Induction of wet-dog shaking in rats by analogues and metabolites of thyrotrophin-releasing hormone (TRH)

The ability of thyrotrophin-releasing hormone (TRH), its metabolites and several analogues to induce wet-dog shaking (WDS) was tested by their injection into the periaqueductal grey region of male rats. TRH and its metabolite deamido-TRH (TRH-OH) both stimulated WDS, though TRH-OH gave a longer duration of response; other TRH metabolites were inactive. Of the TRH analogues studied, RX77368 (pGlu-His-3,3'-dimethyl-ProNH2) was the most potent in this behavioural test system. Both CG3509 and CG3703 were also very active in inducing WDS, as were their deamidated metabolites. The relative stability of the TRH analogues to enzymic degradation in the brain may be related to their enhanced behavioural activity over TRH. The production from these analogues of biologically-active metabolites may also explain the increased activity in stimulating WDS of the parent peptides.     

Conformational properties of 2,3-methanopyroglutamic acid in peptides: NMR and x-ray diffraction studies

The effects of replacing L-pyroglutamic acid with the cyclopropane analogue 2,3-methanopyroglutamic acid (2,3-MeGlp) on conformation and enzymatic stability have been investigated in 2,3-MeGlp-NHMe and the novel thyrotropin releasing hormone (TRH) analogue [2,3-MeGlp1]-TRH by x-ray diffraction and nmr. While 2,3-MeGlp-NHMe adopts a folded conformation (small psi angle) in the solid state, several conformations are available to the molecule in solution. 1H-nmr of the diastereomeric mixture [(+/- )-2,3-MeGlp1]-TRH indicates a close orientation of the pyrrolidone and imidazole rings. The 2,3-MeGlp-His amide bond is considerably more stable to pyroglutamate aminopeptidase than the Glp-His bond in TRH.     

江浙蝮蛇毒酸性与碱性磷脂酶A_2溶液构象变化的差示扫描量热法研究

运用差示扫描量热法,在不同ｐＨ值的缓冲溶液内和各种浓度的碱土族氯化物溶液内,研究了来自江浙蝮蛇（ＡｇｋｉｓｔｒｏｄｏｎｈａｌｙｓＰａｌｌａｓ）毒的酸性与碱性磷脂酶外Ａ２（ＰＬＡ２）的热变性过程。得到表征这两种酶溶液构象变化的热力学参数。依据这些参数研究了两者的溶液构象及其变化。在ｐＨ４．５以下,分子净荷正电的这两种酶在溶液中不形成可热致伸展的有序构象;ｐＨ高于４．５时,Ａｓｐ和Ｇｌｕ的侧链羧基以负离子形式存在有利于有序构象的稳定。Ｈｉｓ是决定ＰＬＡ２活力和热稳定性的重要残基。磷酸根离子和这两种酶有结合作用而降低有序构象的热稳定性。碱土族阳离子除和这两种酶结合外,还以依赖于离子强度的方式复杂地影响酶的溶液构象,但其作用不完全是静电性的,是或多或少地随离子的不同而不同的。计算给出酸性ＰＬＡ２的△Ｈｃｄ．     

Synthesis and conformational studies by 1H- and 13C-NMR spectroscopy of a novel, sterically constrained analogue of thyrotropin-releasing hormone

A novel thyrotropin-releasing hormone (TRH) analogue, [2,4-MePro3]-TRH (2,4-MePro: 2-carboxy-2,4-methanopyrrolidine), has been synthesized using a rapid solid phase peptide synthesis method, and its conformational properties investigated by one- and two-dimensional (2D) nmr spectroscopy and by proton Overhauser measurements. Following a published approach, calibrated interproton Overhauser effects were used together with distance geometry analysis to deduce that the single conformation of the His-2,4-MePro tertiary amide bond is trans in aqueous solution. This conclusion was corroborated by 2D dipolar-correlated (NOESY) spectroscopy. A preferentially extended conformation is indicated by the nmr data, similar to that of TRH. The phi, psi conformational space of 2,4-MePro is, however, significantly different from that of trans proline and the structural consequences of these differences at the C-terminus are discussed. The distribution of histidine side-chain conformations in the TRH analogue was deduced from coupling constants and from the short-range interaction between the imidazole ring and one of the prochiral faces of the 2,4-MePro side chain.     

The linker of calmodulin--to helix or not to helix.

The linker regions of the central helices of calmodulin and of troponin C are observed to be alpha-helices in crystal and in solution. However, these linkers are predicted to be non-helical by standard algorithms. Further, there is strong evidence that when calmodulin interacts with some of its targets this linker helix bends. The linker appears to be delicately balanced between helical and non-helical conformations. A review of this subject suggests that one can anticipate more unpredicted conformations for the central helices of the score of other proteins that have four EF-hand domains.     

Conformational equilibria and free energy profiles for the allosteric transition of the ribose-binding protein

The ribose-binding protein (RBP) is a sugar-binding bacterial periplasmic protein whose function is associated with a large allosteric conformational change from an open to a closed conformation upon binding to ribose. The crystal structures of RBP in open and closed conformations have been solved. It has been hypothesized that the open and closed conformations exist in a dynamic equilibrium in solution, and that sugar binding shifts the population from open conformations to closed conformations. Here, we study by computer simulations the thermodynamic changes that accompany this conformational change, and model the structural changes that accompany the allosteric transition, using umbrella sampling molecular dynamics and the weighted histogram analysis method. The open state is comprised of a diverse ensemble of conformations; the open ribose-free X-ray crystal conformations being representative of this ensemble. The unligated open form of RBP is stabilized by conformational entropy. The simulations predict detectable populations of closed ribose-free conformations in solution. Additional interdomain hydrogen bonds stabilize this state. The predicted shift in equilibrium from the open to the closed state on binding to ribose is in agreement with experiments. This is driven by the energetic stabilization of the closed conformation due to ribose-protein interactions. We also observe a significant population of a hitherto unobserved ribose-bound partially open state. We believe that this state is the one that has been suggested to play a role in the transfer of ribose to the membrane-bound permease complex.     

Characterization and functional expression of cDNAs encoding thyrotropin-releasing hormone receptor from Xenopus laevis.

Thyrotropin-releasing hormone receptor (TRHR) has already been cloned in mammals wherethyrotropin-releasing hormone (TRH) is known to act as a powerful stimulator of thyroid-stimulating hormone (TSH) secretion. The TRH receptor of amphibians has not yet been characterized, although TRH is specifically important in the adaptation of skin color to environmental changes via the secretion of alpha-melanocyte-stimulating hormone (alpha-MSH). Using a dege-nerate PCR strategy, we report on the isolation of three distinct cDNA species encoding TRHR from the brain of Xenopus laevis. We have designated these as xTRHR1, xTRHR2 and xTRHR3. Analysis of the predicted amino acid sequences revealed that the three Xenopus TRHRs are only 54-62% identical and contain all the highly conserved residues constituting the TRH binding pocket. Amino acid sequences and phylogenetic analysis revealed that xTRHR1 is a member of TRHR subfamily 1 and xTRHR2 belongs to subfamily 2, while xTRHR3 is a new TRHR subtype awaiting discovery in other animal species. The three Xeno-pus TRHRs have distinct patterns of expression. xTRHR3 was abundant in the brain and much scarcer in the peripheral tissues, whereas xTRHR1 was found mainly in the stomach and xTRHR2 in the heart. The Xenopus TRHR subtype 1 was found specifically in the intestine, lung and urinary bladder. These observations suggest that the three xTRHRs each have specific functions that remain to be elucidated. Expression in Xenopus oocytes and HEK-293 cells indicates that the three Xenopus TRHRs are fully functional and are coupled to the inositol phosphate/calcium pathway. Interestingly, activation of xTRHR3 required larger concentrations of TRH compared with the other two receptors, suggesting marked differences in receptor binding, coupling or regulation.     

Extrahypothalamic thyrotropin releasing hormone (TRH) -- its distribution and its functions.

Thyrotropin releasing hormone (TRH) is distributed throughout the extrahypothalamic nervous system and spinal cord, in the retina, in the pancreas and gastrointestinal tract, in the placenta, in amniotic fluid, in the adrenals and in frog skin. TRH has been shown to have a variety of effects in the central nervous system, both on isolated neurones and in a number of in vivo situations. TRH interacts with endogenous and exogenous opiates and it has been suggested that endogenous TRH may mediate part of the opiate withdrawal syndrome. The presence of TRH in the retina suggests the possibility that TRH plays a role in the visual process. TRH appears to be integrally related to central thermoregulatory mechanisms. The role of TRH in psychiatric disorders is at present controversial. Recent studies suggest a role for TRH as a modulator of gastrointestinal and pancreatic function. The gastrointestinal actions of TRH include inhibition of gastric acid secretion and alterations in gastic motility. The high concentrations of TRH in the neonatal pancreas suggest a role for TRH in the early development of the pancreas. One of the metabolites of TRH histidyl-proline diketopiperazone, appears to have a number of extrahypothalamic actions and this suggests the need for further exploration of the affects of this compound both on the central nervous system and the gastrointestinal tract. The multiple extrahypothalamic actions of TRH have led to the concept that it is an ubiquitous neurotransmitter that has been co-opted by the pituitary as a releasing factor.     

Induced conformational states of amphipathic peptides in aqueous/lipid environments.

Specific conformational effects have been reported for amphipathic model peptides upon binding of defined hydrophobic domains to nonpolar stationary phases during reversed-phase high performance liquid chromatography (RP-HPLC). Such induced conformations are found to be especially pronounced for peptides that are amphipathic in an alpha-helical conformation. Such induced amphipathic conformations resulted in substantially later elution than predicted using amino acid-based retention coefficients. In the present study, the induced conformational behavior of model peptides observed during RP-HPLC was correlated with their secondary structure as determined by circular dichroism (CD) spectroscopy in both aqueous solution and C18-mimetic environments. The experimental retention times of the peptides studied were found to correlate with their CD spectra in the presence of lipids, whereas a poor correlation was observed with their CD spectra in the presence of trifluoroethanol. A new approach was developed to evaluate the induction of secondary structure in peptides due to interactions at aqueous/lipid interfaces, which involves the measurement of the CD ellipticities of peptides bound to a set of C18-coated quartz plates. An excellent correlation was found in this environment between the RP-HPLC retention times and CD ellipticities of the bound peptides.     

Detection of thyrotropin-releasing hormone (TRH) mRNA by the reverse transcription-polymerase chain reaction in the human normal and tumoral anterior pituitary.

To investigate the presence of TRH mRNA in the human anterior pituitary tissue, total RNA from human normal and tumoral anterior pituitary, hypothalamus (positive control) and muscle tissues (negative control) was reverse transcribed (RT) to the first strand of cDNA. RT products were then amplified by polymerase chain reaction (PCR) using a set of three exon-specific primers (two external 5' and 3' primers and one internal 3' primer) for a target sequence of the TRH gene including an intronic sequence of about 650 base pairs (bp). Southern analysis of the RT-PCR products specifically hybridizing with a 45-mer TRH probe showed two bands of the predicted sizes (399 and 351 bp) far more intense in hypothalamus than in normal and tumoral anterior pituitary tissue. The 399 and 351 bp RT-PCR products contained the BglII enzyme restriction site included in the TRH cDNA sequences spanned by the primers and the two respective digested fragments which were, as predicted, 337 and 289 bp long, hybridized with the TRH probe. Based on these results, we can conclude that the RT-PCR products generated from RNA tissue were the target TRH sequences in the human normal and tumoral anterior pituitary tissue as well as in the hypothalamus. Our data imply TRH gene expression in the human anterior pituitary.     

Conformational analysis of biologically active thyroliberin analogs by two-dimensional NMR spectroscopy]

Preferable conformations of thyrotropin-releasing hormone (TRH, Glp-His-Pro-NH2) and its analogues Glp-Glu(R)-Pro-NH2 (R = NHCH(CH3)CH2Ar), Glp-Gln-Abu-NH2, Dho-Gln-Abu-NH2 in DMSO solution are determined using two-dimensional 1H NMR spectroscopy (delta-J-correlated, COSY and NOESY). Torsion angles psi i and chi i for every amino acid were calculated on the basis of the spin-spin coupling constants 3JNH-C alpha H and 3JC alpha H-C beta H values. The NOESY data were used for selecting the peptide conformations realized in solution. Distances between protons interacting by the dipole mechanism (d-contacts) were calculated using NOE values. These experiments allow one to estimate the torsion angles psi (between C alpha H-CO). TRH has an intramolecular H-bond between NH2-protons and His carbonyl with the torsion angles omega 3 = 180 degrees and psi 3 = 0 degrees. It is formation of this H-bond that apparently promotes the domination of the trans configuration of the His-Pro peptide bond. An intramolecular NH2-C alpha CO (Glp) H-bonding is revealed in other investigated compounds. It is known that a similar conformation of the TRH is realized in the course of its interaction with receptor.     

Conformation of the third domain of turkey ovomucoid in solution. Structural analysis by two-dimensional Overhauser nuclear effect spectroscopy]

The conformations of a polypeptide chain of turkey ovomucoid third domain and its modified form with split reactive site peptide bond Leu-18--Glu-19 have been determined by the literary two-dimensional nuclear Overhauser effect spectroscopy data using an earlier suggested method. It has been found that the polypeptide domain backbone contains an alpha-helical fragment (residues 32-47), five segments having extended conformation (1-5, 11-17, 19-25, 29-31, 48-50) and beta-turn type 1 (26-29). Segments 23-26, 28-31 and 50-51 form an antiparallel beta-structure. Conformational states of the residues entering irregular domain segments have been analysed. Splitting of the reactive site peptide bond Leu-18--Glu-19 is shown to cause insignificant changes in the conformations of a number of amino acid residues except for Val-6 and Asp-7 ones which undergo essential conformational alterations. The conformations of domain in solution and of japanese quail ovomucoid third domain in crystal have been compared. The root-mean-square deviations for phi and psi angles indicate their high similarity. The conformations of turkey ovomucoid third domain and proteinase inhibitor BUSI IIA in solution have been analysed. In spite of moderate (50%) homology of primary structures, some 75% of amino acid residues are shown to have close conformational phi and psi parameters.     

Loop formation in polynucleotide chains. II. Flexibility of the anticodon loop of tRNAPhe

The flexibility of hairpin loops containing n bases (residues) has been examined using a theoretical model [N. L. Marky and W. K. Olson (1982), Biopolymers, 21 , 2329–2344] of oligonucleotide loop closure. The study is based on correlated probabilities of chain separation and terminal residue orientation as outlined previously. The probabilities are calculated using standard statistical mechanical methods as functions of local conformational changes of the chain backbone. Our results for an RNA chain of 9 residues suggest that the anticodon loop is a dynamic structure capable of assuming a variety of different spatial conformations. Free energy values related to the various conformations span a narrow range of values (2–4 kcal/mole) and compare well with experimental observations in aqueous solution. Conformational transitions between the loop conformations are within less than 0.5 kcal/mole in free energy. The different spatial loop conformations and the likely pathways between them may have potential relevance to the molecular translation of the genetic code.     

Calculation of the conformation of glycosphingolipids. 2. GM1- and GM2-gangliosides

The conformations of GM1- and GM2-gangliosides have been predicted by energy minimization techniques including an orbital force field approach. The global energy minimum conformers for these two gangliosides show marked differences, particularly in the relative orientation of the sugar rings. The predicted structures are compared with those postulated from NMR spectroscopy in relation to the formation of a cation-binding site. The minimum energy conformer of GM2-ganglioside is able to form this binding site whereas this conformer of GM1-ganglioside is not. The nature of the specific interaction of gangliosides with activator proteins is discussed.     

Crystal structures of dialkylglycine decarboxylase inhibitor complexes.

The crystal structures of four inhibitor complexes of dialkylglycine decarboxylase are reported. The enzyme does not undergo a domain closure, as does aspartate aminotransferase, upon inhibitor binding. Two active-site conformations have been observed in previous structures that differ in alkali metal ion content, and two active-site conformations have been shown to coexist in solution when a single type of metal ion is present. There is no indication of coexisting conformers in the structures reported here or in the previously reported structures, and the observed conformation is that expected based on the presence of potassium in the enzyme. Thus, although two active-site conformations coexist in solution, a single conformation, corresponding to the more active enzyme, predominates in the crystal. The structure of 1-aminocyclopropane-1-carboxylate bound in the active site shows the aldimine double bond to the pyridoxal phosphate cofactor to be fully out of the plane of the coenzyme ring, whereas the Calpha-CO2(-) bond lies close to it. This provides an explanation for the observed lack of decarboxylation reactivity with this amino acid. The carboxylate groups of both 1-aminocyclopropane-1-carboxylate and 5'-phosphopyridoxyl-2-methylalanine interact with Ser215 and Arg406 as previously proposed. This demonstrates structurally that alternative binding modes, which constitute substrate inhibition, occur in the decarboxylation half-reaction. The structures of d and l-cycloserine bound to the active-site show that the l-isomer is deprotonated at C(alpha), presumably by Lys272, while the d-isomer is not. This difference explains the approximately 3000-fold greater potency of the l versus the d-isomer as a competitive inhibitor of dialkylglycine decarboxylase.     

Multiple conformations of the sea anemone polypeptide anthopleurin-A in solution.

Anthopleurin-A (AP-A) is a member of a family of sea anemone-derived polypeptides that interact with sodium channels in a voltage-dependent manner, producing a positive inotropic effect on the mammalian heart. There has been considerable interest in this molecule as a lead compound for the development of novel therapeutic agents. Earlier attempts to define the 3-dimensional structure of AP-A were complicated by the fact that it was found to exist in 2 conformations in solution. Using 1H- and 13C-NMR spectroscopy, we have now shown that this conformational heterogeneity arises from cis-trans isomerization about the Gly 40-Pro 41 peptide bond and that in the major form of the protein this peptide bond adopts a cis conformation. Furthermore, the increased sensitivity afforded by higher-field NMR has allowed identification of additional minor conformations of AP-A, the origin of which is presently unknown. We believe there will be many more examples of the detection by high-field NMR of previously unobserved minor conformations of proteins in solution.     

Local structure of cytochrome c from horse heart in solution. Conformational analysis using data of two-dimensional nuclear Overhauser effect spectroscopy]

Using the earlier suggested method the calculation of the backbone conformations of horse heart cytochrome c in oxidized (ferricytochrome c) and reduced (ferrocytochrome c) states has been performed by the two-dimensional nuclear Overhauser effect spectroscopy data. For both protein forms the secondary structure elements have been revealed and the conformations of the irregular polypeptide chain segments have been analysed. The similarity of the secondary structures of ferri- and ferrocytochrome c in solution was established from the comparison of their conformations. Small differences between the conformations of two molecule forms are shown to be localized within the polypeptide chain fragments situated in the spatial structure near the heme crevice. The comparison of the dihedral phi and psi angles in the calculated conformations of horse cytochrome C with the corresponding characteristics of X-ray structures of tuna ferri- and ferrocytochrome c made for the oxidized and reduced protein forms using the quantitative criteria testifies the similarity of their conformations in solution and crystal. In is shown that the conformational changes of the separate amino acid residues which take place as the result of the "solution-to-crystal" transition occur on the surface fragments of protein globule and do not lead to essential alterations of the secondary molecule structure.     

The structures of some cobalamins in solution.

Nuclear magnetic resonance spectroscopy has been used to examine the conformations of cobalamins in solution. The perturbation of the resonances in the n.m.r. spectra by lanthanide probes provided particularly valuable information. Except for minor details the conformations are the same as those in the crystalline state. The temperature dependence of the conformation has been reassessed.     

Recognition in cell adhesion. A comparative study of the conformations of RGD-containing peptides by Monte Carlo and NMR methods

Many of the proteins that mediate cell adhesion processes processes-fibronectin, fibrinogen, vitronectin, von Willebrand factor, osteopontin, laminin and various collagens--contain the amino acid sequence Arg-Gly-Asp. Short peptides that include this sequence have been shown to inhibit the interactions of cell adhesion proteins with their receptors and to have dramatic effects on developmental processes involving cellular recognition. In order to determine which conformations are accessible to Arg-Gly-Asp containing peptides, we analyzed tri-, tetra- and pentapeptides using molecular mechanics and Monte Carlo methods, and studied the solution conformations of the pentapeptide Gly-Arg-Gly-Asp-Ser using nuclear magnetic resonance techniques. The Monte Carlo method was used to: (a) identify the low energy conformations of the peptides and (b) evaluate their thermodynamic properties. In the case of the pentapeptide Gly-Arg-Gly-Asp-Gly, the four stable conformations include three with reverse turns and one open structure. The conformations found in this analysis are compatible with the nuclear magnetic resonance (nuclear Overhauser effect) data.