Pyrrolidine Ring Of Proline Research Articles

Cycloalkyl Groups as Building Blocks of Artificial Carbohydrate Receptors: Studies with Macrocycles Bearing Flexible Side-Arms.

The cyclopentyl group was expected to act as a building block for artificial carbohydrate receptors and to participate in van der Waals contacts with the carbohydrate substrate in a similar way as observed for the pyrrolidine ring of proline in the crystal structures of protein-carbohydrate complexes. Systematic binding studies with a series of 1,3,5-trisubstituted 2,4,6-triethylbenzenes bearing various cycloalkyl groups as recognition units provided indications of the involvement of these groups in the complexation process and showed the influence of the ring size on the receptor efficiency. Representatives of compounds that exhibit a macrocyclic backbone and flexible side arms were now chosen as further model systems to investigate whether the previously observed effects represent a general trend. Binding studies with these macrocycles towards β-D-glucopyranoside, an all-equatorial substituted carbohydrate substrate, included 1H NMR spectroscopic titrations and microcalorimetric investigations. The performed studies confirmed the previously observed tendency and showed that the compound bearing cyclohexyl groups displays the best binding properties.

Cycloalkyl Groups as Subunits of Artificial Carbohydrate Receptors: Effect of Ring Size of the Cycloalkyl Unit on the Receptor Efficiency

1,3,5‐Trisubstituted 2,4,6‐triethylbenzenes consisting of isopropyl groups as recognition units have previously been shown to have interesting binding properties towards selected carbohydrates. The design of such artificial carbohydrate receptors was inspired by the mode of action of carbohydrate‐binding proteins, namely by the participation of the isopropyl side chain of valine in the formation of van der Waals interactions with the carbohydrate substrate. This study aimed to investigate how the replacement of the isopropyl groups by other structural motifs, such as cycloalkyl groups of varying sizes (three to seven‐membered rings), influences the binding properties of the acyclic compounds towards carbohydrates. The cyclopropyl moiety represents a cyclic analog of the isopropyl group, whereas the other cycloalkyl units can be regarded as structural elements, which differ from the structure of the isopropyl group by a –(CH2)n‐bridge (n = 1–4) between the two methyl groups. Furthermore, according to the natural interactions, the cyclopentyl group should be able to participate in the formation of van der Waals contacts in a similar way as that observed for the pyrrolidine ring of proline in some protein‐carbohydrate complexes. Such systematic binding studies allow the identification of interesting structure‐activity relationships, which are very useful for the development of artificial carbohydrate receptors with predictable binding properties.

Solution NMR Investigation of Proline-Rich Domains Reveals Mechanism of Modulation of Signal Transduction

Proline-rich domains are abundant in eukaryotic proteome and play an essential role in signal transduction by acting as docking sites for a multitude of signaling molecules. This is partly due to the restrictive backbone mobility afforded by the cyclical pyrrolidine ring of proline, which imposes unique conformational constraints and provides a distinct entropic advantage upon binding to its signaling partners. Despite their importance, proline-rich domains are often unexplored by structural biophysical techniques, including X-ray crystallography and NMR, due to the problems associated with their expression and purification in E. coli and due to their disordered nature. Moreover, as prolines lack the amide protons, they are not amenable to conventional 1H-detected NMR methods. Here, we present our latest results related to one such proline-rich domain - 168 amino acids, containing ∼30% prolines - belonging to a human protein ALIX, a multifaceted adaptor protein involved in apoptosis, endocytic membrane trafficking, cell adhesion, viral budding and tumor growth. We demonstrate new strategies to successfully express this domain in E. coli (∼ 40 mg protein / L of bacterial culture), and new NMR methods to carry out complete backbone resonance assignments of this domain. Biophysical investigation of interactions with its binding partners as well as Src-kinase mediated post-translational modifications using modern NMR methods reveal the mechanistic role of prolines in modulation of signal transduction.

Crystal structures of resorcin[4]arene and pyrogallol[4]arene complexes with proline: A model for proline recognition through C[sbnd]H···π interaction

Resorcin[4]arene (resorcinol cyclic tetramer, abbreviated as RCT) or pyrogallol[4]arene (pyrogallol cyclic tetramer, PCT) form host–guest 1:1 complexes with DL-proline (DL-Pro) or l-proline (L-Pro), [RCT·DL-Pro]·2MeOH·3.5H2O (1) and 2[PCT·L-Pro]·2EtOH·10H2O (2), whose crystal structures have been determined. In each complex, the proline ligand is incorporated into the bowl-shaped cavity of RCT or PCT host molecules through CH … π interactions between alkyl protons of the pyrrolidine ring of proline and π-rings of RCT or PCT, forming an [RCT/PCT·Pro] structural fragment. In the crystal lattice, two [RCT/PCT·Pro] fragments self-associate to form a ligand-mediated dimeric structure, [RCT·D-Pro·L-Pro·RCT] in 1 or [PCT·L-Pro·L-Pro·PCT] in 2. A 1H NMR solution study gave the host‒ligand binding constants of 10.0 ± 1.1 M–1 for the RCT–DL-Pro system and 17.3 ± 1.3 M–1 for the PCT–L-Pro system. These complexes provide a synthetic model for the recognition of the proline residue in proline-containing substrates or inhibitors by enzymes through CH … π interaction. The CSD survey revealed that the absolute value of the torsion angle N–Cα–CO1 (O1 is cis to N) about the carboxyl Cα–C bond of proline is significantly smaller than that of the Cβ–Cα–CO2 (O2 is cis to Cβ) torsion angle.

Study of structural and dynamic characteristics of copper(II) amino acid complexes in solutions by combined EPR and NMR relaxation methods.

Structural features and dynamical behaviour of the copper(ii) bis-complexes with glycine, d-alanine, d-valine, l-serine, l-aspartic acid, l-glutamic acid, l-lysine, l-proline, and sarcosine were studied by combined EPR and NMR relaxation methods. The cis and trans isomers were unambiguously assigned and characterized by EPR data. It was found that addition of a salt background has an influence on the cis-trans isomer equilibrium in favour of the formation of the cis isomer. By comparison of NMRD, DFT computations, and structural data it was shown that only one water molecule is coordinated in the axial position of these complexes. The increased exchange rates of this molecule found for Cu(l-Asp)2(2-), Cu(l-Glu)2(2-), Cu(l-LysH)2(2+), and Cu(l-Pro)2 were attributed to its pushing out by side chain groups of the ligands. By simulation of NMRD profiles an increase of lifetimes of the copper(ii) 2nd coordination sphere water molecules was revealed in the presence of additional carboxylic, alcoholic, or ammonium groups of the ligands, as well as the pyrrolidine ring of proline. The very short lifetimes of the 2nd coordination sphere water molecules (4-13 ps at 298 K) were explained in terms of the Frank-Wen structural model by the existence of cavities which draw in quickly enough water molecules from the 2nd coordination sphere.

Structure of the prolyl-acyl carrier protein oxidase involved in the biosynthesis of the cyanotoxin anatoxin-a

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins biosynthesized from L-proline by a short pathway involving polyketide synthases. Proline is first loaded onto AnaD, an acyl carrier protein, and prolyl-AnaD is then oxidized to 1-pyrroline-5-carboxyl-AnaD by a flavoprotein, AnaB. Three polyketide synthases then transform this imine into anatoxin-a or homoanatoxin-a. AnaB was crystallized in its holo form and its three-dimensional structure was determined by X-ray diffraction at 2.8 Å resolution. AnaB is a homotetramer and its fold is very similar to that of the acyl-CoA dehydrogenases (ACADs). The active-site base of AnaB, Glu244, superimposed very well with that of human isovaleryl-CoA dehydrogenase, confirming previous site-directed mutagenesis experiments and mechanistic proposals. The substrate-binding site of AnaB is small and is likely to be fitted for the pyrrolidine ring of proline. However, in contrast to ACADs, which use an electron-transport protein, AnaB uses molecular oxygen as the electron acceptor, as in acyl-CoA oxidases. Calculation of the solvent-accessible surface area around the FAD in AnaB and in several homologues showed that it is significantly larger in AnaB than in its homologues. A protonated histidine near the FAD in AnaB is likely to participate in oxygen activation. Furthermore, an array of water molecules detected in the AnaB structure suggests a possible path for molecular oxygen towards FAD. This is consistent with AnaB being an oxidase rather than a dehydrogenase. The structure of AnaB is the first to be described for a prolyl-ACP oxidase and it will contribute to defining the structural basis responsible for oxygen reactivity in flavoenzymes.

Gas-Phase Fragmentation of Oligoproline Peptide Ions Lacking Easily Mobilizable Protons

The fragmentation of peptides containing quaternary ammonium group, but lacking easily mobilizable protons, was examined with the aid of deuterium-labeled analogs and quantum-chemical modeling. The fragmentation of oligoproline containing quaternary ammonium group involves the mobilization of hydrogens localized at α- and γ- or δ-carbon atoms in the pyrrolidine ring of proline. The study of the dissociation pattern highlights the unusual proline residue behavior during MS/MS experiments of peptides. Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-013-0585-1) contains supplementary material, which is available to authorized users.

Proline Recognition through C–H···π Interaction within Crystal Structures of Dl-Proline–Calix[4]pyrogallolarene Molecular Complexes

Abstract X-ray analyses of two calix[4]pyrogallolarene complexes with Dl-proline have shown that they form host–guest-like 1:1 complexes in which the pyrrolidine ring of proline is incorporated into the bowl-shaped cavity of calix[4]pyrogallolarene through C–H···π interactions.

The Cytoplasmic Peptidase DPP9 Is Rate-limiting for Degradation of Proline-containing Peptides

Protein degradation is an essential process that continuously takes place in all living cells. Regulated degradation of most cellular proteins is initiated by proteasomes, which produce peptides of varying length. These peptides are rapidly cleaved to single amino acids by cytoplasmic peptidases. Proline-containing peptides pose a specific problem due to structural constrains imposed by the pyrrolidine ring that prevents most peptidases from cleavage. Here we show that DPP9, a poorly characterized cytoplasmic prolyl-peptidase, is rate-limiting for destruction of proline-containing substrates both in cell extracts and in intact cells. We identified the first natural substrate for DPP9, the RU1(34-42) antigenic peptide (VPYGSFKHV). RU1(34-42) is degraded in vitro by DPP9, and down-regulation of DPP9 in intact cells results in increased presentation of this antigen. Together our findings demonstrate an important role for DPP9 in peptide turnover and antigen presentation.

Conformational study of palindromic tripeptides (GPG, IPI and KPK) in HIV-1 protease—A density functional theory study

A comparative study has been carried out on three palindromic tripeptides Gly–Pro–Gly, Ile–Pro–Ile and Lys–Pro–Lys which were present in HIV protein along with their analogues applying density functional computation at B3LYP/6-31G* level of theory. Discrepancy from the structural analysis has been noted for all the systems and it was found to be more for amide capped structure at the C terminal of proline. The puckering amplitude A and Phase angle P of the pyrrolidine ring of proline in the chosen palindromic tripeptides and their analogues were calculated from the endocyclic torsion angles. The minimum energy conformers lying well within the prescribed region of proline were obtained for the derived compounds from potential energy surface scan mentioning that no role has been played by its terminal residues. This is further supported by the simulated amide bands identifying the helical structure for all three palindromic tripeptides signifying the importance of proline. The molecular properties such as stabilization energy, chemical hardness along with dipole moment were calculated and interpreted. The values of Cα-H s and the peptide backbone N–Cα–C O for all the selected conformers specify the three palindromic tripeptides to have a symmetrical achiral structure.

Synthesis of a Val-Pro diaminodiol dipeptide isostere by epoxyamine cyclization.

[reaction: see text] The stereoselective synthesis of a novel proline-containing dipeptide isostere is described. Starting from l-valine, three new contiguous stereocenters are generated by asymmetric induction and epoxide chemistry, while the pyrrolidine ring of proline is introduced in the final step via intramolecular ring opening of the amino acid derived epoxyamine. Proline-containing peptidomimetics are potentially attractive as selective inhibitors of proline-specific enzymes, such as PPIases and retroviral proteases, and as analogues of bioactive peptides.

Conformers of gaseous proline.

Accurate geometries, relative energies, rotational and quartic centrifugal distortion constants, dipole moments, harmonic vibrational frequencies, and infrared intensities were determined from ab initio electronic structure calculations for eighteen conformers of the neutral form of the amino acid L-proline. Only four conformers have notable population at low and moderate temperature. The second most stable conformer is only 2+/-2 kJ mol(-1) above the global minimum, while the third and fourth conformers are nearly degenerate and have an excess energy of 7+/-2 kJ mol(-1) relative to the global minimum. All four conformers have one hydrogen bond: N.HO in the lower energy pair of conformers, and NH.O in the higher energy pair of conformers. The conformer pairs differ only in their ring puckering. The relative energies of the conformers include corrections for valence electron correlation, extrapolated to the complete basis set limit, as well as core correlation and relativistic effects. Structural features of the pyrrolidine ring of proline are discussed by using the concept of pseudorotation. The accurate rotational and quartic centrifugal distortion constants as well as the vibrational frequencies and infrared intensities should aid identification and characterization of the conformers of L-proline by rotational and vibrational spectroscopy, respectively. Bonding features of L-proline, especially intramolecular hydrogen bonds, were investigated by the atoms-in-molecules (AIM) technique.

Further Delineation of the Two Binding Sites (R*n) Associated with Tachykinin Neurokinin-1 Receptors Using [3-Prolinomethionine11]SP Analogues

Two binding sites are associated with neurokinin-1 substance P receptors in both transfected cells and mammalian tissues. To further delineate the interactions between the crucial C-terminal methionine of substance P and these two binding sites, we have incorporated newly designed constrained methionines, i.e. (2S, 3S)- and (2S,3R)-prolinomethionines. The potencies of these C terminus-modified SP analogues to bind both sites and to activate phosphatidylinositol hydrolysis and cAMP formation have been measured, together with those of their corresponding sulfoxides and sulfones. The molecular nature of these two binding sites and their selective coupling to effector signaling pathways are discussed in the light of current models of receptor activation. The less abundant binding site is coupled to G(q/11) proteins, whereas the most abundant one interacts with G(s) proteins in Chinese hamster ovary cells transfected with human neurokinin-1 receptors. The specific orientation of the C-terminal methionine side chain imposed by these constraints shows that macroscopically chi(1) and chi(2) angles of this crucial C-terminal residue are similar in both binding sites. However, slight but significant variations in the rotation around the Cgamma-S bond yield different either stabilizing or destabilizing interactions in the two binding sites. These results highlight the need of such constrained amino acids to probe subtle interactions in ligand-receptor complexes.

Role of the Pyrrolidine Ring of Proline in Determining the Substrate Specificity of cdc2 Kinase or cdk5

To examine structural features of proline which are essential for the proline-directed phosphorylation by cdc2 kinase or cdk5, we prepared the peptide representing the cdc2 kinase phosphorylation site at Ser-55 in vimentin [Ser-Leu-Tyr-Ser-Ser-Ser55-Pro56-Gly-Gly58-Ala-Tyr-NH2], the peptide containing arginine in place of Gly-58, and their derivatives containing various N-methylamino acids or proline homologs in place of Pro-56, and tested them as substrates for the kinases. While substitution of the proline by proline homologs (L-pipecolic acid or L-azetidine-2-carboxylic acid) increased the K(m) value 2- to 4-fold at utmost, substitution by N-methylamino acids (sarcosine, L-N-methylalanine, L-N-methylvaline, or L-N-methylleucine) increased the K(m) value 7- to 40-fold for cdc2 kinase. For cdk5, these substitutions led to parallel effects on the K(m) value to those found for cdc2 kinase; cdk5 recognized the peptides with a proline specificity similar to that for cdc2 kinase. These results suggest that the pyrrolidine ring of proline is important for substrate recognition by cdc2 kinase or cdk5. Molecular dynamics and molecular mechanics simulations indicated that the pyrrolidine ring of proline is optimal to stabilize a beta-turn at the phosphorylation site and that the K(m) values of the peptides for the enzymes might be related to the probability of the turn structure. The results obtained here also suggest that the pyrrolidine ring of proline is required to maintain a high V(max) value for cdc2 kinase or especially for cdk5. These will aid in designing specific substrates or inhibitors for cdc2 kinase or cdk5.

Cyclo(L-prolyl-L-N-methylphenylalanyl). Conformation in solution and in the crystal.

Detailed analysis of the proton and carbon-13 NMR spectra of cyclo(L-prolyl-L-N-methylphenylalanyl) in chloroform and methanol in relation to its nonmethylated analog provided information on the conformation of the title compound in solution as well as on the effect of N-methylation and solvation. The X-ray structure of the title compound in the crystalline state showed the same conformational features as the solution structure. The phenyl group folds over the diketopiperazine ring which resembles a flattened half-chair. Both amide bonds are considerably nonplanar. The pyrrolidine ring of proline shows a strong pucker at the ring junction with the largest chi 5 value hitherto observed.

Hyp3]‐tuftsin ([Hyp3]‐TU) synthesis and biological activity

[Hyp3]-tuftsin (Thr-Lys-Hyp-Arg) has been synthesized by the liquid-phase method. In biological investigations performed on rats antinociceptive and diuretic effects have been determined. It has been suggested that the presence of hydroxyl substituent in pyrrolidine ring of proline slightly modifies antinociceptive TU effect and is responsible for the increased diuretic [Hyp3]-TU activity.

Probing the role of proline in peptide hormones: NMR studies of bradykinin and related peptides

The use of NMR methods to study conformational and dynamic aspects of the proline residues in the nonapeptide bradykinin is reviewed. NMR analyses involve considerations of bistable equilibria which include the cis/trans conformational heterogeneity of the imide bond, the cis'/trans' regions of conformational stability which characterize rotation about the C α—CO bond (dihedral angle Ψ), and the interconversion of the pyrrolidine ring of proline between puckered C γ-endo and C γ-exo conformations. These conformational features are all characterized by different kinetic behavior, are interdependent with peptide bond conformation, and exhibit sensitivity to amino acid substitutions. Thus, the substitution of Gly 6 for Ser 6 increases the fractional cis probability of the sixth peptide bond from 0.1 to 0.35. Substitutions of α-aminoisobutyric acid (AIB) residues for proline introduce conformational constraints analogous to those in cis' proline. Correlations of pyrrolidine ring conformation and dynamics with the cis/trans ratio of the imide bond have also been observed in model systems. Conformational and activity analyses of [AIB 7]-bradykinin provided a stimulus for the development of the first bradykinin antagonist by Stewart and Vavrek (Vavrek RJ and Stewart JM, Competitive antagonists of bradykinin. Peptides 6: 161–164, 1985).

Specificity of prolyl endopeptidase

A series of tetrapeptides, Cbz(Bz)-Gly-X-Leu-Gly, were synthesized and the kinetic parameters, k cat and k cat K m , determined for their hydrolyses by prolyl endopeptidase from Flavobacterium. The peptides with X = N-Me-Ala, Sar and Ala as well as the standard substrate (X = Pro) were found to be good substrates, while those with X = α-aminobutyryl, Hyp, Ser and Gly were poor substrates, and those with X = pipecolyl, α-aminoisobutyryl, N-Me-Val, N-Me-Leu, Hyp(O-Bzl) and Ser(O-Bzl) were not cleaved at all. These results suggest that the specificity-determining site or Sl subsite of the enzyme is designed to fit exactly the proline residue of the substrate with allowance for the residues carrying substituents at the N and/or C α which must not exceed the size of the pyrrolidine ring of proline.

13C NMR relaxation and conformational flexibility of the deoxyribose ring.

13C T1's and NOE's have been measured for all protonated carbons of 2'-deoxy-D-ribose (2'-d-ribose), 2'-deoxyadenosine-5'-monophosphate (5'-dAMP), thymidine-3'-monophosphate (3'-TMP) and thymidine-5'-monophosphate (5'-TMP) in D2O solutions. In all of the deoxy sugars examined, NT1 values for C-2' are significantly larger than the values for the remaining carbons. This result is interpreted in terms of rapid puckering motion of C-2'. By contrast, NT1 values measured in ribose are found to be equal, within experimental error. The results are compared with analogous data obtained for the five membered pyrrolidine ring of proline and with results for DNA itself.

Conformational properties of the sequential polyproline analogs poly(Pro‐Aze‐Pro) and poly(Aze‐Pro‐Aze)

AbstractThe lack of the positive band at around 226 nm in the CD spectra of poly(prolyl‐azetidine‐2‐carbonyl‐proline) in trifluoroethanol and of poly(azetidine‐2‐carbonyl‐prolyl‐azetidine‐2‐carboxylic) acid in F3EtOH and water, the hyperchromism of the absorption maximum at about 202 nm, and the extremely small intensity of the Cβ‐Pro, Cγ‐Pro, and Cβ‐Aze signals for the cis peptide bonds in the 13C nmr spectrum of poly(Pro‐Aze‐Pro) in F3EtOH indicate that both polyproline analogs exist as disordered chains in this solvent, the trans peptide group being maintained. The disordering of the chains is attributed to an increase in the accessible range of ψ due to the reduced dimensions of the square ring of L‐azetidine‐2‐carboxylic acid residue relative to the pyrrolidine ring of proline and to strong interactions of the haloalcohol with the peptide groups of the chains.