Helix formation in poly (N epsilon,N epsilon,N epsilon-trimethyl-L-lysine) and poly (L-lysine): dependence on concentration and molecular weight.

Helix formation in (Lys)n.HClO4 and poly(N epsilon,N epsilon,N epsilon-trimethyl-L-lysine).HClO4 +AD(LysMe3)n.HClO4+BD is dependent on peptide concentration and on molecular weight. For (LysMe3)n.HClO4 of degree of polymerization (DP) 2510 the midpoint of the coil-to-helix transition is 2 mM and for DP of 190 it is 5 mM. For (Lys)n.HClO4 the peptide concentration for half-helix is 30-60 times as high, and is only weakly dependent, if at all, on molecular weight. Helix formation is an intermolecular process. The use of methylated (Lys)n as the perchlorate permits study of the intermolecular coil-helix transition at low concentration, instead of the high concentration (ca. 1-2 M) required for (Lys)n.HBr. At constant peptide concentration helix content increases with added NaClO4. The higher the peptide concentration, the less NaClO4 is needed to induce helix.     

Role of helix nucleation in the kinetics of binding of mastoparan X to phospholipid bilayers

Many antimicrobial peptides undergo a coil-to-helix transition upon binding to membranes. While this conformational transition is critical for function, little is known about the underlying mechanistic details. Here, we explore the membrane-mediated folding mechanism of an antimicrobial peptide, mastoparan X. Using stopped-flow fluorescence techniques in conjunction with a fluorescence resonance energy transfer (FRET) pair, p-cyanophenylalanine (donor) and tryptophan (acceptor), we were able to probe, albeit in an indirect manner, the membrane-mediated folding kinetics of this peptide. Our results show that the association of mastoparan X with model lipid vesicles proceeds with biphasic kinetics. The first step shows a large change in the FRET signal, indicating that the helix forms early in the time course of the interaction, while the second step where a further increase in tryptophan fluorescence is observed presumably reflects deeper insertion of the peptide into the bilayer. Additional kinetic studies on a double mutant of mastoparan X, designed to form a nucleation site for alpha-helix formation through coordination with a metal ion (e.g., Zn2+ or Ni2+), indicate that while the coil-to-helix transition occurs in the first step, it follows the rate-determining docking of the peptide onto the membrane surface. Taken together, these results indicate that the initial association of the peptide with the membrane occurs in a nonhelical conformation, which rapidly converts to a helical state within the anisotropic environment of the bilayer surface.     

Studies of the helix–coil transition of poly-L-lysine in film and solution

Water-insoluble films of poly-L -lysine, crosslinked with formaldehyde, were suspended in aqueous media and their relative lengths measured as a function of pH. A sharp transition of the polymer was observed in the pH range which corresponded with that observed in polylysine solutions by optical rotation or dilatometry. In NaBr and NaCl solutions the coiled form of the polylysine film shrinks with increasing salt concentration, but in NaHCO₃ solution the extent of the contraction is larger, and the coil–helix transition of polylysine occurs at lower pH when NaHCO₃ is added to the medium. If one assumes the formation of amino carbamate in this case, this phenomenon can be well explained. Urea does break up the hydrogen bonds in helical polylysine film, but not completely. This result is interesting compared with that obtained for poly(L -glutamic acid). After the coil–helix transition region was found by film experiments, the volume change associated with the coil-to-helix transition was measured and found to be about 1–l.5 ml. per amino residue after taking electrostatic interaction into consideration. This value is nearly same as that obtained for poly(L -glutamic acid). By contrast, the value for poly-γ-benzyl-L -glutamate was reported to be ?0.077 ml./mole of repeating unit. So it is still necessary to determine the magnitude and direction of the volume change for various kinds of polypeptides.     

Theoretical study of volume changes associated with the helix-coil transition of an alanine-rich peptide in aqueous solution

The changes in the partial molar volume (PMV) associated with the conformational transition of an alanine-rich peptide AK16 from the alpha-helix structure to various random coil structures are calculated by the three-dimensional interaction site model (3D-RISM) theory coupled with the Kirkwood-Buff theory. The volume change is analyzed by decomposing it into contributions from geometry and hydration: the changes in the van der Waals, void, thermal, and interaction volume. The total change in the PMV is positive. This is primarily due to the growth of void space within the peptide, which is canceled in part by the volume reduction resulting from the increase in the electrostatic interaction between the peptide and water molecules. The changes in the void and thermal volume of the coil structures are widely distributed and tend to compensate each other. Additionally, the relations between the hydration volume components and the surface properties are investigated. We categorize coil structures into extended coils with the PMV smaller than helix and general coils with the PMV larger than helix. The pressure therefore can both stabilize and destabilize the coil structures. The latter seems to be a more proper model of random coil structures of the peptide.     

Design, synthesis and structure of an amphipathic peptide with pH-inducible haemolytic activity.

A synthetic, 26-residue peptide having a strong helix forming potential in the protonated state was designed to interact with lipid bilayers in a pH-dependent way. On the basis of this concept a cluster of four glutamic acid residues was inserted in the central region of the amphipathic peptide to promote helix destabilization by mutual charge repulsion at neutral pH. Protonation of these residues might then bring about both a pH-mediated change in hydrophobicity and conformation forming a membrane-active amphiphilic helix. The sequence GLGTLLTLLEFLLEELLEFLKRKRQQamide produced by the design strategy induced pH-triggered lysis of human erythrocytes. A molecular model correlating the lytic activity to the formation of transmembrane pores which were detected by electron microscopy in erythrocyte membranes is discussed. Circular dichroism studies indicated a self-association of the monomeric random coil form with increasing peptide concentration leading to the apparent induction of strong alpha-helix formation (approximately 100% helicity) in the fully aggregated state. However, no pH-dependent helix-random coil transition was observed, implying that interhelical hydrophobic and ionic interactions not only govern the self-association but also decisively influence the conformational stability of the peptide.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

Design and synthesis of a helix heparin-binding peptide.

Elaboration of heparin-protein-binding interactions is necessary to understand how heparin modulates protein function. The heparin-binding domain of some proteins is postulated to be a helix structure which presents a surface of high positive charge density. Thus, a synthetic 19-residue peptide designed to be alpha-helical in character was synthesized, and its interaction with heparin was studied. The peptide was shown to be 75% helix by circular dichroism (CD) spectrometry in neutral pH buffer (at 2 degrees C); helicity increased to nearly 85% under high ionic strength conditions or to nearly 100% in 75% ethanol. Increasing the temperature of the solution caused a change in the spectral envelope consistent with a coil transition of the peptide. The midpoint of the transition (i.e., the temperature at which the helix content was determined to be 50%) was 25 degrees C, and the determined van't Hoff enthalpy change (delta HvH) was 3.2 kcal/mol of peptide. By CD, heparin increases the helix content of the peptide to 100% and increases the apparent thermal stability of the peptide by about 1 kcal/mol. The melting point for the helix/coil transition of the heparin-peptide complex was 50 degrees C. The thermal coefficient of the transition (approximately 300 deg.cm2.dmol-1.degree C-1) was essentially the same for the peptide alone or the peptide-heparin complex. Dissociation of the complex under high ionic strength conditions was also observed in the CD experiment. Biological assays showed less heparin-binding activity than expected (micromolar KD values), but this was attributed to the absence of critical lysyl residues in the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The contribution of residue ion pairs to the helical stability of a model peptide.

Comparative CD measurements were made on the model helical peptides acetylYEAAAKEAXAKEAAAKAamide and acetylYEAAAEKAXAKEAAAKAamide in which X represents a nonaromatic nonionic residue. The former peptide contains three potential i, i + 4 complementary ion pairs at neutral pH, while the latter peptide contains one potential complementary and two potential antagonistic i, i + 4 ion pairs. The effect of pH and ionic strength on the mean residue ellipticity of these peptides was measured at 222 nm and 0 degrees C. These measurements were analyzed assuming a common two-state helix/coil transition and only i, i, + 4 ion-pair interactions. The analyses suggest that the central ion pairs do modulate helical content while the peripheral ion pairs do not, presumably due to the location of the peripheral ion pairs in the frayed ends of the helix. The complementary central ion pair stabilizes the helix by about 0.4 kcal/mole and the antagonistic central ion pair destabilizes the helix by about 0.2 kcal/mole.     

Thermodynamics of the alpha-helix-coil transition of amphipathic peptides in a membrane environment: implications for the peptide-membrane binding equilibrium

Amphipathic alpha-helices are the membrane binding motif in many proteins. The corresponding peptides are often random coil in solution but are folded into an alpha-helix upon interaction with the membrane. The energetics of this ubiquitous folding process are still a matter of conjecture. Here, we present a new method to quantitatively analyze the thermodynamics of peptide folding at the membrane interface. We have systematically varied the helix content of a given amphipathic peptide when bound to the membrane and have correlated the thermodynamic binding parameters determined by isothermal titration calorimetry with the alpha-helix content obtained by circular dichroism spectroscopy. The peptides investigated were the antibiotic magainin 2 amide and three analogs in which two adjacent amino acid residues were substituted by their d-enantiomers. The thermodynamic parameters controlling the alpha-helix formation were found to be linearly related to the helicity of the membrane-bound peptides. Helix formation at the membrane surface is characterized by an enthalpy change of DeltaH(helix) approximately -0.7 kcal/mol per residue, an entropy change of DeltaS(helix) approximately -1.9 cal/molK residue and a free energy change of DeltaG(helix)=-0.14 kcal/mol residue. Helix formation is a strong driving force of peptide insertion into the membrane and accounts for about 50 % of the free energy of binding. An increase in temperature entails an unfolding of the membrane-bound helix. The temperature dependence can be described with the Zimm-Bragg theory and the enthalpy of unfolding agrees with that deduced from isothermal titration calorimetry.     

Analysis of peptides for helical prediction

Two terminally blocked peptides, acetylAETAAAKFLRQHMamide and acetylAETSSSRYLRQHMamide, were obtained by solid-phase synthesis, purified by reversed-phase chromatography, and characterized by fast atom bombardment mass spectrometry. Both peptides were soluble in aqueous solutions and remained monomeric over the concentration range examined. Changes in the temperature, pH, and trifluoroethanol concentration of solutions of each peptide produced changes in the far-ultraviolet circular dichroic spectrum characteristic of a two-state helix/coil transition. The limiting mean residue ellipticity of the coil and helix form of each peptide was estimated by addition of the denaturant guanidinium chloride at elevated temperature and by addition of trifluoroethanol at subzero temperatures, respectively. The midpoint for the thermal transition of the peptide SSSRY is lowered by about 30 degrees C relative to that of peptide AAAKF, in qualitative agreement from predictions based on helix probabilities of amino acid residues. The magnitude of the change observed in the midpoint of the thermal transitions suggests that the effect of single amino acid replacements on helix formation should be experimentally measurable.     

Interaction of a mitochondrial presequence with lipid membranes: role of helix formation for membrane binding and perturbation

The binding of a peptide to a biological membrane is often accompanied by a transition from a random coil structure to an amphipathic alpha-helix. Recently, we have presented a new approach which allows the determination of the thermodynamic parameters of membrane-induced helix formation [Wieprecht et al. (1999) J. Mol Biol. 294, 785]. It involves a systematic variation of the helix content of a given peptide by double D-substitution and a correlation of the binding parameters with the helicity. Here we have used this method to study membrane-induced helix formation for the presequence of rat mitochondrial rhodanese (RHD). The thermodynamic parameters of binding of the peptide RHD and of four of its double D-isomers were determined for 30 nm (SUVs) and 100 nm (LUVs) unilamellar vesicles composed of phosphatidylcholine/phosphatidylglycerol (3:1) using circular dichroism spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry. The incremental changes of the thermodynamic parameters of helix formation were found to be very similar for SUVs and LUVs. Membrane-induced helix formation of RHD entailed a negative enthalpy of Delta H(helix) = -0.5 to -0.6 kcal/mol/residue and was opposed by an entropy of about Delta S(helix) = -1 to -1.4 cal/mol K/residue. The free energy of helix formation, Delta G(helix), was about -0.2 kcal/mol, and helix formation accounted for 50-60% of the total free energy of membrane binding. Dye-release experiments were used to assess the role of helix formation for the membrane perturbation potential of the peptides. While helix formation plays a major role for membrane binding, it appears to have little importance for inducing membrane leakiness.     

Observation of the closing of individual hydrogen bonds during TFE-induced helix formation in a peptide

Helix formation of an S-peptide analog, comprising the first 20 residues of Ribonuclease A and two additional N-terminal residues, was studied by measuring hydrogen bond (H-bond) (h3)J(NC') scalar couplings as a function of 2,2,2-trifluoroethanol (TFE) concentration. The (h3)J(NC') couplings give direct evidence for the closing of individual backbone N-H***O = C H-bonds during the TFE-induced formation of secondary structure. Whereas no (h3)J(NC') correlations could be detected without TFE, alpha-helical (i,i +4) H-bond correlations were observed for the amides of residues A5 to M15 in the presence of TFE. The analysis of individual coupling constants indicates that alpha-helix formation starts at the center of the S-peptide around residue E11 and proceeds gradually from there to both peptide ends as the TFE concentration is increased. At 60% to 90% TFE, well-formed alpha-helical H-bonds were observed for the amides hydrogens of residues K9 to Q13, whereas H-bonds of residues T5 to A8, H14, and M15 are affected by fraying. No intramolecular backbone H-bonds are present at and beyond the putative helix stop signal D16. As the (h3)J(NC') constants represent ensemble averages and the dependence of (h3)J(NC') on H-bond lengths is very steep, the size of the individual (h3)J(NC') coupling constants can be used as a measure for the population of a closed H-bond. These individual populations are in agreement with results derived from the Lifson-Roig theory for coil-to-helix transitions. The present work shows that the closing of individual H-bonds during TFE-induced helix formation can be monitored by changes in the size of H-bond scalar couplings.     

Thermodynamic model of secondary structure for α-helical peptides and proteins

A thermodynamic model describing formation of α-helices by peptides and proteins in the absence of specific tertiary interactions has been developed. The model combines free energy terms defining α-helix stability in aqueous solution and terms describing immersion of every helix or fragment of coil into a micelle or a nonpolar droplet created by the rest of protein to calculate averaged or lowest energy partitioning of the peptide chain into helical and coil fragments. The α-helix energy in water was calculated with parameters derived from peptide substitution and protein engineering data and using estimates of nonpolar contact areas between side chains. The energy of nonspecific hydrophobic interactions was estimated considering each α-helix or fragment of coil as freely floating in the spherical micelle or droplet, and using water/cyclohexane (for micelles) or adjustable (for proteins) side-chain transfer energies. The model was verified for 96 and 36 peptides studied by ¹H-nmr spectroscopy in aqueous solution and in the presence of micelles, respectively ([set I] and [set 2]) and for 30 mostly α-helical globular proteins ([set 3]). For peptides, the experimental helix locations were identified from the published medium-range nuclear Overhauser effects detected by ¹H-nmr spectroscopy. For sets 1, 2, and 3, respectively, 93, 100, and 97% of helices were identified with average errors in calculation of helix boundaries of 1.3, 2.0, and 4.1 residues per helix and an average percentage of correctly calculated helix—coil states of 93, 89, and 81%, respectively. Analysis of adjustable parameters of the model (the entropy and enthalpy of the helix—coil transition, the transfer energy of the helix backbone, and parameters of the bound coil), determined by minimization of the average helix boundary deviation for each set of peptides or proteins, demonstrates that, unlike micelles, the interior of the effective protein droplet has solubility characteristics different from that for cyclohexane, does not bind fragments of coil, and lacks interfacial area. © 1997 John Wiley & Sons, Inc. Biopoly 42: 239–269, 1997     

Position-dependence of stabilizing polar interactions of asparagine in transmembrane helical bundles

Recent studies with model peptides and statistical analyses of the crystal structures of membrane proteins have shown that buried polar interactions contribute significantly to the stabilization of the three-dimensional structures of membrane proteins. Here, we probe how the location of these polar groups along the transmembrane helices affect their free energies of interaction. Asn residues were placed singly and in pairs at three positions within a model transmembrane helix, which had previously been shown to support the formation of trimers in micelles. The model helix was designed to form a transmembrane coiled coil, with Val side chains at the "a" positions of the heptad repeat. Variants of this peptide were prepared in which an Asn residue was introduced at one or more of the "a" positions, and their free energies of association were determined by analytical ultracentrifugation. When placed near the middle of the transmembrane helix, the formation of trimers was stabilized by at least -2.0 kcal/mol per Asn side chain. When the Asn was placed at the interface between the hydrophobic and polar regions of the peptide, the substitution was neither stabilizing nor destabilizing (0.0 +/- 0.5 kcal/mol of monomer). Finally, it has previously been shown that a Val-for-Asn mutation in a water-soluble coiled coil destabilizes the structure by approximately 1.5 kcal/mol of monomer [Acharya, A., et al. (2002) Biochemistry 41, 14122-14131]. Thus, the headgroup region of a micelle appears to have a conformational impact intermediate between that of bulk water and the apolar region of micelle. A similarly large dependence on the location of the polar residues was found in a statistical survey of helical transmembrane proteins. The tendency of different types of residues to be buried in the interiors versus being exposed to lipids was analyzed. Asn and Gln show a very strong tendency to be buried when they are located near the middle of a transmembrane helix. However, when placed near the ends of transmembrane helices, they show little preference for the surface versus the interior of the protein. These data show that Asn side chains within the apolar region of the transmembrane helix provide a significantly larger driving force for association than Asn residues near the apolar/polar interface. Thus, although polar interactions are able to strongly stabilize the folding of membrane proteins, the energetics of association depend on their location within the hydrophobic region of a transmembrane helix.     

Conformational analysis of a mitochondrial presequence derived from the F1-ATPase beta-subunit by CD and NMR spectroscopy.

Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood. We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the presequence for the beta-subunit of F1-ATPase, a mitochondrial matrix protein. Although this peptide is essentially unstructured in water, alpha-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE). In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and alpha-helical conformations, with a significant population of alpha-helix throughout the entire peptide. The helix is somewhat more stable in the N-terminal part of the presequence (residues 4-10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV. Addition of increasing amounts of TFE causes the alpha-helical content to increase even further, and the TFE titration data for the presequence peptide of the F1-ATPase beta-subunit are not consistent with a single, cooperative transition from random coil to alpha-helix. There is evidence that helix formation is initiated in two different regions of the peptide. This result helps to explain the redundancy of the targeting information contained in the presequence for the F1-ATPase beta-subunit.     

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.

An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side-chain interactions favor alpha-helix formation, while the 3(10)-helix population is maximized when weaker i, i + 4 side-chain interactions are present.     

Interaction of poly(α-L-glutamic acid) and sodium poly(α-L-glutamate) with solvent components in water–2-chloroethanol mixtures

The preferential interaction of sodium poly(α-L -glutamate) and poly(α-L -glutamic acid) with the solvent components in water/2-chloroethanol mixtures has been determined using density-increment measurements. The degree of preferential interaction was deduced from the density increments at constant molality of 2-chloroethanol and at constant chemical potential of 2-chloroethanol. Sodium poly(α-L -glutamate) and poly(α-L -glutamic acid) are both preferentially hydrated in the whole range of solvent composition. A dehydration process occurs during the 2-chloroethanol-induced coil-to-helix transition of sodium poly(α-L -glutamate). This dehydration process was attributed to the release of some moles of water from the neighborhood of the peptide bond during the nucleation of the helix. After the conformational transition, sodium poly(α-L -glutamate) is solvated by one 2-chloroethanol molecule. The location of water and 2-chloroethanol molecules in the different parts of the residue (more polar and less polar portions) is also discussed.     

Solvent effects on the helix–coil transition in polypeptides

A simple way to incorporate the solvent–peptide interaction in any available theory of the helix–coil transition is developed. The competition between the intramolecular hydrogen bonding and the solvent–polymer hydrogen bonding is considered in multi-component solvents where some of the components have hydrogen-bonding capacity. Molecular averages are computed by using the theory of Lifson and Roig. The experimental data of Yang are analyzed, and the range of acceptable values of the equilibrium constants of hydrogen bond formation is deduced. The enthalpy of the transition in multicomponent solvents is calculated.     

Electrostatic interactions in a peptide--RNA complex

Parallel experimental measurements and theoretical calculations have been used to investigate the energetics of electrostatic interactions in the complex formed between a 22 residue, alpha-helical peptide from the N protein of phage lambda and its cognate 19 nucleotide box B RNA hairpin. Salt-dependent free energies were measured for both peptide folding from coil to helix and peptide binding to RNA, and from these the salt-dependence of binding pre-folded, helical peptide to RNA was determined ( partial differential (DeltaG degrees (dock))/ partial differential log[KCl]=5.98(+/-0.21)kcal/mol). (A folding transition taking place in the RNA hairpin loop was shown to have a negligible dependence on salt concentration.) The non-linear Poisson-Boltzmann equation was used to calculate the same salt dependence of the binding free energy as 5.87(+/-0.22)kcal/mol, in excellent agreement with the measured value. Close agreement between experimental measurements and calculations was also obtained for two variant peptides in which either a basic or acidic residue was replaced with an uncharged residue, and for an RNA variant with a deletion of a single loop nucleotide. The calculations suggest that the strength of electrostatic interactions between a peptide residue and RNA varies considerably with environment, but that all 12 positive and negative N peptide charges contribute significantly to the electrostatic free energy of RNA binding, even at distances up to 11A from backbone phosphate groups. Calculations also show that the net release of ions that accompanies complex formation originates from rearrangements of both peptide and RNA ion atmospheres, and includes accumulation of ions in some regions of the complex as well as displacement of cations and anions from the ion atmospheres of the RNA and peptide, respectively.