranes by differential scanning calorimetry. One dipeptide, Z-Ser-Leu-NH₂, reduces measles virus cytopathic effect (CPE) in Vero cells. The mechanism by which this peptide reduces the CPE is not known, although some peptides which raise the bilayer to hexagonal phase transition temperature of phospholipids inhibit membrane fusion.Abbreviations Z carbobenzoxy - DEPE dielaidoylphosphatidylethanolamine - DSC differential scanning calorimetry - HPLC high pressure liquid chromatography - CPE cytopathic effect To whom correspondence should be addressed.