Structures of neuropeptide γ from goldfish and mammalian neuropeptide γ, as determined by 1H NMR spectroscopy

Abstract: Neuropeptide γ belongs to tachykinin families which have a common C‐terminal amino acid sequence (Phe‐X‐Leu‐Met‐NH₂) and which induce various biological responses including salivation, hypotension, and contraction of gastrointestinal, respiratory, and urinary smooth muscle. In the present study, we present the solution structures of neuropeptide γ (NPγ) from gold fish (G‐NPγ) and mammalian NPγ (M‐NPγ), as determined by nuclear magnetic resonance (NMR) spectroscopy in 50% trifluoroethanol (TFE)/water (1 : 1, v/v) solution and 200 mm sodium dodecyl sulfate (SDS) micelles. In aqueous TFE solution, G‐NPγ has a α‐helical conformation in the region of His¹²–Met²¹ and a short helix in the N‐terminal region, and has a β‐turn from Arg⁹ to Arg¹¹ in between. In aqueous TFE solution, M‐NPγ also has α‐helical conformations both in the C‐terminal region and the N‐terminal region and a β‐turn from His⁹ to Arg¹¹ in between. In SDS micelle, the structure of G‐NPγ contains a stable α‐helix from His¹² to Met²¹ and a β‐turn from Arg⁹ to Arg¹¹, while M‐NPγ has a short helix from Ser¹⁶ to Met²¹. The region from His¹² to Met²¹ corresponds to the amino acid sequence of neurokinin A. Neuropeptide γ may act as a precursor of neurokinin A and the post‐translational processing of this peptide involves the enzymatic attack of the basic β‐turn region from residue 9 to residue 11 in the middle. From our relaxation study, it could be suggested that in fish system G‐NPγ induces the biological actions corresponding to those of substance P in mammalian system. The structures of G‐NPγ and M‐NPγ contain α‐helical structures at the C‐terminus and this helix seems to promote the affinity for NK₁ and/or NK₂ receptor.     

Context‐dependent conformation of diethylglycine residues in peptides

Abstract: Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C₅) or helical conformations. The conformations of three tetrapeptides Boc‐Xxx‐Deg‐Xxx‐Deg‐OMe (Xxx = Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C₅ conformation. NMR evidence supports a folded β‐turn conformation involving Deg(2)‐Gly(3), stabilized by a 4 → 1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)–Deg(2) adopting a distorted type II/II′ conformation, while the Deg(2)–Pro(3) segment adopts a type III/III′ structure. A lone water molecule is inserted into the potential 4 → 1 hydrogen bond of the Gly(1)–Deg(2) β‐turn.     

Synthetic protein design: construction of a four‐stranded β‐sheet structure and evaluation of its integrity in methanol–water systems

Abstract: The characterization of a four‐stranded β‐sheet structure in a designed 26‐residue peptide Beta‐4 is described. The sequence of Beta‐4 (Arg‐Gly‐Thr‐Ile‐Lys‐^Dpro‐Gly‐Ile‐Thr‐Phe‐Ala‐^DPro‐Ala‐Thr‐Val‐Leu‐Phe‐Ala‐Val‐^DPro‐Gly‐Lys‐Thr‐Leu‐Tyr‐Arg) was chosen such that three strategically positioned ^DPro‐Xxx segments nucleate type II′β‐turns, which facilitate hairpin extension. A four‐stranded β‐sheet structure is determined in methanol from 500 MHz ¹H NMR data using a total of 100 observed NOEs, 11 dihedral restraints obtained from vicinal J_CαH‐NH values and 10 hydrogen bonding constraints obtained from H/D exchange data. The observed NOEs provide strong evidence for a stable four‐stranded sheet and a nonpolar cluster involving Ile⁸, Phe¹⁰, Val¹⁵ and Phe¹⁷. Circular dichroism studies in water–methanol mixtures provide evidence for melting of the β‐sheet structure at high water concentrations. NMR analysis establishes that the four‐stranded sheet in Beta‐4 is appreciably populated in 50% (v/v) aqueous methanol. In water, the peptide structure is disorganized, although the three β‐turn nuclei appear to be maintained.     

Role of azaamino acid residue in β‐turn formation and stability in designed peptide

Abstract: The structural perturbation induced by C^αH→N^α exchange in azaamino acid‐containing peptides was predicted by ab initio calculation of the 6‐31G* and 3‐21G* levels. The global energy‐minimum conformations for model compounds, For‐azaXaa‐NH₂ (Xaa = Gly, Ala, Leu) appeared to be the β‐turn motif with a dihedral angle of φ = ± 90°, ψ = 0°. This suggests that incorporation of the azaXaa residue into the i + 2 position of designed peptides could stabilize the β‐turn structure. The model azaLeu‐containing peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, which is predicted to adopt a β‐turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using ¹H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (φ_i+1, ψ_i+1) (φ_i+2, ψ_i+2)] of the Phe‐azaLeu fragment in the model peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, as [(?59^°, 127^°) (107^°, ?4^°)]. This solution conformation supports a βII‐turn structural preference in azaLeu‐containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i + 2 position in synthetic peptides is expected to provide a stable β‐turn formation, and this could be utilized in the design of new peptidomimetics adopting a β‐turn scaffold.     

Aromatic interactions in tryptophan‐containing peptides: crystal structures of model tryptophan peptides and phenylalanine analogs*

Abstract: The crystal structures of the peptides, Boc‐Leu‐Trp‐Val‐OMe ( 1) , Ac‐Leu‐Trp‐Val‐OMe ( 2a and 2b), Boc‐Leu‐Phe‐Val‐OMe ( 3 ), Ac‐Leu‐Phe‐Val‐OMe ( 4 ), and Boc‐Ala‐Aib‐Leu‐Trp‐Val‐OMe ( 5 ) have been determined by X‐ray diffraction in order to explore the nature of interactions between aromatic rings, specifically the indole side chain of Trp residues. Peptide 1 adopts a type I β‐turn conformation stabilized by an intramolecular 4→1 hydrogen bond. Molecules of 1 pack into helical columns stabilized by two intermolecular hydrogen bonds, Leu(1)NH…O(2)Trp(2) and IndoleNH…O(1)Leu(1). The superhelical columns further pack into the tetragonal space group P4₃ by means of a continuous network of indole–indole interactions. Peptide 2 crystallizes in two polymorphic forms, P2₁ ( 2a ) and P2₁2₁2₁ ( 2b ). In both forms, the peptide backbone is extended, with antiparallel β‐sheet association being observed in crystals. Extended strand conformations and antiparallel β‐sheet formation are also observed in the Phe‐containing analogs, Boc‐Leu‐Phe‐Val‐OMe ( 3 ) and Ac‐Leu‐Phe‐Val‐OMe ( 4 ). Peptide 5 forms a short stretch of 3₁₀‐helix. Analysis of aromatic–aromatic and aromatic–amide interactions in the structures of peptides, 1 , 2a , 2b are reported along with the examples of 14 Trp‐containing peptides from the Cambridge Crystallographic Database. The results suggest that there is no dramatic preference for a preferred orientation of two proximal indole rings. In Trp‐containing peptides specific orientations of the indole ring, with respect to the preceding and succeeding peptide units, appear to be preferred in β‐turns and extended structures.     

Insights into the determinants of β‐sheet stability: 1H and 13C NMR conformational investigation of three‐stranded antiparallel β‐sheet‐forming peptides

Abstract: In a previous study we designed a 20‐residue peptide able to adopt a significant population of a three‐stranded antiparallel β‐sheet in aqueous solution (de Alba et al. [1999]Protein Sci. 8, 854–865). In order to better understand the factors contributing to β‐sheet folding and stability we designed and prepared nine variants of the parent peptide by substituting residues at selected positions in its strands. The ability of these peptides to form the target motif was assessed on the basis of NMR parameters, in particular NOE data and ¹³C_α conformational shifts. The populations of the target β‐sheet motif were lower in the variants than in the parent peptide. Comparative analysis of the conformational behavior of the peptides showed that, as expected, strand residues with low intrinsic β‐sheet propensities greatly disfavor β‐sheet folding and that, as already found in other β‐sheet models, specific cross‐strand side chain–side chain interactions contribute to β‐sheet stability. More interestingly, the performed analysis indicated that the destabilization effect of the unfavorable strand residues depends on their location at inner or edge strands, being larger at the latter. Moreover, in all the cases examined, favorable cross‐strand side chain–side chain interactions were not strong enough to counterbalance the disfavoring effect of a poor β‐sheet‐forming residue, such as Gly.     

Artificial ion channels formed by a synthetic cyclic peptide

Abstract: A new cyclic peptide 1 having an (llld )₃ configuration pattern was designed that is capable of forming artificial transmembrane ion channels by self‐assembly of planar peptide rings, with hydrophilic groups arrayed in the interior of the channel. Ion permeability in the presence of the synthetic peptide 1 , cyclo[‐Trp‐Dap‐Leu‐d ‐Ala‐Trp‐Ser‐Val‐d ‐Ala‐Trp‐Ser‐Ile‐Gly‐] (Dap: l ‐diaminopropionic acid), was observed in lipid bilayer membranes. The pH dependence of ionic conductance showed that the β‐amino group of Dap may play a role in the conductance of the peptide channels. Fourier‐transform infrared and circular dichroism data imply that, in a membrane, a stack of cyclic peptides is formed in which the inter peptide H bonds form a kind of β‐structure analogous to that in the gramicidin A dimer and distinct from the H‐bonding pattern of the β‐barrels.     

Experimental studies and potential-energy calculations of the blocked tetrapeptide Ac-Lys-Gln-Gly-Ile-NMA from the third loop of a short-chain snake venom neurotoxin

The conformational space of the tetrapeptide Ac-Lys-Gin-Gly-Ile-NMA from the β-bend in the third loop of a short-chain snake venom neurotoxin was investigated with the aid of energy calculations. It was shown that this peptide has a preference for an α-helical conformation. This result was compared with the experimentally determined conformations, as observed using NMR and CD spectroscopy. With NMR spectroscopy a random-coil conformation of the peptide is indicated in H₂O, DMSO and TFE. The results from the CD experiments suggest that the peptide exists as a random coil in water, but a small population of 3-helical conformations is present in TFE. These results indicate that additional long-range interactions also play a role in the conformation of this tetrapeptide in the protein.     

NMR analysis of a potent decapeptide agonist of human C5a anaphylatoxin

A NMR investigation in H₂0, TFE and DMSO of a conformationally constrained, potent decapeptide agonist of human C5a, YSFKDMPLaR (C5a₆₅-₇₄, Y65, F67, P71, d -Ala73) showed that its N-terminal region (YSFKD) exhibited an extended backbone conformation in H₂O and a more twisted conformation in both TFE/H₂O (30:70, v/v; referred to as TFE) and DMSO. The C-terminal region (MPLaR) of the peptide adopted compact, turn-like structures. In H₂O, the C-terminal region adopted a type II β-turn or a distorted type V/II β-turn involving residues PLaR. In the distorted type V/II β-turn, Leu72 exhibited a conformation typical of a type V β-turn, whereas D -Ala73 exhibited a conformation typical of a type II β-turn. The distorted type V/II β-turn overlapped with an inverse γ-turn involving residues MPL. In DMSO, the C-terminal region had the analogous inverse y-turn and the V/II γ-turn found in H₂O. In many of the DMSO structures, two inverse γ-turns in the MPL and PLa positions formed a double-inverse γ-turn. None of the turns observed in H₂O were present in TFE. However, in TFE, the PLa residues formed an inverse γ-turn. Overall, the turn-like structural motifs in the C-terminal region of the peptide in both H₂O and DMSO (but not in TFE) agreed with the biologically important conformations obtained earlier by the structure-function analysis of a panel of C5a agonist peptides. These motifs may represent key structural elements important for C5a agonist activity and may be used to design the next generation of C5a agonist and antagonist analogues. © Munksgaard 1998.     

Amphipathic control of the 310‐/α‐helix equilibrium in synthetic peptides

Abstract: A series of short, amphipathic peptides incorporating 80% C^α,C^α‐disubstituted glycines has been prepared to investigate amphipathicity as a helix‐stabilizing effect. The peptides were designed to adopt 3₁₀‐ or α‐helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mm SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3₁₀‐helix in peptides containing amino acids with large side‐chains, as with 1‐aminocyclohexane‐1‐carboxylic acid (Ac₆c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3₁₀‐helix.     

Probing opioid receptor–ligand interactions by employment of indolizidin‐9‐one amino acid as a constrained Gly2‐Gly3 surrogate in a leucine‐enkephalin mimic

Abstract: The relationship between the conformation and biological activity of Leu‐enkephalin was studied using (2S,6R,8S)‐9‐oxo‐8‐N‐(Boc)amino‐1‐azabicyclo[4.3.0]nonane‐2‐carboxylic acid [(2S,6R,8S)‐ 1 , I⁹AA] as a constrained Gly²‐Gly³ dipeptide surrogate. [I⁹AA]^2,3‐Leu‐enkephalin 12 was assembled using solid‐phase peptide synthesis on Merrifield resin with TBTU as the coupling reagent. The in vitro assays indicated that [I⁹AA]^2,3‐Leu‐enkephalin 12 exhibited affinities for the µ‐ and δ‐opioid receptors that were three orders of magnitude lower than that of Leu‐enkephalin, as well as partial agonist character for both receptors. In in vivo assays for spinal analgesia, the indolizidinone analog 12 showed significantly enhanced duration of action, indicating an increased metabolic stability. Conformational analysis was performed using NMR and CD spectroscopy. The amide temperature coefficients and ³J_NH‐CαH coupling constants for 12 could not support a hydrogen‐bonded β‐turn structure; however, its CD spectrum indicated a turn conformation. Incorporation of indolizidinone amino acid 1 into Leu‐enkephalin thus provided additional support for the importance of a turn conformation for the biological activity of the native peptide.     

Synthesis,biology, NMR and conformation studies of the topographically constrained δ‐opioid selective peptide analogs of [β‐iPrPhe3]deltorphin I

Abstract: Replacement of Phe³ in the endogenous δ‐opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid β‐isopropylphenylalanine (β‐iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand‐binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [β‐iPrPhe³]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe³ residue in [β‐iPrPhe³]deltorphin I provided the most desirable biological properties with binding affinity (IC₅₀ = 2 n m ), bioassay potency (IC₅₀ = 1.23 n m in MVD assay) and exceptional selectivity for the δ‐opioid receptor over the µ‐opioid receptor (30 000). Further conformational studies based on two‐dimensional NMR and computer‐assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr¹ and (2S,3R)‐β‐iPrPhe³ residues adopt trans side‐chain conformations, and the linear peptide backbone favors a distorted β‐turn conformation.     

Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

Conformational analysis by NMR and distance geometry techniques of a peptide mimetic of the third helix of the Antennapedia homeodomain*

Abstract: The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well‐known helix‐turn‐helix motif. The recognition helix penetrates the DNA major groove, gives specific protein–DNA contacts and forms direct, or water‐mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42–53, where residues W48 and F49, not involved in the protein–DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well‐defined secondary structure in TFE and in TFE/water (90/10, v/v) with an α‐helix encompassing residues 4–9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted α‐helical conformation involving residues 4–8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the α‐helical conformation even if separated from the rest of the molecule.     

The conformational and biological analysis of a cyclic anti‐obesity peptide from the C‐terminal domain of human growth hormone

Abstract: The three‐dimensional solution structure of anti‐obesity drug (AOD), a 15‐residue, disulfide‐bonded, cyclic peptide, cyclo(6,13)‐H₂N‐Leu‐Arg‐Ile‐Val‐Gln‐Cys‐Arg‐Ser‐Val‐Glu‐Gly‐Ser‐Cys‐Gly‐Phe‐OH, derived from the C‐terminal domain of the human growth hormone (hGH) (residues 177–191) was determined using two‐dimensional ¹H NMR spectroscopy. AOD stimulates lipolysis and inhibits lipogenesis, in vitro, in rodent, porcine and human adipose tissues. These biological effects suggest that AOD is a potential therapeutic candidate for the treatment of obesity. Conformational studies of AOD were conducted in aqueous solution and in water/dimethylsulfoxide mixtures. In general, spectral quality was superior in the water/dimethylsulfoxide mixtures. The cyclic region of AOD in water/dimethylsulfoxide adopts type I β‐turns at residues Ser⁸‐Val⁹‐Glu¹⁰‐Gly¹¹ and Ser¹²‐Cys¹³‐Gly¹⁴‐Phe¹⁵, each preceded by loop‐like structures. Comparison of the conformation of this peptide with residues 177–191 in the native hGH protein X‐ray crystal structure indicates that the synthetic peptide retains some structural similarity to the intact protein. This study provides evidence that the C‐terminal region of hGH is a specific functional domain of the multifunctional hGH protein.     

Solution stability of salmon calcitonin at high concentration for delivery in an implantable system

Abstract: Salmon calcitonin solutions (50 mg/mL and 100 mg/mL) were placed on stability at 37°C for 1 year in a variety of solvent systems including water, ethanol, glycerol, propylene glycol (PG) and dimethyl sulfoxide (DMSO). Calcitonin degradation was monitored by RP‐HPLC and size‐exclusion chromatography. DMSO and pH 3.3 solutions provided optimum stability. Conformational stability was also monitored by FTIR over the 1 year time course and compared with chemical and physical stability. After 12 months at 37°C, four major conformations were observed: a β‐sheet conformation (pH 3.3, pH 5.0, 70% DMSO and 70% glycerol), an aggregate conformation (pH 7.0 water), a strong α‐helical conformation (70% EtOH, 70% PG) and a weak α‐helical conformation (100% DMSO). No correlation between structure and chemical stability was observed in which both the β‐sheet structure (pH 3.3, water) and a loose α‐helical structure (100% DMSO) demonstrated good stability. However, some correlation was observed between structure and physical stability, where co‐solvents inducing an α‐helical structure resulted in a decrease in gelation. These two structural states associated with improved stability and minimal gelation, indicated that gelation can be reduced or eliminated by the use of pharmaceutically acceptable co‐solvents. Finally, salmon calcitonin (50 mg/mL) was formulated in 100% DMSO and delivered from a DUROS® implant over 4 months. Delivery at a target dose of 18 µg/day calcitonin at 37°C was confirmed.     

Determination of structural elements related to the biological activities of a potent decapeptide agonist of human C5a anaphylatoxin

Abstract: The structural features related to the biologic activities of a potent, response-selective decapeptide agonist of human C5a, YSFKPMPLaR (C5a_65–74, Y65, F67, P69, P71, d -Ala73), were identified by NMR analysis in H₂O, DMSO and TFE. This investigation showed that the KPM residues in H₂O and the SFKPM residues in DMSO exhibited an extended backbone conformation, whereas a twisted conformation was found in this region in TFE. In H₂O, the C-terminal region (PLaR) adopted a distorted type II β-turn or a type II/V β-turn. In the type II/V β-turn, Leu72 exhibited a conformation typical of a type II β-turn, whereas d -Ala73 exhibited a conformation characteristic of a type V β-turn. Furthermore, a γ-turn involving residues LaR overlapped with the type II/V β-turn. In DMSO, the C-terminal region had the analogous turn-like motif (type II/V β-turn overlapping with γ-turn) found in H₂O. In TFE, no β-turn motifs were formed by the PLaR residues. These turn-like motifs in the C-terminal region of the peptide in both H₂O and DMSO were in agreement with the biologically important conformations predicted earlier by a structure–function analysis of a related panel of decapeptide analogs. The motifs determined by the NMR analysis of YSFKPMPLaR in H₂O and DMSO may represent structural elements important for C5a agonist activity and thus can be used to design the next generation of C5a agonist, partial agonist and antagonist analogs.     

Preferred conformations of a linear RGD tripeptide

Abstract: Conformational study of RGD tripeptides in the nonhydrated and hydrated states was carried out using an empirical potential function ECEPP/3 and the hydration shell model in order to investigate preferred conformations and factors responsible for their stability. RGD tripeptides in the nonhydrated and hydrated states can be interpreted as existing as an ensemble of feasible conformations rather than as a single dominant conformation from the analysis of distributions of backbone conformations, hydrogen bonds and β‐turns. The different distributions of conformations for the neutral and zwitterionic RGD tripeptides in both states may indicate that the conformation of the RGD tripeptide is liable to depend on solvent polarity and pH values. β‐Turn populations for the neutral tripeptide in both states are reasonably consistent with NMR measurements on linear RGD‐containing peptides. The degradation of RGD tripeptide seems to be attributed mainly to the hydrogen bonds between the Asp side‐chain and the backbone of Asp residue or C‐terminal NHMe group, rather than to the flexible backbones of Gly and Asp residues.     

Reactivity toward deamidation of asparagine residues in β‐turn structures

Mimetics of β‐turn structures in proteins have been used to calibrate the relative reactivities toward deamidation of asparagine residues in the two central positions of a β‐turn and in a random coil. N‐Acetyl‐Asn‐Gly‐6‐aminocaproic acid, an acyclic analog of a β‐turn mimic undergoes deamidation of the asparaginyl residue through a succinimide intermediate to generate N‐acetyl‐Asp‐N‐Gly‐6‐aminocaproic acid (6‐aminocaproic acid, hereafter Aca) and N‐acetyl‐l ‐iso‐aspartyl (isoAsp)‐Gly‐Aca (pH 8.8, 37 °C) ≈ 3‐fold faster than does the cyclic β‐turn mimic cyclo‐[L‐Asn‐Gly‐Aca] with asparagine at position 2 of the β‐turn. The latter compound, in turn, undergoes deamidation ≈ 30‐fold faster than its positional isomer cyclo‐[Gly‐Asn‐Aca] with asparagine at position 3 of the β‐turn. Both cyclic peptides assume predominantly β‐turn structures in solution, as demonstrated by NMR and circular dichroism characterization. The open‐chain compound and its isomer N‐acetyl‐Gly‐Asn‐Aca assume predominantly random coil structures. The latter isomer undergoes deamidation 2‐fold slower than the former. Thus the order of reactivity toward deamidation is: asparagine in a random coil ≈ 3× asparagine in position 2 of a β‐turn ≈ 30× asparagine in position 3 of a β‐turn.