Novel sulfur and selenium containing bis-α-amino acids from 4-hydroxyproline

The synthesis of new substituted prolines carrying at C-4 a second α-amino acid residue is reported. The amino acid, l-cysteine or l-selenocysteine, is linked to the proline ring through the sulfur or the selenium atom, respectively. The products were prepared with different stereochemistry at C-4, in few and clean high-yielding steps, with suitable protections for solid phase applications. The introduction of both sulfur and selenium atoms at C-4 of the proline ring seems to enhance significantly the cis geometry at the prolyl amide bond.     

Flexible-geometry conformational energy maps for the amino acid residue preceding a proline.

Previously calculated conformational energy maps suggest that the alpha-helical conformation for the residue preceding a proline is disfavored relative to the extended conformation by more than 7 kcal/mol. In known protein structures this conformation is observed, however, to occur for about 9% of all prolines. In addition, introduction or removal of prolines at theoretically unfavorable positions in proteins and peptides can have modest effects on stability and structure. To investigate the discrepancy between calculation and experiment, we have determined how the conformation of the proline affects the calculated energy. We have also explored the effect of bond length and bond angle relaxation on the conformational energy map. The conformational energy of the preceding residue is found to be unaffected by the conformation of the proline, but the effect of allowing covalent bond relaxation is dramatic. If bond lengths and angles, and dihedral angles within the pyrrolidine ring, are allowed to relax, a calculated energy difference between the alpha and beta conformations of 1.1 kcal/mol is obtained, in reasonable agreement with experiment. The detailed shape of the calculated energy surface is also in excellent agreement with the observed conformational distributions in known protein structures.     

Novel 3-amino-2-hydroxy acids containing protease inhibitors. Part 1: Synthesis and kinetic characterization as aminopeptidase P inhibitors

Novel, potent inhibitors of aminopeptidase P, containing a 3-amino-2-hydroxy acid and a proline or a proline analogues, have been prepared. One part of the bestatin-derived inhibitors was found to inhibit APP from Escherichia coli and from rat intestine according to a mixed-type mechanism, with Ki values up to 1.26 microM. The other compounds, 3-amino-2-hydroxy acyl prolines of a different configuration, inhibit APP competitively, according to a slow-binding mechanism, with Ki values in the nanomolar up to the micromolar range.     

Conformational studies of proline-, thiaproline- and dimethylsilaproline-containing diketopiperazines.

As proline plays an important role in biologically active peptides, many analogues of this residue have been developed to modulate the proportion of cis and trans conformers. A correlation between the pyrrolidine ring shape and structural properties of proline has been established. Diketopiperazine (DKP) is the model of choice to study the influence of the proline ring modification. In this contribution, cyclo(Gly-Pro) and two analogues cyclo(Sip-Pro) and cyclo(Thz-Pro) have been studied with proton NMR. We showed that both analogues with heteroatoms in gamma position, silicon and sulfur respectively, display a more rigid five-member ring. The usual flexibility of proline ring is restrained in both cases and only the two C(beta)-exo and C(beta)-endo conformations are observed.     

Gating of shaker-type channels requires the flexibility of S6 caused by prolines

The recent crystallization of a voltage-gated K+ channel has given insight into the structure of these channels but has not resolved the issues of the location and the operation of the gate. The conserved PXP motif in the S6 segment of Shaker channels has been proposed to contribute to the intracellular gating structure. To investigate the role of this motif in the destabilization of the alpha-helix, both prolines were replaced to promote an alpha-helix (alanine) or to allow a flexible configuration (glycine). These substitutions were nonfunctional or resulted in drastically altered channel gating, highlighting an important role of these prolines. Combining these mutations with a proline substitution scan demonstrated that proline residues in the midsection of S6 are required for functionality, but not necessarily at the positions conserved throughout evolution. These results indicate that the destabilization or bending of the S6 alpha-helix caused by the PXP motif apparently creates a flexible "hinge" that allows movement of the lower S6 segment during channel gating and opening.     

Substrate recognition by proline permease in Salmonella

Summary. Proline transport is required for catabolism of proline as a carbon, nitrogen, and energy source, and for accumulation of proline during adaptation to osmotic stress. These physiological processes are widespread in nature, and play essential roles in the virulence of both prokaryotic and eukaryotic pathogens. In enteric bacteria, the major proline permease is encoded by the putP gene. To identify the structural features required for substrate recognition by PutP, we assayed the transport and toxicity of a variety of natural and synthetic derivatives of proline. The results indicate that the substrate binding site of proline permease consists of a hydrophobic pocket that accommodates C3, C4, and C5 of the pyrrolidine ring. Both 4- and 5-membered rings fit into the substrate binding pocket, but 6-membered rings are excluded. Analogs with substituents on the C4 position are also excluded. In addition, the binding site includes a hydrophilic region that recognizes the imino and carbonyl groups. A free carboxyl group is not required. Taken together, these results may be used to design new synthetic inhibitors of proline transport that can effectively block proline uptake by microbial pathogens. Received November 1, 2000 Accepted December 1, 2000     

High affinity Grb2-SH3 domain ligand incorporating Cbeta-substituted prolines in a Sos-derived decapeptide

Peptide ligands that disrupt MAPK pathways are of great interest for a better understanding of these signalling cascades and represent therefore an attractive target to control cell degenerative processes. In that context, selective disruption of the upstream Grb2/Sos complex in the Ras/MAPK cascade has focused extensive work. The Sos PPII decapeptide, which interacts with the Grb2-SH3 domains, has been modified in various positions and the best inhibitors designed so far are either dimeric ligands or peptoid analogues of the VPPPVPPRRR sequence. We report the synthesis of new Grb2 ligands in which the key Val5 residue has been replaced by a cis C(beta)-substituted proline. Both fluorescence and ITC assays have been employed to measure the affinity of these substituted peptides for a recombinant Grb2 protein. Whereas proline in position 5 completely abolished the binding potency, a cis C(beta)-methyl-L-proline restored the affinity. Other cis C(beta)-proline substituents led to a complete loss of binding potency. Combining the best modifications: a cis C(beta)-methylproline 5, N-acetylation, C-carboxamide and dimerization yielded a 560-fold affinity enhancement compared to the wild-type VPPPVPPRRR sequence. This study shows that C(beta)-substituted prolines may constitute a new alternative for PPII ligands, combining entropy and enthalpy beneficial effects.     

Synthetic biology of proteins: tuning GFPs folding and stability with fluoroproline

Background

Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the C^γ-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. C^γ-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides.

Methodology/Principal Findings

In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the C^γ-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines.

Conclusions/Significance

We discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the C^γ-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and existing protein engineering methods can be envisioned as a promising strategy for the design of complex tailor-made proteins and even cellular structures of superior properties compared to the native forms.     

Proline transport in Salmonella typhimurium: putP permease mutants with altered substrate specificity.

The putP gene encodes a proline permease required for Salmonella typhimurium LT2 to grow on proline as the sole source of nitrogen. The wild-type strain is sensitive to two toxic proline analogs (azetidine-2-carboxylic acid and 3,4-dehydroproline) also transported by the putP permease. Most mutations in putP prevent transport of all three substrates. Such mutants are unable to grow on proline and are resistant to both of the analogs. To define domains of the putP gene that specify the substrate binding site, we used localized mutagenesis to isolate rare mutants with altered substrate specificity. The position of the mutations in the putP gene was determined by deletion mapping. Most of the mutations are located in three small (approximately 100-base-pair) deletion intervals of the putP gene. The sensitivity of the mutants to the proline analogs was quantitated by radial streaking to determine the affinity of the mutant permeases for the substrates. Some of the mutants showed apparent changes in the kinetics of the substrates transported. These results indicate that the substrate specificity mutations are probably due to amino acid substitutions at or near the active site of proline permease.     

Dermorphin tetrapeptide analogues with 2',6'-dimethylphenylalanine (Dmp) substituted for aromatic amino acids have high mu opioid receptor binding and biological activities

To investigate the value of the 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid substitution, we prepared analogues of the mu opioid receptor-selective dermorphin tetrapeptide Tyr-D-Arg-Phe-betaAla-NH(2) (YRFB) in which Dmp or its D-isomer replaced Tyr(1) or Phe(3). Replacing Phe(3) with Dmp essentially tripled mu receptor affinity and the receptor's in vitro biological activities as determined with the guinea pig ileum (GPI) assay but did not change delta receptor affinity. Despite an inversion of the D configuration at this position, mu receptor affinity and selectivity remained comparable with those of the L-isomer. Replacing the N-terminal Tyr residue with Dmp produced a slightly improved mu receptor affinity and a potent GPI activity, even though the substituted compound lacks the side chain phenolic hydroxyl group at the N-terminal residue. Dual substitution of Dmp for Tyr(1) and Phe(3) produced significantly improved mu receptor affinity and selectivity compared with the singly substituted analogues. Subcutaneous injection of the two analogues, [Dmp(3)]YRFB and [Dmp(1)]YRFB, in mice produced potent analgesic activities that were greater than morphine in the formalin test. These lines of evidence suggest that the Dmp residue would be an effective aromatic amino acid surrogate for both Tyr and Phe in the design and development of novel opioid mimetics.     

Biosynthesis of trans-4-hydroxy-L-proline by Streptomyces griseoviridus.

Radioisotopic experiments have revealed that free trans-4-hydroxy-L-proline is an intermediate synthesized from L-proline during formation of the peptide-bound cis-4-hydroxy-D-proline residue in the antibiotic, etamycin. This conclusion was based on the fact that 1) both radiolabeled L-proline and trans-4-hydroxy-L-proline are precursors of the bound D-imino acid as noted previously by Hook and Vining ((1973) J. Chem. Soc. Chem. Commun. 185-186; (1973) Can. J. Biochem. 51, 1630-1637), 2) the unlabeled trans isomer specifically inhibited the incorporation of radiolabel from proline into the antibiotic, 3) the 14C-hydroxyimino-acid was isolated from the intracellular pool and medium following incubations with L-[14C]proline during antibiotic biosynthesis and when etamycin synthesis was blocked by D-leucine. By means of chromatographic and enzymatic analyses, it was established that the free imino acid possesses the trans-L configuration.     

Binding of spin-labeled galactosides to the lactose permease of Escherichia coli

A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1 -yl )]aminophenyl pyrrolidine (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand. The equilibrium dissociation constant (K(D)) is about 23 microM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS. It is concluded that NN binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label.     

Synthesis and structure-activity relationships of 5-substituted pyridine analogues of 3

In an effort to probe the steric influence of C5 substitution of the pyridine ring on CNS binding affinity, analogues of 1 substituted with a bulky moiety--such as phenyl, substituted phenyl, or heteroaryl-were synthesized and tested in vitro for neuronal nicotinic acetylcholine receptor binding affinity. The substituted analogues exhibited Ki values ranging from 0.055 to 0.69 nM compared to a Ki value of 0.15 nM for compound 1. Assessment of functional activity at subtypes of neuronal nicotinic acetylcholine receptors led to identify several agonists and antagonists.     

Prolines are not essential residues in the "barrel-stave" model for ion channels induced by alamethicin analogues.

In the "barrel-stave" model for voltage-gated alamethicin channels in planar lipid bilayers, proline residues, especially Pro14, are assumed to play a significant role. Taking advantage of a previous synthetic alamethicin analogue in which all eight alpha-aminoisobutyric acids were replaced by leucines, two new analogues were prepared in order to test the effects of Pro14 and Pro2 substitutions by alanines. The alpha-helical content of the three analogues in methanol solution remains predominant (between 63 and 80%). Macroscopic conductance experiments show that a high voltage dependence is conserved, although the apparent mean number of monomers forming the channels is significantly reduced when the substitution occurs at position 14. This is confirmed in single-channel experiments which further reveal faster fluctuations for the modified analogues. These results demonstrate that, although prolines, especially Pro14, are favorable residues for alamethicin-like events, they are not absolute prerequisites for the development of highly voltage-dependent multistate conductances.     

Conformation-activity relationships of cyclic dermorphin analogues

A theoretical conformational analysis (molecular mechanics study) of nine cyclic tetrapeptides, structurally related to the highly mu-receptor-selective dermorphin analogue H-Tyr-D-Orn-Phe-Asp-NH2, was performed. These compounds display considerable diversity in their mu-receptor affinity and selectivity. A systematic search and subsequent energy minimization in absence of the exocyclic Tyr1 residue and Phe3 side chain revealed the constrained nature of the 11-13-membered ring structures contained in these analogues. No more than four low-energy conformers (within 2 kcal/mol of the lowest energy conformation) were found in each case. After attachment of the Tyr1 moiety and Phe3 side chain to the "bare" low-energy ring structures, a systematic search and energy minimization of these exocyclic moieties resulted in a limited number of low-energy conformational families for all compounds. Five analogues with high mu-receptor affinity--H-Tyr-D-Orn-Phe-Asp-NH2, H-Tyr-D-Orn-Phe-D-Asp-NH2, H-Tyr-D-Asp-Phe-Orn-NH2, H-Tyr-D-Asp-Phe-A2bu-NH2 (A2 bu: alpha, gamma-diaminobutyric acid) and H-Tyr-D-Cys-Phe-Cys-NH2--all showed a tilted stacking interaction between the Tyr1 and Phe3 aromatic rings in the lowest or second lowest energy conformation found. The same kind of stacking was not possible in low-energy conformers of the four analogues with poor affinity for the mu-receptor [H-Tyr-L-Orn-Phe-Asp-NH2, H-Tyr-D-Orn-D-Phe-Asp-NH2, H-Tyr-D-Orn-Phe(NMe)-Asp-NH2 [Phe(NMe): N alpha-methylphenylalanine], and H-Tyr-D-Orn-Phg-Asp-NH2 (Phg: phenylglycine)].(ABSTRACT TRUNCATED AT 250 WORDS)     

THE DISSOCIATION CONSTANTS OF RACEMIC PROLINE AND CERTAIN RELATED COMPOUNDS

1. It has been experimentally shown that breaking of the pyrrolidine ring of proline by nitrous acid is not a factor of sufficient magnitude to account for the amino nitrogen which is usually found in proline preparations. 2. A method for the preparation of racemic proline is described. The product was found to be free from amino nitrogen. 3. The dissociation constants of racemic proline and of certain structurally related compounds were determined.     

Structural basis for the lower affinity of the insulin-like growth factors for the insulin receptor

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A "quadruple insulin" analogue ([Phe(A8), Ser(A10), Thr(B5), Gln(B16)]Ins) showed affinity as IGF-I for the insulin receptor, and a "sextuple insulin" analogue ([Phe(A8), Ser(A10), Thr(A18), Thr(B5), Thr(B14), Gln(B16)]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a "quadruple IGF-I" analogue ([His(4), Tyr(15), Thr(49), Ile(51)]IGF-I) and a "sextuple IGF-II" analogue ([His(7), Ala(16), Tyr(18), Thr(48), Ile(50), Asn(58)]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.     

Regulation of the major proline permease gene of Salmonella typhimurium.

The structural gene for the major proline permease is located in a tight cluster with genes coding for the proline degradative enzymes, proline oxidase and pyrroline-5-carboxylic acid dehydrogenase. Expression of the permease is regulated in parallel with the two degradative enzymes, and all three functions are subject to catabolite repression. Regulatory mutants (putC) have constitutively high levels of all three activities, suggesting that all are regulated by a single mechanism.