QUANTITATIVE EVALUATION OF γ-TURN CONFORMATION IN PROLINE-CONTAINING PEPTIDES USING13 C N.M.R.

The dependence of the ¹³C shift difference of proline carbons C_β and C_γ on the dihedral angle ø has been studied using the model peptide acetyl-d -proline N-methylamide. The shift difference Δ_βγ is shown to be correlated with the percent cis isomer about the acetylproline bond, both factors depending strongly on the degree of intermolecular hydrogen bonding. Both the fraction of trans peptide bond and the fractional γ-turn conformation increase as the sample concentration is decreased in CDCl₃. Δ_βγ values have been used to evaluate the fractional γ-turn probabilities in a number of cyclic and linear peptides including thyrotropin releasing factor and bradykinin. Using this parameter, it is concluded that in bradykinin the γ-turn probability is low in D₂O and not strongly temperature dependent. In contrast, studies of a model peptide for the portion of bradykinin believed to adopt a γ-turn conformation are consistent with an increased γ-turn probability in less polar solvents. Data for X-Pro-Y peptides (Y = imino acid) indicate significantly reduced values of Δ_βγ, and this appears to be a useful basis for assigning the Pro C_β resonances corresponding to this sequence.     

Conformational studies of cyclic tetrapeptides

The conformations of chlamydocin and cyclo (Ala-Aib-Phe-D-Pro)(Ala⁴-chlamy-docin) in chloroform have been investigated by nuclear magnetic resonance, infrared and circular dichroism spectroscopy. The data obtained from these experiments establish an all transoid, bis γ-turn conformation for both compounds in chloroform with the following torsional angles (± 20d?): Ala⁴4-chlamydocin: Aib, Ø+ 60d?, Ψ– 50d?; ω+ 160d?; Phe Ø– 120d?, Ψ+ 120d?, ω– 160d?; D-Pro Ø+ 60d?, Ø– 55d?, ω+ 160d?; Ala Ø– 110d?, Ψ+ 110d?, ω– 160d?. Chlamydocin adopts a closely related conformation in neat chloroform. Nuclear Overhauser Effect (NOE) data are utilized to assign amide bond geometries in the cyclic tetrapeptide ring system.     

CD-n.m.r. study of the solution conformation of bradykinin analogs containing α-aminoisobutyric acid

The conformation in aqueous solution of several α-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and ¹H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro² and/or Pro³ in BK stabilizes a degree of β-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB⁷]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a β-turn involving their common Arg¹-Pro²-Pro³-Gly⁴ moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis¹ Pro and the propensity for a β-turn at the N-terminus of the peptide.     

Circular dichroism and fluorescence spectroscopic analyses of a proline-rich glycoprotein from human parotid saliva

A proline-rich glycoprotein (PRG) was isolated from human parotid saliva and examined by circular dichroism and fluorescence spectroscopy. Addition of guanidine hydrochloride to PRG labeled with an extrinsic dansyl probe had no effect on the fluorescence spectra's 511 nm lambda-max location. Thermodynamic calculations supported the contention that PRG has no significant tertiary structure. Circular dichroism results for PRG were simulated by computer and a secondary structure composed of 70% random coil and 30%β-form conformation was predicted. Circular dichroism of PRG failed to detect either poly-L-proline type I or II structures. Deglycosylation of PRG had no measurable effect on the circular dichroism spectrum, indicating that the carbohydrate side chains had little influence on PRG secondary structure. Based upon mathematical calculations, β-turns were predicted around three glycosylated Asn residues of PRG. These collective data suggest that PRG is composed of a disordered polypeptide chain with at least three of the N-linked Asn residues participating in some type of β-turn.     

Testing for cis‘ proline with α-aminoisobutyric acid substitution

Substitution of Pro residues with AIB (α-aminoisobutyric acid) residues in peptides provides a means of evaluating the presence of cis' proline conformations both in solution and, using bioassay data, in a receptor complex. ¹ H n.m.r. has been used to probe the DMSO solution conformation of all seven of the possible AIB/Pro isomers of bradykinin. AIB substitution for Pro² and/or Pro³ appears to stabilize a type III β-turn involving the N-terminal residues, but not an incipient 3₁₀ helix suggested by model peptides. These substitutions are correlated with low biological potencies, suggesting that such conformational features may be incompatible with receptor complexation. Alternatively, AIB⁷ -bradykinin analogs exhibit a variety of long range shift perturbations relative to bradykinin. The data suggests that bradykinin can adopt several folded conformations, including β-turns involving both Ser⁶-Pro⁷-Phe⁸-Arg⁹ and Phe⁵-Ser⁶-Pro⁷-Phe⁸. The relatively high biological activities of the AIB⁷-BK suggest that the complexed form of the peptide is characterized by a cis' Pro⁷ conformation.     

Synthesis and conformational study of two cyclic analogues of somatostatin containing an AMPA-spacer unit

Two cyclic somatostatin analogues containing the active sequence Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰ and a meta- or para-(aminomethyl) phenylacetic acid (AMPA) spacer unit, have been synthesized. A conformational study using 2D n.m.r. techniques (COSY, NOESY) reveals that the conformation of the meta-AMPA analogue has some analogy with the bio-active conformation proposed earlier by Veber and colleagues, while in the para-AMPA analogue an equilibrium exists between a β-II' turn and a γ-turn structure. Both analogues show no GH-inhibition or LH-inhibition in in vitro assays.     

Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His-Phe-Arg-Trp of α-Melanotropin by NMR

Solution conformation of cyclo(Gly¹-His²-Phe³-Arg⁴-Trp⁵-Gly⁶) and its d -Phe analog corresponding to the message sequence [Gly-α-MSH_5-10] of α-MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)-d₆ solution and in a DMSO-d₆/H₂O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only -NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross-peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the d -analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low-energy conformers with two γ-turns, a γ-turn and a β-turn, two β-turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a γ-bend at Gly⁶, two γ-bends at Phe³ and Gly⁶ and a conformer with a single β-turn and a γ-bend for the l -Phe analog. On the other hand, a conformation with two fused β-turns around the two tetrads His²-d -Phe³-Arg⁴-Trp⁵ and Trp⁵-Gly⁶-Gly¹-His² dominates the equilibrium mixture for the d -Phe analog. For the d -Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.     

Structure of cyclic peptides: the crystal and solution conformation of cyclo(Phe-Phe-Aib-Leu-Pro)

A solid-state and solution conformation analyses of the cyclopentapeptide cyclo(Phe-Phe-Aib-Leu-Pro) has been carried out by X-ray diffraction and nuclear magnetic resonance techniques. The structure of the hexagonal crystals, grown from a methanol solution [a=b= 16.530(4) Å, c= 21.356(9) Å, space group P6₅, Z = 6], shows the presence of one intramolecular N-H?O=C hydrogen bond with the formation of a γ-turn (C₇). The Aib³ residue, at the center of the γ-turn, presents unexpected values of the torsion angles [φ= 70.5° and ψ= -73.8°], which have been observed only once before for this helicogenic residue. A cis peptide bond occurs between Leu⁴ and Pro⁵; all other peptide bonds are trans. The overall conformation for the cyclopentapeptide with one cis-peptide bond on one side and an intramolecular γ-turn on the opposite side results in an equatorial topology of the side-chains of the Phe¹, Phe² and Leu⁴ residues. Indeed, the C^α-C^βand C^β-C^γ bonds of these residues lie approximately in the mean plane of the cyclic ring system. The structure is compared with data in the literature on cyclic pentapeptides. In addition the Pro-Phe-Phe moiety shows a conformation similar to that observed in other larger cyclic bioactive peptides, which indicates a reduced number of conformations for this sequence. The solution study was carried out in three different solvent systems: chloroform, acetonitrile and methanol in the temperature interval 220–300 K. In all three solvents the room temperature spectra show that the peptide is conformationally nonhomogeneous. In acetonitrile at low temperatures it is possible to reduce the conformational equilibrium to two predominant conformers which differ for the cis-trans isomerism of the Leu⁴-Pro⁵ peptide bond.     

The evaluation of type I and type II β-turn mixtures

Circular dichroism (CD) and¹ H-{¹H}NOE spectra were obtained for Piv-Pro-Ser-NHCH₃(1),[Piv-(CH₃)₃-C-CO], Boc-Pro-Ser-NHCH₃ (2) and Boc-Val-Ser-NHCH₃ (3), to determine the solution conformation of these p-turn models. In the crystal, 1 and 3 adopt an ideal type I β-turn, while 2 is characterized by a semifolded backbone geometry incorporating a cis Boc-Pro tert-amide bond. The predominance of a β-turn conformation in solution was suggested for models 1-3 on the basis of ¹H-{¹H}NOE data. In a nonpolar solvent the prevailing trans rotamer form (>80%) of 2 has a β-turn conformation according to heteronuclear NOE measurement. Positive ¹H-{¹H} NOEs were detected between the H^α(Pro)/NH(Ser), Hα(Ser)/NH(Ser) and NH(NHCH₃3)/HN(Ser) protons in the trans Boc-Pro rotamer form of 2 at -20° in CDCl₃. Similar positive homonuclear NOE enhancements were also observed on the appropriate proton signals in other models, such as Boc-Val-Ser-NHCH₃ (3). Boc-Val-D-Ser-NHCH₃ (4) and Boc-Pro-D-Ser-NHCH₃ (5), in various solvents. The ¹H- {¹H)NOE experiments carried out in CD₃CN clearly showed that besides the type I (or III) β-turn structure, one of the main conformations of models 1-5 is close to the type II β-turn backbone geometry in a nonpolar solvent. Unexpectedly, the conformational mixture of models 1-3 were characterized by class C (helix-like) CD spectra, although class C spectra are generally only correlated with the type I β-turn conformation. These acyclic models are the first carefully investigated examples of -L-L- triamide systems, containing a significant amount of a type II β-turn, as well as the type I p-turn and, however, yielding a class C circular dichroism spectra. The CD spectra recorded for 3 and 4 in acetonitrile were ‘calibrated’ using the ¹H-{¹H}NOE data. Such a “calibration”, as well as the semi-quantitative CD and NMR comprehensive analyses, demonstrated that class C, class B, as well as class C’ CD spectra may be obtained from the linear combination of the same two-component spectra, with different conformational weights. Therefore, it is suggested that the extraction of the conformational components of such models, simply on the basis of their CD spectra, must be made with caution.     

Synthesis and solution conformation of c(D-Trp-D-Cys(SO3-Na+)-Pro-D-Val-Leu), a potent endothelin-A receptor antagonist

The endothelin family of polypeptides are known to exert potent physiological effects which include cardiovascular regulation. The solution conformation and dynamics of c(D-Trp-D-Cys(SO₃-Na⁺)-Pro-D-Val-Leu), a potent endothelin-A receptor-selective antagonist, were characterized in aqueous solution by NMR spectroscopy and molecular modeling. NMR-derived conformational constraints were combined with computer-assisted molecular modeling using distance geometry calculations and energy minimization. The pentapeptide backbone is shown to adopt a single conformation in solution comprising a type II β-turn and an inverse γ-turn, with each residue in the trans conformation. Molecular dynamics were explored using relaxation measurements and low-temperature studies, and indicate that the peptide backbone is highly constrained with little conformational mobility present.     

Peptides related to the active fragment of “proline rich polypeptide”, an immunoregulatory protein of the ovine colostrums

The preferred solution conformation of the PRP-hexapeptide (Tyr-Val-Pro-Leu-Phe-Pro) and of some of its structural analogues was investigated by NMR- spectroscopy, spectrofluorimetry and computer simulation technic. It was found that the preferred conformation is characterized by cis′-conformation of Pro³ and the γ-turn on the Leu⁴-residue: for Val² and Phe⁵ a β-structure seems to be privileged. In such a conformation Val² and Leu⁴ residues occupy exactly the same positions in space as residues i and i+ 3 in an α-helix. It suggests that the PRP-hexapeptide can interact with receptor protein inducing or stabilizing its helical conformation by “knobs into holes” packing.     

Studies on the peptide corresponding to residues 34–47 of bovine factor X

Calcium binding studies of a 14-residue peptide corresponding to the 37–46 sequence of bovine factor X were performed using calcium ion selective electrode titrations and equilibrium dialysis. The presence of γ-carboxyglutamic acid residues at positions 36 and 40 coupled with the assumption that the peptide would bind calcium ions also prompted an investigation of possible secondary conformational changes in the peptide by use of circular dichroism spectroscopy. Equilibrium dialysis revealed a single relatively weak calcium binding site (log K_a= 2.39); an ion selective electrode experiment confirmed this result (log K_a= 2.17). The peptide maintained a random coil conformation throughout the calcium ion titrations as measured by circular dichroism.     

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.     

Purification and conformation of ribosomal protein L25 from E. coli ribosome

Ribosomal protein L25 from the large subunit of E. coli ribosomes has been purified using a new procedure involving a 2 m LiCl extraction followed by phosphocellulose chromatography in 6 m urea elution buffer. The conformation of purified L25 was studied employing circular dichroism and ultraviolet absorption spectroscopy in reconstitution buffer. The analysis of the far u.v. circular dichroism spectrum of L25 indicates L25 contains ～ 16%α-helix and ～ 19%β-structure. The conformation of L25 was also studied using the predictive methods of Chou & Fasman and Maxfield & Scheraga. Both of these methods predict approximately three times the percent α-helix present in L25 as compared with that determined from the analysis of the circular dichroism spectrum. A structure for L25 is predicted which contains two positively charged binding domains and is consistent with published binding data on the interaction of 5S RNA and L25. The large difference in the %α-helix as determined from the analysis of the circular dichroism spectrum and the predictive techniques is suggested to result from the denaturing effects of 6 m urea used in the preparation of ribosomal proteins.     

Vibrational analysis of peptides,polypeptides, and proteins

The normal modes have been calculated for three kinds of low energy γ-turn structures resulting from recent conformational energy calculations by Némethy. Frequencies have been computed for a γ-turn, a mirror-related γ-turn, and an inverse γ-turn of CH₃-CO-(L-Ala)_n-NH-CH₃, with n = 3 and n = 5, and for certain ¹⁴C and ¹⁵N derivatives of the n = 3 molecule. Correlations are evident between amide frequencies and γ-turn structures, and it is found that only amide I modes of peptide groups in the turn are relatively insensitive to the lengths of attached chains.     

苦豆子多糖溶液构象的研究

目的研究苦豆子多糖溶液的构象特征以及在不同因素影响下的溶液行为。方法采用碘–碘化钾反应观察苦豆子多糖链的分支情况和刚果红试验研究构象特征;采用圆二色谱(CD)法研究在不同浓度、温度和添加钙离子等条件下苦豆子多糖构象的变化。结果碘–碘化钾反应结果显示苦豆子多糖有较长的侧链和较多的分支;刚果红试验中苦豆子多糖具有多股螺旋结构;浓度、温度以及添加金属离子使苦豆子多糖溶液构象发生变化。结论通过碘–碘化钾反应、刚果红试验和圆二色谱法的结合可更加全面地了解苦豆子多糖溶液的构象。     

Simulations of conformers of tuftsin and a cyclic tuftsin analog

The conformational properties of the configurational isomers of tuftsin, a linear tetrapeptide with the sequence Thr-Lys-Pro-Arg, were investigated with six 1 ns molecular dynamics simulations in explicit water and in a 1.0 M NaCl solution. The average conformation of the cis isomer is a type VI β-turn. Our results indicate that water-peptide hydrogen bonding, in addition to intramolecular hydrogen bonds, stabilizes the cis conformer. The trans isomer is neither a β- nor a γ-turn. Results are compared with parallel studies on a cyclic analog of tuftsin, cyclo(Thr-Lys-Pro-Arg-Gly). The addition of salt does not influence the backbone conformation of the peptide. Differences between the structures are confined to the side-chain orientations of the Lys and Arg residues. © Munksgaard 1995.     

猪毛菜的化学成分与药理作用研究进展

目的：建立体外抗癌天然药物筛选模型,评价丹参素的抗癌活性。方法：以遗传物质DNA为靶标,溴化乙锭（EB）为致癌物质,丹参素为试药,利用荧光光谱检测丹参素对EB-DNA体系的荧光猝灭效应;利用圆二色谱技术检测丹参素对EB-DNA体系的干扰作用。结果：丹参素能明显抑制EB-DNA体系的荧光强度,且具有浓度依赖性;丹参素能够有效阻断EB嵌入DNA的碱基对中,维持DNA分子的正常构象。结论：丹参素抑制致癌剂对DNA链的嵌合,从而起抗癌活性。该模型可应用于抗癌功效的天然药物的筛选中。     

Backbone modifications in cyclic pep tides Conformational analysis of a cyclic pseudopentapeptide containing a thiomethylene ether amide bond replacement

NMR and X-ray crystallographic studies have shown that cyclic pentapeptides of the general structure cyclo(D-Xxx-Pro-Gly-Pro-Gly) possess β- and γ-turn intramolecular hydrogen bonds. As part of our continuing series surveying the compatibility of various amide bond replacements on peptide structure, we have synthesized cyclo(D-Phe-Proψ [CH₂S]Gly-Pro-Gly). The pseudopeptide was prepared by solid phase methods and cleaved from the resin by a new procedure involving phase transfer catalysis using K₂CO₃ and tetrabutylammonium hydrogen sulfate. Cyclization was carried out with the use of DPPA, HOBt, and DMAP to afford the product in 69% yield. The conformational behavior of the pseudopeptide was analyzed by ¹H and ¹³C (1D and 2D) NMR techniques. The backbone modification replaced the amide bond that is involved in a γ-turn intramolecular hydrogen bond in the all-amide structure. In CDCl₃, the pseudopeptide adopted the same all-trans conformation as its parent, although the remaining β-turn hydrogen bond was weaker according to Δδ/ΔT_NH measurements. In DMSO-d₆, the all-trans conformer and a second conformer were observed in a ratio of 55:45. These conformers, which slowly inter converted on the NMR time scale, could be separately assigned; peaks due to chemical exchange were readily distinguishable by the ROESY technique as reported earlier by others. ¹³C and ROESY experiments suggested the minor conformer contained one cis amide bond at the Gly¹-Pro² position. Thus, both the location and type of amide surrogate are important determinants affecting the compatibility of the replacement with a particular conformational feature.     

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.