Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme

Quenched fluorescence peptides were used to investigate the substrate specificity requirements for recombinant wild-type angiotensin I-converting enzyme (ACE) and two full-length mutants bearing a single functional active site (N- or C-domain). We assayed two series of bradykinin-related peptides flanked by o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp), namely, Abz-GFSPFXQ-EDDnp and Abz-GFSPFRX-EDDnp (X = natural amino acids), in which the fluorescence appeared when Abz/EDDnp are separated by substrate hydrolysis. Abz-GFSPFFQ-EDDnp was preferentially hydrolyzed by the C-domain while Abz-GFSPFQQ-EDDnp exhibits higher N-domain specificity. Internally quenched fluorescent analogues of N-acetyl-SDKP-OH were also synthesized and assayed. Abz-SDK(Dnp)P-OH, in which Abz and Dnp (2,4-dinitrophenyl) are the fluorescent donor-acceptor pair, was cleaved at the D-K(Dnp) bond with high specificity by the ACE N-domain (k(cat)/K(m) = 1.1 microM(-)(1) s(-)(1)) being practically resistant to hydrolysis by the C-domain. The importance of hydroxyl-containing amino acids at the P(2) position for N-domain specificity was shown by performing the kinetics of hydrolysis of Abz-TDK(Dnp)P-OH and Abz-YDK(Dnp)P-OH. The peptides Abz-YRK(Dnp)P-OH and Abz-FRK(Dnp)P-OH which were hydrolyzed by wild-type ACE with K(m) values of 5.1 and 4.0 microM and k(cat) values of 246 and 210 s(-)(1), respectively, have been shown to be excellent substrates for ACE. The differentiation of the catalytic specificity of the C- and N-domains of ACE seems to depend on very subtle variations on substrate-specific amino acids. The presence of a free C-terminal carboxyl group or an aromatic moiety at the same substrate position determines specific interactions with the ACE active site which is regulated by chloride and seems to distinguish the activities of both domains.     

Determination of chemical properties of individual histidine and tyrosine residues of concanavalin A by competitive labeling with 1-fluoro-2,4-dinitrobenzene

A selective peptide-mapping procedure was devised to purify peptides containing histidine or tyrosine residues from proteolytic digests of concanavalin A (Con A). The protein was modified with maleic anhydride followed by 1-fluoro-2,4-dinitrobenzene (Dnp-F) and then digested with thermolysin. The resulting labeled peptides were separated by high-performance liquid chromatography, and the Dnp-histidine and Dnp-tyrosine peptides were identified by their spectral characteristics. From their amino acid compositions, the labeled peptides could all be assigned within the known sequence. Peptides representing five of the six histidines and all seven tyrosines were obtained. With the same peptide-mapping procedure, the chemical properties (pK and reactivity) of these residues were determined. Samples of concanavalin A at various pH values were labeled with trace amounts of [3H]Dnp-F, in the presence of Gln-Gly as an internal standard. To each sample was added an aliquot of a mixture of [14C]Dnp-Gln-Gly and [14C]Dnp-maleyl-Con A. Portions of each sample were removed, [14C]Dnp-Ala-Ala and epsilon-[14C]Dnp-lysine were added, and the mixtures were hydrolyzed. The various Dnp amino acid derivatives were purified by HPLC. The remainder of each [3H]Dnp sample was maleylated, dinitrophenylated, and digested with thermolysin and separated by HPLC as above. From the 3H/14C ratios of the Dnp amino acid derivatives and the Dnp peptides relative to the ratio of the internal standard, pK and reactivity data were obtained for (a) the average behavior of the lysine, histidine, and tyrosine residues and (b) the individual behavior of the N-terminal alanine residue and the five histidine and seven tyrosine residues in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides for defining substrate specificity of the angiotensin I-converting enzyme and development of selective C-domain substrates

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides were used for the analyses of the S(3) to S(1)' subsites of the somatic angiotensin I-converting enzyme (ACE). Substrate specificity of ACE catalytic domains (C- and N-domains) was assessed in an effort to design selective substrates for the C-domain. Initially, we defined the S(1) specificity by preparing a library with the general structure Abz-GXXZXK(Dnp)-OH [Abz = o-aminobenzoic acid, K(Dnp) = N(epsilon)-2,4-dinitrophenyllysine, and X is a random residue], where Z was successively occupied with one of the 19 natural amino acids with the exception of Cys. The peptides containing Arg and Leu in the P(1) position had higher C-domain selectivity. In the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH, Arg was fixed at P(1) so we could define the C-domain selectivity of the S(1)', S(2), and S(3) subsites. On the basis of the results from these libraries, we synthesized peptides Abz-GVIRFK(Dnp)-OH and Abz-GVILFK(Dnp)-OH which contain the most favorable residues for C-domain selectivity. Systematic reduction of the length of these two peptides resulted in Abz-LFK(Dnp)-OH, which demonstrated the highest selectivity for the recombinant ACE C-domain (k(cat)/K(m) = 36.7 microM(-1) s(-1)) versus the N-domain (k(cat)/K(m) = 0.51 microM(-1) s(-1)). The substrate binding of Abz-LFK(Dnp)-OH with testis ACE using a combination of conformational analysis and molecular docking was examined, and the results shed new light on the binding characteristics of the enzyme.     

Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L

The S1 and S2 subsite specificity of recombinant human cathepsins X was studied using fluorescence resonance energy transfer (FRET) peptides with the general sequences Abz-Phe-Xaa-Lys(Dnp)-OH and Abz-Xaa-Arg-Lys(Dnp)-OH, respectively (Abz=ortho-aminobenzoic acid and Dnp=2,4-dinitrophenyl; Xaa=various amino acids). Cathepsin X cleaved all substrates exclusively as a carboxymonopeptidase and exhibited broad specificity. For comparison, these peptides were also assayed with cathepsins B and L. Cathepsin L hydrolyzed the majority of them with similar or higher catalytic efficiency than cathepsin X, acting as an endopeptidase mimicking a carboxymonopeptidase (pseudo-carboxymonopeptidase). In contrast, cathepsin B exhibited poor catalytic efficiency with these substrates, acting as a carboxydipeptidase or an endopeptidase. The S1' subsite of cathepsin X was mapped with the peptide series Abz-Phe-Arg-Xaa-OH and the enzyme preferentially hydrolyzed substrates with hydrophobic residues in the P1' position.     

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC⁰-AngI and TOAC¹-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe⁸-His⁹ peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr⁴ fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.     

Identification of essential tyrosine and lysine residues in angiotensin converting enzyme: evidence for a single active site

Inactivation of rabbit lung angiotensin converting enzyme (ACE) by 1-fluoro-2,4-dinitrobenzene (Dnp-F) has been shown to be due primarily to the modification of a tyrosine residue [Bünning, P., Kleeman, S.G., & Riordan, J.F. (1990) Biochemistry (preceding paper in this issue)]. Rabbit testicular ACE is also inactivated by Dnp-F. The specific site of modification has been identified by peptide mapping of tryptic digests of the Dnp-modified protein. Two principal 340-nm-absorbing peaks, not observed with protein modified in the presence of inhibitor, have been characterized. Amino acid and sequence analyses show that these peptides contain two distinct residues that have been selectively modified. The sequence of the major (greater than 90% of the total) modified peptide is YVEFTNK with the Dnp group on tyrosine. The sequence of the second, minor peptide is KVQDLQR with the Dnp group on lysine. Identical peptides were obtained from Dnp-modified rabbit lung ACE. These modified amino acids correspond to residues 200 and 118, respectively, in testicular ACE (human enzyme numbering). Both peptides are present only in the carboxy-terminal half-domain of lung ACE, corresponding to residues 776 and 694, respectively. These results indicate that the Dnp-F sensitive, catalytically functional active site is located in the "testicular" half of lung ACE.     

Selective isolation of free and blocked amino-terminal peptides from enzymatic digestion of proteins

A general method for the selective isolation of free and blocked amino-terminal peptides from proteins is described. The rationale behind the methodology is based on the reasoning that if a protein, which has all its free amino groups blocked by citraconylation, is digested with a protease, all peptides, except those derived from the amino terminus, will have a free amino group. Reaction of such a digest with 1-fluoro-2,4-dinitrobenzene (Dnp-F) followed by removal of citraconyl groups by acid treatment and removal of dinitrophenyl (Dnp) groups from histidine and tyrosine side chains by thiolysis will result in dinitrophenylation of all alpha-amino groups of peptides generated from internal cleavages, leaving only peptides derived from the amino terminus without a Dnp group. The strong adsorption of Dnp groups to polystyrene is used to selectively elute the underivatized amino-terminal peptides from such a column. It is also demonstrated how selective isolation of amino-terminal peptides can be used to determine whether a protein has a free or blocked amino terminus.     

CD exciton chirality method for determination of the absolute configuration of beta-hydroxy-alpha-amino acid derivatives

The absolute configuration of beta-hydroxy-alpha-amino acids was studied by CD exciton chirality method using 7-diethylaminocoumarin-3-carboxylate as a red-shifted chromophore. The CD spectra of bischromophoric derivatives of (S)-serine and (2S,3R)-threonine methyl esters (2 and 7) were compared with those of acyclic vic-aminoalcohols and diols (3--6 and 8--9). This study indicates that the polar carboxylate group of beta-hydroxy-alpha-amino acids makes them a unique subclass of vic-aminoalcohols. By combining the data of CD and NMR coupling constants, we are able to correlate their preferred conformer B and positive CD to the corresponding absolute configuration.     

Induction of an antibody response in vitro against synthetic antigens in guinea pigs. I. Culture and assay conditions

Guinea pig spleen and lymph node cells were found to produce anti-2,4-dinitrophenyl (Dnp) oligolysine PFC in vivo against 2,4-dinitrophenyl-β-alanyl glycyl glycyl (Dagg-SRBC) but not against trinitrophenyl-SRBC target indicator cells. Furthermore, when sensitized spleen cells or their purified B-cell fractions were cocultured with primed peritoneal exudate lymphocytes (PEL) but not splenic T cells they were able to generate a secondary PFC response in vitro to the synthetic antigens, Dnp oligolysines. PFC were not induced in vitro if these same cultures were pulsed with short-chain peptides (five lysines) or the complex antigen, dinitrophenyl-bovine γ-globulin (DnpBGG). Con A was able to substitute for PEL in triggering spleen cells to mount a secondary in vitro PFC response to homologous Dnp oligolysines. More importantly, the Con A-aided spleen cell cultures were not induced above background values when challenged in vitro with heterologous Dnp oligolysines. This study suggests that spleen cells may lack a nonspecific signal for the development of a secondary in vitro PFC response.     

Amino acid sequencing by gas chromatography-mass spectrometry using perfluoro-dideuteroalkylated peptide derivatives: B. Interpretation of the mass spectra

The mass spectra of the O-trimethylsilylated trifluoro-dideuteroethyl polyamino alcohols, produced by LiAlD₄-reduction and O-trimethylsilylation of N-trifluororacetyl oligopeptide methyl esters, are evaluated. Characteristic mass spectra of derivatives are shown which are derived from peptides containing all protein amino acids including Arg, His, Trp, Gln, Asn and carboxyl terminal amides as well as modified Cys-residues. The mass spectra of these derivatives can be easily interpreted in terms of the amino acid sequence of the original peptides since they contain abundant and intensity-balanced sequence-determining ions.     

Chiral discrimination phenomena in mixed-ligand copper(II) complexes with amino acids and 1,3-dicarbonyl compounds

Circular dichroism (CD) spectra of individual mixed-ligand copper(II) complexes of 1,3-dicarbonyl compounds, (1S)- or (1R)-3-hydroxymethylene camphor, (1S)-3-trifluoroacetyl camphor, or (1R)-2-hydroxymethylene menthone, and α-amino acids, alanine, valine, proline, or their N-alkyl derivatives, were calculated from CD spectra of equilibrium solutions containing the above constituents in methanol or ethylene dichloride. Diastereomeric mixed-ligand complexes incorporating identical dicarbonyl but enantiomeric N-alkyl-α-amino acid ligands exhibit quasi-enantiomeric CD spectra. Unsubstituted amino acids, on the contrary, will make no decisive contributions to the net optical activity spectrum of the mixed-ligand complexes. Formation constants of diastereomeric mixed-ligand complexes have been calculated from data on disproportionation of the latter into corresponding equally paired complexes. Enantioselectivity was demonstrated to amount to up to 700 cal/mol. Possible steric structures of mixed-ligand complexes are discussed. © 1993 Wiley-Liss, Inc.     

The relative order of helical propensity of amino acids changes with solvent environment

A model peptide of sequence Ac-Y-VAXAK-VAXAK-VAXAK-NH(2), where X is substituted with one of nineteen amino acids (P excluded), was synthesized and titrated with methanol to study helical propensity as a function of solvent environment. The CD spectra of these peptides are largely random coil in 2 mM sodium phosphate buffer (pH 5.5) and show a conformational change to alpha-helix with increasing methanol content. Singular value decomposition was used to correct the CD spectra for the absorbing side chains of W, Y, F, C, and M, and this correction can be substantial. With correction both W and F become good helix formers. The free energy for helix propagation was calculated using the Lifson-Roig statistical model for each of the nineteen amino acids at each point in their titration. The results show that the rank order of helical propensity for the nineteen amino acids changes with solvent environment. This result will be particularly important if proteins undergo hydrophobic collapse before secondary structures are formed, because amino acids can then see different solvent environments as the secondary structures are formed. Related amino acids are found to have interesting correlations in the shape of their titration curves. This finding provides one explanation for the limiting 70% accuracy in predicting secondary structure from sequence, since the helical propensities used are calculated for an average solvent environment. Proteins 2000;39:132-141.     

Sequence-dependence of secondary structure formation: conformational studies of host-guest peptides in alpha-helix and beta-structure supporting media

A newly designed host–guest approach is introduced as a experimental tool to explore the relationship between the sequence of peptides and their secondary structure. From the CD spectra of the host–guest peptides studied, a tentative scale for the α-helix potential in 2,2,2-trifluorethanol of guest amino acids is delineated. The conformational preferences are also examined in β-structure supporting media (solid state, CH₂Cl₂, CH₃OH, H₂O) using ir-absorption and CD techniques. Scales for the β-forming tendency of guest amino acid residues in the different media are delineated. It is shown that the preferred conformation of the host–guest peptides is a function of the medium, the chain length, and the protecting groups. Given the fact that conformational effects are important in peptide synthesis, the tentative scales may serve as a guideline to predict secondary structures of side-chain-protected or -deprotected peptides in a given solvent, complementing the well-known empirical conformational prediction parameters.     

Glycosylation of synthetic peptides breaks helices. Phosphorylation results in distorted structure.

Two proposed glycosylation sites are located within T cell epitopes of rabies virus glycoprotein, namely VVEDEGCTNLSGF (VF13; amino acids 29-41) and GKAYTIFNKTLM (GM12; amino acids 312-323). To explore the effects on peptide conformation due to post-translational modifications, we synthesized glycosylated and phosphorylated versions of the two peptides and compared their structures with the native peptide using CD and FT-IR spectroscopy. After the modifications, i.e., glycosylation on Asn with one or two N-acetyl-glucosamine or glucose residues or phosphorylation on Ser, the low to medium degree of helicity of the unmodified peptides disappears as indicated by CD measurements in water-trifluoroethanol mixtures. Incorporation of one sugar moiety into either peptide resulted with a high probability in a type I (III) beta-turn formation with almost identical spectra for the different peptides. Elongation of the carbohydrate in GM12 only slightly enhanced this effect. In contrast, phosphorylation of VF13 caused distorted conformation of the peptide backbone. This novel and direct demonstration of a change in secondary structure by glycosylation (or phosphorylation) might be an important element in determining peptide antigen structure and function.     

Combined effect of the DeltaPhe or DeltaAla residue and the p-nitroanilide group on a didehydropeptides conformation

Two series of dehydropeptides of the general formulae Boc-Gly-X-Phe-p-NA, Boc-Gly-Gly-X-Phe-p-NA, Gly-X-Gly-Phe-p-NA.TFA, and Boc-Gly-X-Gly-Phe-p-NA, with X = Delta(Z)Phe and DeltaAla, were studied with NMR in DMSO and CDCl(3)-DMSO, and with CD in MeOH, MeCN, and TFE. The NMR spectra measured in DMSO suggest that peptides with the DeltaPhe residue next to Phe are folded whereas peptides with Gly between DeltaPhe and Phe are less ordered. NMR spectra of DeltaAla-containing peptides indicate that these peptides are flexible and their conformational equilibria are populated by many different conformations. The CD spectra show that conformational properties of the peptides studied are distinctly influenced by a mutual position of the dehydroamino acid residue and the p-NA group. They indicate that all dehydropeptides with the DeltaPhe residue, Boc-Gly-DeltaAla-Phe-p-NA, and Boc-Gly-Gly-DeltaAla-Phe-p-NA adopt ordered conformations in all solvents studied, presumably of the beta-turn type. The last two peptides exhibit surprising chiroptical properties. Their spectra show exciton coupling-like couplets in the region of the p-NA group absorption. This shape of CD spectra suggests a rigid, chiral conformation with a fixed disposition of the p-NA group. The CD spectra indicate that Boc-Gly-DeltaAla-Gly-Phe-p-NA and Gly-DeltaAla-Gly-Phe-p-NA.TFA are unordered, independently of the solvent.     

Relationship between Bitterness of Peptides and their Chemical Structures

Various peptides and derivatives of peptides and amino acids were synthesized and tasted, systematically, to elucidate the relationship between bitterness and chemical structures of peptides.

We have found that: 1. Peptides become more bitter than the original amino acids when their amino and carboxyl groups are blocked and when peptide bond is formed. 2. A peptide molecule with a high content of amino acids with hydrophobic side chains will develop bitter taste. 3. The amino acids in a peptide chain independently contribute to bitterness regardless of amino acid sequences and configuration.     

Interaction of dinitrophenyl groups bound to bovine serum albumin with univalent fragments of anti-dinitrophenyl antibody

Two lysine residues of bovine serum albumin reacted with 1-fluoro-2,4-dinitrobenzene with apparent second-order rate constants approx. 500-times greater than those observed in similar reactions with low-molecular-weight lysine derivatives. A series of dinitrophenyl (Dnp)-bovine serum albumins were prepared and their ability to bind univalent fragments of anti-Dnp antibody was measured by fluorescence-quenching titrations. Compared with the Dnp group of the free hapten, 6-N-Dnp-aminohexanoate, the majority of the protein-bound Dnp groups were unavailable to the antibody at pH8.0. When the same Dnp-albumins were titrated at pH3.0 the availability of the Dnp groups increased approx. 3-fold. Dnp-albumins were treated with pepsin at pH3.0 and Dnp-containing fragments isolated by chromatography on DE-52 DEAE-cellulose. Fluorescence-quenching titrations showed that the Dnp groups on the fragments behaved like the free hapten with respect to quenching efficiency, although with an increased dissociation constant. The association between the Dnp-albumins and the antibody was measured also by difference-spectral titrations at high protein concentrations. Antibody binding was increased under these conditions, but the Dnp group of mono-Dnp-albumin remained unavailable to antibody. We propose that the reactive lysine residues are located in clefts between the globular sub-domains of the single polypeptide chain. Dnp groups attached to these lysine residues are fully exposed to the solvent, but binding of the macromolecular probe, anti-Dnp antibody, is sterically hindered by the adjacent surface of the albumin molecule.     

Application of (S)‐TBMB carboxylic acid‐based on‐line HPLC‐exciton CD analysis to acyclic vicinal alcohols and amino alcohols

Applications of the on‐line HPLC‐exciton CD analysis using (S)‐2‐tert‐butyl‐2‐methyl‐1,3‐benzodioxole‐4‐carboxylic acid [(S)‐TBMBC‐OH] that can simultaneously determine the enantiomeric compositions and the absolute configuration of cyclohexane‐1,2‐diols and diamines as well as acyclic vicinal diols and amino alcohols were studied. Di‐O‐ or di‐N,O‐(S)‐TBMBC derivatives of acyclic terminal vicinal diols, 2‐hydroxy‐1‐amines, and nonterminal vicinal diols gave symmetrical exciton CD spectra between enantiomers, indicating their absolute configurations. However, Di‐N,O‐(S)‐TBMBC derivatives of 2‐amino‐1‐ols did not always give symmetrical exciton CD spectra between enantiomers, but their 2‐phthalimido‐1‐O‐(S)‐TBMBC derivatives gave symmetrical exciton CD spectra, indicating their absolute configurations. All these (S)‐TBMBC derivatives were separated by normal‐phase HPLC and unequivocally determined by the on‐line HPLC‐exciton CD analysis without recourse to reference samples. Chirality 11:149–159, 1999. © 1999 Wiley‐Liss, Inc.     

Synthesis by conventional methods of human growth hormone peptide fragments. I.

The synthesis of two protected peptides which correspond to positions 139-146 and 147-156 of the HGH primary structure is described. These peptides prepared by the stepwise procedure, are: Boc-Phe-Lys(Z)-Gln-Thr(Bzl)-Ser(Bzl)-Lys(Z)-Phe-OMe and Boc-Asp(OBzl)-Asn-Ser(Bzl)-His(Dnp)-Asn(OBzl)-Asp(OBzl)-Ala-Leu-OBzl. All protected intermediates were isolated and characterized for homogeneity.     

CD Spectroscopy of Peptides and Proteins Bound to Large Unilamellar Vesicles

Circular dichroism (CD) spectroscopy is an essential tool for determining the conformation of proteins and peptides in membranes. It can be particularly useful for measuring the free energy of partitioning of peptides into lipid vesicles. The belief is broadly held that such CD measurements can only be made using sonicated small unilamellar vesicles (SUVs) because light scattering associated with extruded large unilamellar vesicles (LUVs) is unacceptably high. We have examined this issue using several experimental approaches in which a chiral object (i.e., peptide or protein) is placed both on the membrane and outside the membrane. We show that accurate CD spectra can be collected in the presence of LUVs. This is important because SUVs, unlike LUVs, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. Our data reveal that undistorted CD spectra of peptides can be measured at wavelengths above 200 nm in the presence of up to 3 mM LUVs and above 215 nm in the presence of up to 7 mM LUVs. We introduce a simple way of characterizing the effect on CD spectra of light scattering and absorption arising from suspensions of vesicles of any diameter. Using melittin as an example, we show that CD spectroscopy can be used to determine the fractional helical content of peptides in LUVs and to measure their free energy of partitioning of into LUVs.