SMS 201-995, an octapeptide somatostatin analogue. Assignment of the 1H 500 MHZ n.m.r. spectra and conformational analysis of SMS 201-995 in dimethylsulfoxide

The possible conformations of SMS 201-995, an active analogue of somastostatin, have been studied in dimethylsulfoxide solution by 500 MHz proton n.m.r. spectroscopy. The assignments have been made by use of 2D-correlated methods to detect long-range coupling connectivities in aromatic residues and between the alpha protons of consecutive residues. NOESY experiments enabled us to correlate amide and alpha protons of neighbouring amino acid residues, which indicate a less flexible situation than in water. Measurements of temperature coefficients of the amide protons, of NH-C alpha H coupling constants and NOE effects are in favour of one predominant conformation with a beta turn, of type II', involving amino acids Phe3 to Thr6.     

Hexapeptide analogue of somatostatin, Sandoz 201-456. Conformational study in dimethylsulfoxide by 1D and 2D n.m.r. methods

The conformational properties of the somatostatin analogue 201-456 (1) have been studied by high field n.m.r. in DMSO. This analogue is the base structure of nine derivates synthesized by Bauer et al. and shows a very low biological activity, although derived structures such as SMS 201-995 (2) are very potent. Our study has shown an important difference between the most stable conformation of the two compounds: although the beta turn type II' structure at the Phe3-Trp4-Lys5 level is present in both analogues, an important conformational change appears at the cystine bridge. In SMS 201-995 the beta turn/beta sheet conformation is stabilized by the additional amino-acids D-Phe1 and Thr8 (ol) through intramolecular H-bonds.     

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 beta-II' turn and a gamma-turn structure. Both analogues show no GH-inhibition or LH-inhibition in in vitro assays.     

Conformational analysis of N-terminal O-glycopeptide sequences of interleukin-2]

The preferred conformations of eight O-glycopeptide sequences from the N-terminus of interleukin-2 containing two to ten amino acids, monoglycosylated at Thr3 with a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group, were determined by means of n.m.r. spectroscopic methods. The preferred conformation of the N-terminal sequence, L-Ala-L-Pro-[alpha-D-GalpNAc-(1----3)]-L-Thr-L-Ser, including the O-glycosidically linked 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group is not substantially influenced by the linkage of additional amino acids at the C-terminal end. Extended conformations were observed for all peptide units. Measurements of the relaxation times of the 13C atoms showed that the 2-acetamido-2-deoxy-D-galactose bound to the central amino acids has the lowest mobility, whereas the terminal amino acid residues and peptide side-chains are flexible. Calculations with the force-field program AMBER yielded conformations of minimized energies that were in good agreement with the n.m.r. spectroscopic data. This was only true when n.m.r. parameters that can be used as starting values for the calculations were available. Comparison with a nonglycosylated, N-terminal tetrapeptide sequence analog did not suggest changes in the peptide conformation when Thr3 is glycosylated with a 2-acetamido-2-deoxy-alpha-D-galactopyranosyl group.     

SMS 201-995, a very potent analogue of somatostatin. Assignment of the 1H 500 MHz n.m.r. spectra and conformational analysis in aqueous solution

The conformations of a cyclic analogue of somatostatin, SMS 201-995, have been studied by n.m.r. spectroscopy at 500 MHz in aqueous solution. Assignments were made by use of 2D-correlated methods, especially by detecting long-range connectivities in order to identify the aromic amino-acid and long-range couplings between alpha protons of consecutive residues. Measurements of temperature coefficients of amide protons and of NH-C alpha H coupling constants enabled us to conclude that in water the molecule is rather flexible, with no evidence for a beta turn structure involving Thr6. An equilibrium involving two gamma turn conformations stabilized respectively by Cys2-D-Trp4 and Phe3-Lys5 hydrogen bonds, is responsible for the large upfield shift observed for the Lys5 gamma protons and is compatible with the measured JNH-C alpha H coupling constants.     

Identification of residues essential for progesterone binding to uteroglobin by site-directed mutagenesis

In order to identify amino acids directly involved in progesterone binding to rabbit uteroglobin we have mutated Phe 6, Tyr 21 and Thr 60 by site-directed mutagenesis of the uteroglobin cDNA. These residues have been postulated previously to participate in progesterone binding. High-level expression of the mutated uteroglobin cDNAs in Escherichia coli yields recombinant protein mutants that, like natural uteroglobin, form stable dimers, suggesting that the tertiary structure of the protein has not been altered. Substitution of Phe 6 by Ser or Ala does not change the progesterone binding characteristics. In contrast, replacement of Tyr 21 by Phe or Ala, drastically decreases progesterone binding. In addition, replacement of Thr 60 by Ala reduces the affinity for progesterone by a factor of three. These data suggest a direct interaction of progesterone with these two amino acids and support the idea of direct hydrogen bonding of the carbonyl (C3 and C20) of progesterone with the hydroxyl groups of Tyr 21 and Thr 60, respectively.     

Conformational studies on somatostatin. II. The C-terminal hexapeptide fragment

The results of a conformational study on the C terminal hexapeptide of Somatostatin are presented. Semi-empirical energy calculations and high resolution NMR methods have been used to obtain information on the conformational properties of SRIF9-14 in [2H6]dimethylsulfoxide and 2H2O. It is concluded from the energy calculations that the peptide has an averaged conformation in which semi extended and folded structures are important. Only some of the folded conformations can explain the chemical shift differences between the amino acid residues Thr10 and Thr12 as a ring current shift by the Phe11 aromatic ring on Thr10. The nonequivalence is more pronounced in dimethyl-sulfoxide (0.23--0.15 ppm) where it decreases with increasing temperature towards the temperature independent value in 2H2O (0.03 ppm). This suggests that the folded conformations are somewhat predominant in dimethylsulfoxide solutions. In 2H2O the semi extended and folded structures are statistically equally important and the peptide is more flexible. A comparison with a study on the smaller fragments SRIF10-12 and SRIF10-13 which have similar conformational properties, demonstrates the usefulness of the fragment approach in conformational studies of peptides.     

Topological role of cytochrome P450 2D6 active site residues

Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates.     

Synthesis of a cyclic retro analogue of somatostatin suitable for photoaffinity labelling

Cyclic somatostatin analogues containing the modified retro sequence of the amino acids Phe7 to Phe11 of the natural compound have been found to exhibit high activity for cytoprotection of rat hepatocytes against cell poisons such as phallotoxins and galactosamine. Cyclo(-Phe(p-NH(1-14C)Ac)-Thr-Lys(CO(p-N3)C6H4)-Trp-Phe-D-Pro), a photoreactive and radioactive analogue of one of the most active cyclohexapeptides, was synthesized by a combination of solid phase technique and classical solution peptide synthesis. This peptide labels the same proteins in rat liver cell membrane that are modified by photolysable derivatives of bile acids, phalloidin and antamanide.     

Synthesis and conformational study of a cyclic hexapeptide analogue of somatostatin: cyclo(Phe-D-Trp-Lys-Thr-o-AMPA)

The active sequence Phe7-D-Trp8-Lys9-Thr10 of somatostatin has been cyclized through o-(aminomethyl)phenylacetic acid, a spacer molecule, designed to mimic a Gly-Gly dipeptide containing a cis-constrained peptide bond. The resulting analogue shows no GH-inhibition. A 2D n.m.r. study reveals conformations different from the proposed bio-active one and still sensitive to the medium (solvent).     

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe. Beta-turn conformation of a sequence related to an active chemotactic peptide analog

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and 1H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II beta-turn in the solid state with Met and Aib as the corner residues (phi Met = -51.8 degrees, psi Met = 139.5 degrees, phi Aib = 58.1 degrees, psi Aib = 37.0 degrees). A single, weak 4----1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N---O 3.25 A, N-H---O 128.4 degrees). 1H n.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagnetic radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCl3 and (CD3)2SO. Nuclear Overhauser effects observed between Met C alpha H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II beta-turns in these solvents.     

Conformational modifications of cyclic hexapeptide somatostatin analogs

A model for the bioactive conformation of the highly active cyclic hexapeptide somatostatin analog cyclo-(Pro-Phe-D-Trp-Lys-Thr-Phe) has been proposed. As a test of this model, several compounds containing lactam and N-Me amino acid conformational modifications in the Thr-Phe-Pro-Phe beta turn were synthesized. The N-Me alanine and sarcosine substitutions for proline gave highly active analogs, while lactam dipeptides in place of Phe-Pro decreased potency. 1H n.m.r. and CD spectra of these analogs illustrate the conformational effects in solution of these modifications. The results provide additional support for the proposed conformational model.     

Protein engineering of alcohol dehydrogenase--1. Effects of two amino acid changes in the active site of yeast ADH-1

One of the promises held out by protein engineering is the ability to alter predictably the properties of an enzyme to enable it to find new substrates or catalyse existing substrates more efficiently, such manipulations being of interest both enzymologically and, potentially, industrially. It has been postulated that in yeast alcohol dehydrogenase (YADH-1) certain amino acids such as Trp 93 and Thr 48 constrict the active site due to their bulky side chains and thus impede catalysis of molecules larger than ethanol. To study effects of enlarging the active site we have made two changes into YADH-1, replacing Trp 93 with Phe and Thr 48 with Ser. Kinetic experiments showed that this enzyme had marked increases in reaction velocity for the n-alcohols propanol, butanol, pentanol, hexanol, heptanol, octanol and cinnamyl alcohol compared to the parent, agreeing with the prediction that expanding the active site should facilitate the oxidation of larger alcohols. The substrate affinities were slightly reduced in the altered enzyme, possibly due to its having reduced hydrophobicity at Phe 93.     

癌肿与氨基酸代谢的研究

研究了癌肿与氨基酸代谢的关系。这些癌肿包括喉癌HepⅡ细胞 ,急性非淋巴细胞白血病和急性淋巴细胞白血病 ,结果表明 :( 1 )喉癌细胞株培养过程中亮氨酸、赖氨酸、丝氨酸、天冬酰胺、异亮氨酸、甘氨酸以及苏氨酸等水平明显降低 ,而色氨酸水平明显增加 ,说明喉癌细胞的生长繁殖必须依赖以上 7种氨基酸同时释放了色氨酸 ;( 2 )急性非淋巴细胞白血病 (ANLL)患者血浆中的谷氨酸、甘氨酸、亮氨酸、苯丙氨酸、酪氨酸和色氨酸等水平明显升高 ,而苏氨酸、组氨酸、丙氨酸等水平明显降低 ,这些结果与国际报道相一致 ;( 3)经治疗后 ,ANLL患者血浆中甘氨酸、色氨酸和苯丙氨酸等水平明显降低 ,而丙氨酸、组氨酸等水平明显升高 ,表明肿瘤细胞处在无氧代谢。患者经治疗后色氨酸和苯丙氨酸水平降低和组氨酸水平的升高对患者预后是有益的 ;( 4)急性淋巴细胞白血病患者血浆中苯丙氨酸、赖氨酸、色氨酸和酪氨酸水平提高 ,这些氨基酸能促进肿瘤生长 ,而门冬酰胺、谷氨酰胺以及天冬氨酸水平降低 ,说明这 3种氨基酸为肿瘤生长所必须。此外还发现ALL患者外周淋巴细胞中精氨酸水平增加 ,精氨酸对癌肿细胞有直接杀伤作用。     

Responses of the pancreatic digestive enzyme secretion to various combinations of amino acids and cholecystokinin in chicks (Gallus domesticus)

1. Effect of amino acid administration on pancreatic secretion of digestive enzymes, amylase, trypsinogen and chymotrypsinogen was studied after wing vein injection of an amino acid (AAs) mixture (Thr, Lys, Phe, Leu, Ile, Glu, Val, His, and Met) or combinations of selected amino acids, i.e. Thr + Phe + Ile, Thr + Phe, Thr + Ile or Phe + Ile, in the presence of cholecystokinin (CCK) in chicks. 2. Time course changes of enzyme output were similar in all treatment groups having a peak within 10-30 min, except for Phe + Ile that resulted in delayed induction of the enzyme release as shown by significant increases in the last 20 min compared with those in the rest. 3. When increases in enzyme outputs for the first 30 min were compared, it was shown that the three enzyme responses brought about by the administration of the AAs mixture was almost entirely accounted for by the combined injection of Thr + Phe. 4. Neither Thr + Ile nor Phe + Ile was as effective as Thr + Phe in inducing the output of these pancreatic enzymes. 5. The present results suggest that Thr and Phe may have a specific regulatory role in the secretion of pancreatic digestive enzymes in chicks when administered simultaneously.     

Metabonomics and population studies: age-related amino acids excretion and inferring networks through the study of urine samples in two Italian isolated populations

The study of two different Italian isolated populations was combined with a metabonomic approach to better understand tubular handling of amino acids. Levels of amino acids and metabolites have been analyzed by Nucleic Magnetic Resonance and expressed as ratio vs urinary creatinine concentration (mmol/mol). For most of the amino acids there is an age-related U shape pattern of excretion, with the peaks during childhood and old age, and a significant reduction in the adult age. Hierarchical cluster analysis has clearly identified three groups clustering the same amino acids: His, Thr and Ala (group one); Gly and Phe (group two) and a third larger one. Results have been further confirmed by factor and regression analysis, and used to confirm and, in some cases, infer new amino acids networks. As a matter of facts, the identification of strong evidences for clustering of urine excretion of several neutral amino acids suggests the predominant impact of relevant and common transporters     

Peptide conformation. 48. Conformation and biological activity of proline containing cyclic retro-analogues of somatostatin

Six cyclic retro-analogues of the peptide hormone somatostatin have been synthesized using the solid phase technique. The peptides cyclo(-Xaa1-Phe2-Thr3-Lys4-Ybb5-Phe6-) and cyclo(-Phe1-Xaa2-Thr3-Lys4-Ybb5-Phe6-) with Xaa = D- or L-Pro and Ybb = D- or L-Trp were cyclized via the azide method. The conformations of the cyclic hexapeptides in DMSO-d6 solution were determined by a number of homo- and heteronuclear two-dimensional n.m.r.-techniques including 2D rotating frame NOE-spectroscopy. Two-step coherence transfers, ROE and chemical exchange, are observed for the first time in ROESY spectra. The backbone conformation of the all-trans cyclopeptides consists of a beta-turn containing the Pro residue in the position i + 1. These retro-analogues of somatostatin exhibit a high activity in the inhibition of cholate and phalloidin uptake by liver cells (cytoprotective effect); however, the hormonal activities of the natural hormone are completely suppressed. The constitutional and conformational requirements for the cytoprotective activity are discussed.     

Molecular basis for selectivity of high affinity peptide antagonists for the gastrin-releasing peptide receptor.

Few gastrointestinal hormones/neurotransmitters have high affinity peptide receptor antagonists, and little is known about the molecular basis of their selectivity or affinity. The receptor mediating the action of the mammalian bombesin (Bn) peptide, gastrin-releasing peptide receptor (GRPR), is an exception, because numerous classes of peptide antagonists are described. To investigate the molecular basis for their high affinity for the GRPR, two classes of peptide antagonists, a statine analogue, JMV594 ([d-Phe(6),Stat(13)]Bn(6-14)), and a pseudopeptide analogue, JMV641 (d-Phe-Gln-Trp-Ala-Val-Gly-His-Leupsi(CHOH-CH(2))-(CH(2))(2)-CH(3)), were studied. Each had high affinity for the GRPR and >3,000-fold selectivity for GRPR over the closely related neuromedin B receptor (NMBR). To investigate the basis for this, we used a chimeric receptor approach to make both GRPR loss of affinity and NMBR gain of affinity chimeras and a site-directed mutagenesis approach. Chimeric or mutated receptors were transiently expressed in Balb/c 3T3. Only substitution of the fourth extracellular (EC) domain of the GRPR by the comparable NMBR domain markedly decreased the affinity for both antagonists. Substituting the fourth EC domain of NMBR into the GRPR resulted in a 300-fold gain in affinity for JMV594 and an 11-fold gain for JMV641. Each of the 11 amino acid differences between the GRPR and NMBR in this domain were exchanged. The substitutions of Thr(297) in GRPR by Pro from the comparable position in NMBR, Phe(302) by Met, and Ser(305) by Thr decreased the affinity of each antagonist. Simultaneous replacement of Thr(297), Phe(302), and Ser(305) in GRPR by the three comparable NMBR amino acids caused a 500-fold decrease in affinity for both antagonists. Replacing the comparable three amino acids in NMBR by those from GRPR caused a gain in affinity for each antagonist. Receptor modeling showed that each of these three amino acids faced inward and was within 5 A of the putative binding pocket. These results demonstrate that differences in the fourth EC domain of the mammalian Bn receptors are responsible for the selectivity of these two peptide antagonists. They demonstrate that Thr(297), Phe(302), and Ser(305) of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-pi interactions and hydrogen bonding are important for their high affinity interaction.     

Conformational study of O-glycosylated threonine containing peptide models

1H n.m.r. studies of Z-Thr-OMe, Z-Thr-Ala-OMe, Z-Ala-Thr-OMe and their glycosylated derivatives indicate the possibility of an intramolecular hydrogen bond between Thr N alpha H and the N-acetyl carbonyl of the carbohydrate moiety, 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-alpha-D-galactopyranose (AcGalNAc). This is especially true in the case of Z-Thr(AcGalNAc)-Ala-OMe, suggesting that the strength of this hydrogen bond is dependent on the neighboring amino acids on the carbonyl terminal side of Thr. The existence of such a hydrogen bond implies a conformation in which the carbohydrate moiety is restricted to an orientation with its plane roughly perpendicular to the peptide backbone. In such an orientation, steric problems will be minimized in the case of clustered O-glycosidically linked Thr(Ser) residues as found in human erythrocyte glycophorin. A locked orientation of the carbohydrate moiety with respect to the peptide backbone may also play a conformational role in antifreeze glycoproteins.