Lipid-induced ordered conformation of some peptide hormones and bioactive oligopeptides: predominance of helix over beta form

The conformation of several naturally occurring peptide hormones and bioactive oligopeptides in phospholipid solutions was studied by circular dichroism. Phosphatidylcholine induced a partial helix in human gastrin I at neutral pH, but phosphatidylserine did not unless the five consecutive glutamic acid residues in gastrin were protonated. Reduced somatostatin with two lysines and substance P with one arginine and one lysine were partially helical in phosphatidylserine, but not phosphatidylcholine, solution. Both lipids induced a helical conformation in glucagon and its COOH-terminal fragment (19-29) probably because the helical segment is primarily located at the uncharged COOH terminus. Thus, polypeptides with a helix-forming potential can have the helical conformation only when the peptides carry no charge or charges opposite to those on the polar head of the lipid. Renin substrate, which has potentials for the beta form and beta turn, seemed to form a mixture of the two conformations in phosphatidylserine solution. Angiotensin I with a strong probability for the beta form adopted the beta form in phosphatidylserine solution and sleep peptide with no structure-forming potential remained unordered in lipid solutions. The helix usually predominated over the beta form in lipid solutions if the peptide has potentials for both conformations. This could account for the preponderance of helices in bacteriorhodopsin of the purple membrane, which according to its amino acid sequence would have favored the beta form.     

Stabilization of helical structure in two 17-residue amphipathic analogues of the C-terminal peptide of cytochrome C

The conformations of two 17-residue peptide analogues derived from the C-terminal sequence of pigeon cytochrome c (native sequence = KAERADLIAYLKQATAK) were examined in aqueous and lipid environments by CD spectroscopy. The two analogues, KKLLKKLIAYLKQATAK (K peptide) and EELLEELIAYLKQATAK (E peptide), were made amphipathic with respect to helical segregation by substituting a 6-residue sequence at the N-terminus of the native peptide. Their structures were compared to the native peptide under aqueous conditions of varying pH and temperature, and in the presence of liposomes composed of phosphatidylcholine and phosphatidylserine in the ratio of 9:1. The results indicated that the native peptide remains unstructured under all the conditions examined even though this region of the native molecule is surface exposed and helical. The E peptide, however, was helical under aqueous conditions at 25 degrees C from pH 2-10 with a maximum helicity at pH 4 (54% helix from analysis of CD data). The ellipticity of the E peptide at pH 4 and 8 was concentration dependent, indicating an aggregation phenomenon. In studies in which the CD spectrum was measured at different temperatures, the E peptide became more helical at lower temperatures at pH 4 but not at pH 8. Upon interaction with a lipid membrane in the form of liposomes, there appeared to be a slight destabilization in the structure of the E peptide. The K peptide in an aqueous environment behaved like the native peptide in that it was structureless at all pHs and temperatures examined. In the presence of liposomes, however, this peptide had a high helical content (75% helix from analysis of CD data). These findings suggest that while stabilization of the helix dipole with negative charges at the N-terminus are important in inducing helical conformation in the E peptide, hydrophobic interactions created during aggregation appear to provide the principal stabilizing force. The results with the K peptide demonstrate that the positive N-terminal sequence of this peptide is able to interact with the negatively charged head groups in the phospholipid membrane in such a fashion as to stabilize a helical structure that is not apparent in an aqueous environment alone.     

Structure and membrane orientation of IAPP in its natively amidated form at physiological pH in a membrane environment

Human islet amyloid polypeptide is a hormone coexpressed with insulin by pancreatic beta-cells. For reasons not clearly understood, hIAPP aggregates in type II diabetics to form oligomers that interfere with beta-cell function, eventually leading to the loss of insulin production. The cellular membrane catalyzes the formation of amyloid deposits and is a target of amyloid toxicity through disruption of the membrane's structural integrity. Therefore, there is considerable current interest in solving the 3D structure of this peptide in a membrane environment. NMR experiments could not be directly utilized in lipid bilayers due to the rapid aggregation of the peptide. To overcome this difficulty, we have solved the structure of the naturally occurring peptide in detergent micelles at a neutral pH. The structure has an overall kinked helix motif, with residues 7-17 and 21-28 in a helical conformation, and with a 3(10) helix from Gly 33-Asn 35. In addition, the angle between the N- and C-terminal helices is constrained to 85°. The greater helical content of human IAPP in the amidated versus free acid form is likely to play a role in its aggregation and membrane disruptive activity.     

Conformational switching and fibrillogenesis in the amyloidogenic fragment of apolipoprotein a-I

The N-terminal portion of apolipoprotein A-I corresponding to the first 93 residues has been identified as the main component of apolipoprotein A-I fibrils in a form of systemic amyloidosis. We have been able to characterize the process of conformational switching and fibrillogenesis in this fragment of apolipoprotein A-I purified directly from ex vivo amyloid material. The peptide exists in an unstructured form in aqueous solution at neutral pH. The acidification of the solution provokes a collapse into a more compact, intermediate state and the transient appearance of a helical conformation that rapidly converts to a stable, mainly beta-structure in the fibrils. The transition from helical to sheet structure occurs concomitantly with peptide self-aggregation, and fibrils are detected after 72 h. The alpha-helical conformation is induced by the addition of trifluoroethanol and phospholipids. Interaction of the amyloidogenic polypeptide with phospholipids prevents the switching from helical to beta-sheet form and inhibits fibril formation. The secondary structure propensity of the apolipoprotein A-I fragment appears poised between helix and the beta-sheet. These findings reinforce the idea of a delicate balance between natively stabilizing interactions and fatally stabilizing interactions and stress the importance of cellular localization and environment in the maintenance of protein conformation.     

Bovine pancreatic polypeptide (bPP) undergoes significant changes in conformation and dynamics upon binding to DPC micelles

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08ns derived from the 15N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4-34.The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined alpha-helical region comprising residues 17-31. The average RMSD value for residues 17-31 is 0.22(+/-0.09)A. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4-12 and low values of the generalized order parameter S(2). The membrane-peptide interface was investigated by micelle-integrating spin-labels and H,2H exchange measurements. It is formed by those residues which make contacts between the C-terminal alpha-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y(4) receptor.     

Helical formation of a 13-residue C-peptide analogue of ribonuclease A in sodium dodecyl sulfate solution

The conformation of a 13-residue C-peptide analogue of ribonuclease A——in surfactant solutions was studied by CD. The CD spectrum of the peptide in excess NaDodSO₄ solution was typical for a helical conformation; the spectrum appeared to be virtually independent of pH (2.5–6) and temperature (3–25°C). Analysis of the CD data indicated a helicity of about 65–70% with no α-sheet and β-turn; this corresponded to 8 or 9 residues in the helical form or slightly more than two turns of α-helix. This compares with an average of about one turn of α-helix for the C-peptide analogue in water at pH 4.7 and 7°C. The conformation of the peptide in cationic surfactant, dodecyl ammonium chloride, and nonionic surfactant, dodecyl heptaoxyethylene ether, solution resembled that in water. We concluded that the C-peptide analogue can develop a maximum helicity close to the corresponding segment in ribonuclease A in hydrophobic environment provided by the clustering of NaDodSO₄ molecules to the cationic side groups of the peptide, except that the end effects may destabilize two or three residues each at both ends of the helix. Thus, in the interior of a protein molecule this hydrophobic effect may overshadow the charged-group effect than can be explained by the helix dipole model for the helical segments on the exterior of the protein molecule.     

CD80 binding polyproline helical peptide inhibits T cell activation

The critical role played by the CD28/CD152-CD80/CD86 costimulatory molecules in mediating T cell activation and suppression provides attractive targets for therapeutic strategies. CD28 and CD152 share a conserved polyproline motif in the ligand-binding region. Similar proline-rich regions in globular domains preferentially adopt a polyproline type II (PP) helical conformation and are involved in transient (II)protein-protein interactions. Interestingly, in the human CD80-CD152 complex, Pro(102) of CD152 restricts the preceding proline to PP(II) helix in the binding orientation in relation to the shallow binding pocket of CD80. Peptide agents derived from binding sites of receptors that mimic the bioactive conformation have been shown to block receptor-ligand interactions. Contact preferences of the interface amino acids at the protein-protein interaction sites and the propensity of each residue to form PP(II) helix were integrated in the design of novel peptide agents referred to as CD80 competitive antagonist peptides. Structural and functional studies suggest potential therapeutic value for select CD80 competitive antagonist peptides.     

Chain conformation in polyretropeptides III: Design of a 310 helix using α,α-dialkylated amino acids and retropeptide bonds

Computer simulations have been used to design a polypeptide with a 3₁₀ helix conformation. The study has been been performed taking advantage of the intrinsic helix forming tendency of α-Aminoisobutyric acid. In order to avoid the formation of the α helix, which is the other common helical conformation adopted by α-Aminoisobutyric acid-based peptides, retropeptide bonds have been included in the sequence. Thus, retropeptides are not able to form the intramolecular hydrogen bonding interactions characteristic of the α helix. The influences of both the peptide length and the solvent have been examined and compared with those of the polypeptide without retropeptide bonds. Proteins 29:575–582,1997. © 1997 Wiley-Liss, Inc.     

Structural analysis of the human cannabinoid receptor one carboxyl‐terminus identifies two amphipathic helices

Recent research has implicated the C‐terminus of G‐protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C‐tail has proven a formidable task. Here, a peptide corresponding to the full‐length C‐tail of the human CB1 receptor (residues 400–472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an α‐helical conformation in negatively charged and zwitterionic detergents (48–51% and 36–38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self‐association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled (¹⁵N‐ and ¹³C‐) C‐terminus in dodecylphosphocholine (DPC) identified two amphipathic α‐helical domains. The first domain, S401‐F412, corresponds to the helix 8 common to G protein‐coupled receptors while the second domain, A440‐M461, is a newly identified structural motif in the distal region of the carboxyl‐terminus of the receptor. Molecular modeling of the C‐tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 565–573, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Exploring deltorphin II binding to the third extracellular loop of the delta-opioid receptor

The third extracellular loop of the human delta-opioid receptor (hDOR) is known to play an important role in the binding of delta-selective ligands. In particular, mutation of three amino acids (Trp(284), Val(296), and Val(297)) to alanine significantly diminished delta-opioid receptor affinity for delta-selective ligands. To assess the changes in conformation accompanying binding of the endogenous opioid peptide deltorphin II to the delta-opioid receptor at both the receptor and ligand levels as well as to determine points of contact between the two, an in-depth spectroscopic study that addressed these points was initiated. Fragments of the delta-opioid receptor of variable length and containing residues in the third extracellular loop were synthesized and studied by NMR and CD spectroscopy in a membrane-mimetic milieu. The receptor peptides examined included hDOR-(279-299), hDOR-(283-299), hDOR-(281-297), and hDOR-(283-297). A helical conformation was observed for the longest receptor fragment between Val(283) and Arg(291), whereas a nascent helix occurred in a similar region for hDOR-(281-297). Further removal of N-terminal residues Val(281) and Ile(282) abolished helical conformation completely. Binding of the delta-selective ligand deltorphin II to hDOR-(279-299) destabilized the helix at the receptor peptide N terminus. Dramatic changes in the alpha-proton chemical shifts for Trp(284) and Leu(286) in hDOR-(279-299) also accompanied this loss of helical conformation. Large upfield displacement of alpha-proton chemical shifts was observed for Leu(295), Val(296), and Val(297) in hDOR-(279-299) following its interaction with deltorphin II, thus identifying a gain in beta-conformation at the receptor peptide C terminus. Similar changes did not occur for the shorter peptide hDOR(281-297). A hypothesis describing the conformational events accompanying selective deltorphin II binding to the delta-opioid receptor is presented.     

Conformational analysis of a set of peptides corresponding to the entire primary sequence of the N-terminal domain of the ribosomal protein L9: evidence for stable native-like secondary structure in the unfolded state

There is considerable interest in the structure of the denatured state and in the role local interactions play in protein stability and protein folding. Studies of peptide fragments provide one method to assess local conformational preferences which may be present in the denatured state under native-like conditions. A set of peptides corresponding to the individual elements of secondary structure derived from the N-terminal domain of the ribosomal protein L9 have been synthesized. This small 56 residue protein adopts a mixed alpha-beta topology and has been shown to fold rapidly in an apparent two-state fashion. The conformational preferences of each peptide have been analyzed by proton nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. Peptides corresponding to each of the three beta-stands and to the first alpha-helix are unstructured as judged by CD and NMR. In contrast, a peptide corresponding to the C-terminal helix is remarkably structured. This 17 residue peptide is 53 % helical at pH 5.4, 4 degrees C. Two-dimensional NMR studies demonstrate that the helical structure is distributed approximately uniformly throughout the peptide, although there is some evidence for fraying at the C terminus. Detailed analysis of the NMR spectra indicate that the helix is stabilized, in part, by a native N-capping interaction involving Thr40. A mutant peptide which lacks Thr40 is only 32 % helical. pH and ionic strength-dependent studies suggested that charge charge interactions make only a modest net contribution to the stability of the peptide. The protein contains a trans proline peptide bond located at the first position of the C-terminal helix. NMR analysis of the helical peptide and of a smaller peptide containing the proline residue indicates that only a small amount of cis proline isomer (8 %) is likely to be populated in the unfolded state.     

CD and NMR investigations on trifluoroethanol-induced step-wise folding of helical segment from scorpion neurotoxin.

A 14 amino acid residue peptide from the helical region of Scorpion neurotoxin has been structurally characterized using CD and NMR spectroscopy in different solvent conditions. 2,2,2-Trifluoroethanol (TFE) titration has been carried out in 11 steps from 0 to 90% TFE and the gradual stabilization of the conformation to form predominantly alpha-helix covering all of the 14 residues has been studied by 1H and 13C NMR spectroscopy. Detailed information such as coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each TFE concentration has been extracted and analysed to derive the step-wise preferential stabilization of the helical segments along the length of the peptide. It was found that there is a finite amount of the helical conformation in the middle residues 5-11 even at low TFE concentrations. It was also observed that > 75% TFE (v/v) is required for the propagation of the helix to the N and C termini and for correct packing of the side chains of all of the residues. These observations are significant to understanding the folding of this segment in the protein and may throw light on the inherent preferences and side chain interactions in the formation of the helix in the peptide.     

Secondary structure of polypeptide hormones of the anterior lobe of the pituitary gland

The specific secondary structure of a number of polypeptide hormones of the pituitary gland anterior lobe and their fragments were studied by CD in the peptide bond absorption region and by ir spectroscopy. The state of objects was examined in solvents of different polarity over a wide temperature range as well as in the solid state at different relative humidities. The predominant conformational state of a number of hormones in aqueous solution is shown to represent a left-handed helix of the poly(L -proline) II type. The reversible melting process of the left-handed helical conformation when heated in an aqueous solution appears to be noncooperative. Lowering the temperature stabilizes the left-handed structure. The transition mode of the left-handed form to the α-, and the β-forms on changing the solvent conditions was also studied. Contributions of peptide chromophores and of the aromatic amino acid side-group chromophores with CD bands in the region under study were determined by analysis of CD spectra. The data obtained allow correlating the conformation of separate fragments in the hormone chain with functional activity.     

Fluoroalcohols as structure modifiers in peptides and proteins: hexafluoroacetone hydrate stabilizes a helical conformation of melittin at low pH.

The effect of hexafluoroacetone hydrate (HFA) on the structure of the honey bee venom peptide melittin has been investigated. In aqueous solution at low pH melittin is predominantly unstructured. Addition of HFA at pH approximately 2.0 induces a structural transition from the unstructured state to a predominantly helical conformation as suggested by intense diagnostic far UV CD bands. The structural transition is highly cooperative and complete at 3.6 M (50% v/v) HFA. A similar structural transition is also observed in 2,2,2 trifluoroethanol which is complete only at a cosolvent concentration of approximately 8 M. Temperature dependent CD experiments support a 'cold denaturation' of melittin at low concentrations of HFA, suggesting that selective solvation of peptide by HFA is mediated by hydrophobic interactions. NMR studies in 3.6 M HFA establish a well-defined helical structure of melittin at low pH, as suggested by the presence of strong NH/NHi+1 NOEs throughout the sequence, along with many medium range helical NOEs. Structure calculations using NOE-driven distance constraints reveal a well-ordered helical fold with a relatively flexible segment around residues T10-G11-T12. The helical structure of melittin obtained at 3.6 M HFA at low pH is similar to those determined in methanolic solution and perdeuterated dodecylphosphocholine micelles. HFA as a cosolvent facilitates helix formation even in the highly charged C-terminal segment.     

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 3₁₀‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH₂ was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and ¹³C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/3₁₀‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]₂₂₂/[θ]₂₀₇, as a reporter of α‐/3₁₀‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

The duplication of an eight-residue helical stretch in Staphylococcal nuclease is not helical: a model for evolutionary change

A common method of evolutionary change is gene duplication, followed by other events that lead to new function, decoration of folds, oligomerization, or other changes. As part of a study on the potential for evolutionary change created by duplicated sequences, we have carried out a crystallographic study on a mutant of Staphylococcal nuclease in which residues 55-62 have been duplicated in a wild-type variant termed PHS. In the parental protein (PHS) these residues form the first two turns of a helix running from residue 54 to 68 (hereafter designated as helix I). The crystal structure of the mutant is very similar to that of the parental, with helix I being unaltered. The duplicated residues are accommodated by expanding an existing loop N-terminal to helix I. In addition, circular dichroism (CD) studies have been carried out on a parental peptide containing helix I with six flanking residues at each terminus (residues 48-74) and on the same peptide expanded by the duplication, as a function of 2,2,2-trifluoroethanol (TFE) concentration. Each peptide possesses only modest helical propensity in solution. Our data, which is different from what was observed in T4 lysozyme, show that the conformation of the duplicated sequence is determined by a balance of sequential and longer-range effects. Thus duplicating sequence need not mean duplicating structure. Proteins 2000;40:465-472.     

An alpha-helical peptide model for electrostatic interactions of proteins with DNA. The N terminus of RecA

A series of synthetic peptides have been studied as models for non-specific protein-DNA interactions. In an alpha-helical conformation, the charged amino acid residues of the N-terminal 24 residues of RecA protein are asymmetrically distributed; at neutral pH there is a +4 charge on one face of the helix and a -3 charge on the other face. Modeling suggests that the positive face of the helix can bind five DNA phosphate groups by electrostatic interactions. Circular dichroism (c.d.) spectra indicate that the analogous peptide, Rec24 (AIDENKQKALAAALGQIEKQFGKG-amide), is largely unstructured in water but becomes highly helical in the presence of DNA. Peptide titrations of fluorescent etheno-DNA confirm that the changes in the c.d. spectrum of the peptide are associated with binding, although a dependence of the c.d. signal on the degree of DNA saturation is observed, indicating that peptide can be bound in more than one conformation. At saturation the peptide binds to 5.0(+/- 0.5) DNA phosphate groups as predicted and the electrostatic nature of the binding is confirmed by a strong dependence on salt concentration. A "mutant" peptide where an acidic glutamate residue replaces an alanine on the basic face of the Rec24 helix exhibits weaker binding to single-stranded DNA, also consistent with the electrostatic nature of the proposed peptide-DNA interaction. Extending Rec24 by ten amino acid residues, where the additional residues do not participate in the helical motif, does not noticeably affect binding. Thus, we show experimentally that an asymmetric charge distribution on an alpha-helix can represent an important element for binding nucleic acids.     

Degradation of N5-(2-hydroxyethyl)-L-glutamine and L-glutamic acid homopolymers and copolymers by papain

The rate of degradation of poly[N5-(2-hydroxyethyl)-L-glutamine] (PHEG), poly(L-glutamic acid) (PGA) and poly[HEG-co-GA] random copolymers by papain was measured in the pH range 4.0-7.5, employing the gel permeation chromatography method. The effect of the degree of ionization on the polymer conformation was measured by circular dichroism (c.d.). PHEG, which is uncharged, had a random coil conformation and an almost constant degradation rate within the whole pH interval. The ionization of PGA increased with increasing pH and was accompanied by conformational transition from helix to random coil. The hydrolysis of PGA by papain depended on pH with the optimum at about pH 5, indicating that both the high content of helix (at pH less than 5) and increasing charge density (at pH greater than 5), decreased the degradation rate. Contrary to PGA, pH profiles of the degradation rate of poly[HEG-co-GA] copolymers are monotonous and do not decrease at pH less than 5. In the copolymers the HEG residues act as a helix breaker and limit the formation of helical conformation. The role of structural features of a macromolecular substrate, i.e. the charge, helical conformation and the nature of amino acid residues, in the interaction between enzyme and polymer is discussed.     

Spectroscopic characterization of poly(Glu-Ala)

Infrared linear dichroism and ultraviolet circular dichroism speetroscopy have been used to distinguish four conformational forms of the ionizable sequential polypeptide poly(Glu-Ala). Two of these conformations, the α helix and the β form, were observed for the unionized polypeptide in solution. The α helix appeared immediately upon neutralization of the side-chain carboxyl functions, whereas the β form was observed after the neutralized solution had been standing for several days. The β form was also observed for films cast from either high or low pH solutions. Ionization of the glutamyl residues resulted in a circular dichroism spectrum which has previously been observed for charged homopolymers and appears to result from an extended helical conformation. Further, heating either the α helical or the charged extended helix resulted in a transition to a disordered chain. These results are consistent with the results of conformational calculations presented elsewhere.     

Pituitary adenylate cyclase activating polypeptide (PACAP) with 27 residues. Conformation determined by 1H NMR and CD spectroscopies and distance geometry in 25% methanol solution.

The conformation of pituitary adenylate cyclase activating polypeptide with 27 residues (PACAP27) has been determined by two-dimensional NMR and CD spectroscopies and distance geometry in 25% methanol. Residues 9-20 and 22-25 have well-defined conformations but other residues do not show ordered conformations. The conformation of residues 9-20 is composed of three distinct regions of beta turn-like conformation (residues 9-12), alpha helix (residues 12-14) and the looser helical conformation (residues 15-20), while residues 22-24 form alpha helix. PACAP27 has a 2 helices separated by a disordered region similar to a VIP analog reported by Fry et al. but is distinct from the VIP analog in the position of the first helix, which is shifted by 2 residues toward the C-terminus, and in the form of the second helix [Fry, D.C., Madison, V.S., Bolin, D.R., Greeley, D.N., Toome, V. and Wegrzynski, B.B. (1989) Biochemistry 28, 2399-2409].