Biomedical subjects

M Hollósi

Publications and source records attributed to M Hollósi.

At least 37 records · Page 2Linked to original sources

PubMed1995

Mapping the intersubunit region of influenza virus hemagglutinin: comparative CD and FTIR spectroscopic studies on multiple antigenic peptides.

An A/PR/8/34 (IHN1) influenza virus hemagglutinin (HA)-specific monoclonal antibody (Z38) was found to react with the solid phase adsorbed influenza virus expressing uncleaved (HA0) molecules but not to bind to virus particles bearing enzymatically cleaved hemagglutinin. Synthetic peptides corresponding to the uncleaved HA0 317-341 intersubunit region of subtypes H1-H3 (IP1-IP3) or comprising either the C-terminal 317-329 amino acids of HA1 (CP1) or the N-terminal 330-341 of HA2 (fusion peptide, FP) subunits of cleaved HA were used to characterize the fine specificity of Z38 mAb. Circular dichroism and Fourier-transform infrared spectroscopy showed that, compared to IP2 and IP3 comprising the H2 and H3 subtype fragments of the intact intersubunit region, IP1 has relatively low helicity but a tendency to adopt beta-turns in trifluoroethanol. The immunological and conformational properties of multiple antigenic peptides (MAPs) containing four copies of CP1 were also studied. Based on the appearance of an infrared component band at 1637 cm-1 (beta-turn band), the CP1 arms of MAPs also contain repeats of beta-turns. However, it is only the MAP1-FP construct comprising also the fusion peptide, which binds Z38 mAb as strongly as IP1 does. This puts emphasis on the role of the fusion region in modifying conformation and consequently the ability of peptides to elicit an antibody response. The results obtained for peptide conformation also suggests a beta-turn(s)/beta-sheet/beta-turn/beta-sheet conformational motif in the recognition by the hemagglutinin subtype-specific Z38 monoclonal antibody or by peptide-induced polyclonal antibodies.

Amino Acid Sequence↗

PubMed1995

Complexes of aluminium with peptide ligands: a Fourier transform IR spectroscopic study.

Aluminium has been recognized to be a neurotoxic agent and a risk factor in Alzheimer's disease and other neuronal dysfunctions. CD spectroscopic studies on two synthetic fragments of the human neurofilament protein midsized subunit (NF-M), and their alanine-for-serine-substituted and/or serine-phosphorylated derivatives showed the formation of stable, citric acid resistant complexes of Al3+ with peptide ligands [M. Hollósi, Z. M. Shen, A. Perczel, and G. D. Fasman (1994) Proc. Natl. Acad. Sci. USA, vol. 9, pp. 4902-4906]. In the case of Ser-phosphorylated fragments, a beta-sheet inducing effect of Ca2+ and Al3+ ions was observed. However, the serine-containing parent peptides, NF-M13 (KSPVPKSPVEEKG) and NF-M17 (EEKGKSPVPKSPVEEKG), failed to show CD spectral changes reflecting beta-sheet formation upon addition of Al3+ ions. On the basis of the amide I region of the Fourier transform ir spectra, in trifluoroethanol, the peptide backbone of NF-M17 and NF-M17 (A6A11) shows marked changes in the presence of Al3+. The most significant spectral differences are seen in the carboxyl region (> 1700 cm-1). The high-frequency component bands above 1760 cm-1 in both spectra belong to the C = O of undissociated CF3COOH. Another strong band at 1710 cm-1 which appears only in the spectrum of NF-M17 (A6A11)(NF-M17 with Ser6 and Ser11 replaced by Ala) can be assigned to the side chain or C-terminal COOH groups. The differential protonation state of the carboxyl groups in the two peptides suggests the formation of Al3+ complexes of different structure and stability.(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminum↗

PubMed1994

Conformational mapping of amyloid peptides from the putative neurotoxic 25-35 region.

The secondary structure of amyloid beta A(25-35) and its deletion analogues was studied by circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy and molecular dynamics calculation. Data of our comparative CD and FTIR measurements in trifluoroethanol suggest that beta A(25-35)NH2 has a preferred beta-sheet conformation. Contrary to this beta A(31-35)NH2 tends to adopt a beta-turn conformation. Based on the comparable neurotoxic effect of beta A(25-35)NH2 and beta A(31-35)NH2 the neurotoxicity likely involves the same 31-35 core sequence and the "biologically active conformation" is a beta-turn rather than a beta-sheet structure.

Amino Acid Sequence↗

PubMed1994

Study of Al3+ binding and conformational properties of the alanine-substituted C-terminal domain of the NF-M protein and its relevance to Alzheimer's disease.

NF-M13 [H-(Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly)-OH], NF-M17 [H-(Glu-Glu-Lys-Gly-Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly) -OH], and their phosphorylated derivatives, representing the C-terminal phosphorylation domain of the neurofilament protein midsize subunit, have four possible binding sites for metal ions: the COO- group of glutamate, the OH group of the serine residue, the PO3H- group of phosphoserine (when present), and the COO- at the terminus of the peptide chain. The CD titration of the phosphorylated neurofilament fragments with Al3+ and Ca2+ yielded a significant conformational change that resulted in conformations containing high beta-pleated-sheet contents, which precipitate on standing (intermolecular complex). Al3+ binding to the unphosphorylated NF-M13 and NF-M17 did not exhibit this behavior. Several alanine analogues of the parent NF-M17 peptide were synthesized in order to determine the relationship between metal ions and possible binding sites. CD titration of analogues with Ca2+ indicated that the critical residues of NF-M17 for Ca(2+)-induced conformational changes, from random to beta-pleated sheet, are the N-terminal serine or both phosphorylated serines. Al(3+)-induced conformational changes suggest that the critical sites of NF-M17 yielding the beta-pleated-sheet structure are the four glutamates or phosphorylated serines, especially the C-terminal SerP. On the basis of the titration data, it is very likely that analogues with a serine in position 11 form a stable intramolecular complex with Al3+ that, however, does not result in the adoption of the beta-conformation. Back-titration with citric acid fails to reverse the Al(3+)-induced conformational changes of the phosphorylated peptides. The above results, especially the possible formation of intramolecular and intermolecular Al3+ complexes, may have relevance to the molecular mechanism, through which the neurotoxin Al3+ gives rise to the formation of neurofilament tangles.

Alanine↗

PubMed1994

The 90 kDa heat shock protein (hsp90) induces the condensation of the chromatin structure.

The 90 kDa heat shock protein (hsp90) is a member of the "chaperone-complex" of steroid receptors believed to be partially or transiently localized in the cell nucleus. Demonstrating that hsp90 has an ATP binding site and autophosphorylating activity we have observed that histones, especially histone H1, are able to modulate the autophosphorylation of hsp90 [Csermely, P. and Kahn, C.R. (1991) J. Biol. Chem. 266, 4943-4950]. Our present data suggest a direct interaction of hsp90 with histones, showing that hsp90 is able to bind histone-agarose and enhances the binding of histones to DNA. Circular dichroism spectra of rat liver chromatin indicate that hsp90 induces a tighter, condensed state of the chromatin structure which is resistant against disruption by high salt treatment. Interactions of hsp90 with the chromatin may be important in regulating the transcriptional activity of steroid receptors and other transcription factors.

Animals↗

PubMed1994

Stable intrachain and interchain complexes of neurofilament peptides: a putative link between Al3+ and Alzheimer disease.

The etiologic role of Al3+ in Alzheimer disease has been controversial. Circular dichroism (CD) spectroscopic studies on two synthetic fragments of human neurofilament protein mid-sized subunit (NF-M), NF-M13 (KSPVPKSPVEEKG) and NF-M17 (EEKGKSPVPKSPVEEKG), and their alanine-substituted and/or serine-phosphorylated derivatives were carried out in an attempt to find a molecular mechanism for the effect of Al3+ to induce aggregation of neuronal proteins or their catabolic fragments. Al3+ and Ca2+ ions were found to induce beta-pleated sheet formation in the phosphorylated fragments. The cation sensitivity depended on the length and charge distribution of the sequence and site of phosphorylation. Al3+-induced conformational changes were irreversible to citric acid chelation, whereas Ca(2+)-induced conformational changes were reversible with citric acid. Studies of the alanine derivatives demonstrated which residues affected Al3+ or Ca2+ binding. Peptides containing at least one free (nonphosphorylated) serine residue were shown to form an intramolecular Al3+ complex, rather than an intermolecular one. In the intramolecular (intrachain) complex, the ligand function of the deprotonated serine hydroxyl was delineated [(Al.pepH-1)-type complex]. Ca2+ ions did not show a tendency for intramolecular complexing. The potential role of Al3+ in Alzheimer disease tangle and plaque formation is strongly suggested.

Aluminum↗

PubMed1994

CD and Fourier transform ir spectroscopic studies of peptides. II. Detection of beta-turns in linear peptides.

Comparative CD and Fourier transform ir (FTIR) spectroscopic data on N-Boc protected linear peptides with or without the (Pro-Gly) beta-turn motif (e.g., Boc-Tyr-Pro-Gly-Phe-Leu-OH and Boc-Tyr-Gly-Pro-Phe-Leu-OH) are reported herein. The CD spectra, reflecting both backbone and aromatic contributions, were not found to be characteristic of the presence of beta-turns. In the amide I region of the FTIR spectra, analyzed by self-deconvolution and curve-fitting methods, the beta-turn band showed up between 1639 and 1633 cm-1 in trifluoroethanol (TFE) but only for models containing the (Pro-Gly) core. This band was also present in the spectra in chloroform but absent in dimethylsulfoxide. These findings, in agreement with recent ir data on cyclic models and 3(10)-helical polypeptides and proteins in D2O [see S. J. Prestrelski, D. M. Byler, and M. P. Thompson (1991), International Journal of Peptide and Protein Research, Vol. 37, pp. 508-512; H. H. Mantsch, A. Perczel, M. Hollósi, and G. D. Fasman (1992), FASEB Journal, Vol. 6, p. A341; H. H. Mantsch, A. Perczel, M. Hollósi, and G. Fasman (1992), Biopolymers, Vol. 33, pp. 201-207; S. M. Miick, G. V. Martinez, W. R. Fiori, A. P. Todd, and G. L. Millhauser (1992), Nature, Vol. 359, pp. 653-655], suggest that the amide I band, with a major contribution from the acceptor C = O of the 1<--4 intramolecular H bond of beta-turns, appears near or below 1640 cm-1, rather than above 1660 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

T cell recognition of the posttranslationally cleaved intersubunit region of influenza virus hemagglutinin.

The influenza virus hemagglutinin is synthesized as a single polypeptide chain, but upon maturation it will posttranslationally be modified by a host cell related trypsin-like enzyme. The enzymatic cleavage attacks the so-called intersubunit region of the molecule giving rise to covalently linked HA1 and HA2 subunits. An I-Ed-restricted T cell epitope was identified in the highly conserved intact intersubunit region of the influenza virus hemagglutinin. T cell recognition of a 25-mer synthetic peptide comprising the intact intersubunit region does not require further processing and the elimination of the intervening Arg residue coupling the fusion peptide to the C-terminal segment of HA1 does not abolish the T cell activating capacity. The fine specificity pattern of a T cell hybridoma similar to that of the polyclonal T cell response demonstrates that a single T cell receptor is able to recognize peptides of different sizes representing not only the uncleaved but also the cleaved form of this hemagglutinin region. Based on specificity studies the epitope was localized to the C-terminal 11 amino acids of the HA1 subunit. The cross-reactivity of peptide-primed T cells with influenza virus infected antigen-presenting cells shows that fragments comprising the identified epitope of the intersubunit region can be generated as a result of natural processing of the hemagglutinin molecule. As antigen-presenting cells are lacking the enzyme which is responsible for the posttranslational modification of newly synthesized hemagglutinin molecules, the role of immature viral proteins in immune recognition is discussed.

Amino Acid Sequence↗

PubMed1994

The intersubunit region of the influenza virus haemagglutinin is recognized by antibodies during infection.

The influenza virus haemagglutinin has an important role in the infectious cycle of the virus and carries multiple B and T cell epitopes. It is synthesized as a single polypeptide chain but viral infectivity depends on its post-translational enzymatic cleavage. The cleavage site of a trypsin-like enzyme responsible for this modification is found in the most conserved intersubunit region of the molecule. In this study the role of this region in antibody recognition was investigated. Synthetic peptides comprising the intact and cleaved forms of the intersubunit segment were used to examine the specificity of virus- or peptide-induced antibodies. The immune response elicited by viral infection resulted in the appearance of antibodies capable of neutralizing the virus without interfering with its binding to the receptor. A monoclonal antibody (MoAb) of such functional properties was shown to recognize the intact intersubunit region both in the uncleaved haemagglutinin molecule and in a 25-mer synthetic peptide comprising the intact intersubunit region. Specificity and functional studies revealed the conformation-dependent recognition of the C-terminal segment of the haemagglutinin 1 subunit by this MoAb. The binding of the antibody was shown to inhibit the trypsin-mediated cleavage of the haemagglutinin molecule and the membrane fusion event. The enzymatic cleavage of the haemagglutinin was demonstrated to abolish antibody recognition of the infective virus suggesting an escape mechanism mediated by the functional destruction of this highly conserved region. The synthetic peptide corresponding to the intact intersubunit region is characterized by an ordered structure and is able to elicit an antibody response in BALB/c mice while its subfragments are nonimmunogenic. Furthermore, this peptide elicited a protective immune response demonstrated by in vivo experiments.

Amino Acid Sequence↗

PubMed1993

FT-IR spectroscopy indicates that Ca(2+)-binding to phosphorylated C-terminal fragments of the midsized neurofilament protein subunit results in beta-sheet formation and beta-aggregation.

The Fourier-transform infrared (FT-IR) spectra in trifluoroethanol (TFE) of phosphorylated peptides, NF-M17(Ser6P) and NF-M17(Ser11P) representing the C-terminal repeating domain of the midsized neurofilament protein subunit (NF-M) have been measured. In the absence of Ca2+ ions both phosphopeptides adopt predominantly aperiodic conformation with minor amounts of beta-turns, 3(10) helix or beta-pleated sheet. Addition of Ca(ClO4)2 results in opaque solutions, and in the case of the Ser6P peptide, precipitation. The infrared spectra of the supernatants reflect the presence of unordered and beta-sheet structure. The infrared spectrum of the solid Ca(2+)-complex of NF-M17 (Ser6P) in a KBr pellet shows amide component bands at 1654 (alpha-helix or loops) and 1626.5 cm-1 (beta-sheet). The high intensity of the beta-sheet component suggests extensive beta-aggregation. The data reported herein give an infrared spectroscopic support to our previous findings that upon Ca2+ binding, phosphorylated NF-M fragments undergo a marked conformational change which gives rise to partial beta-sheet formation and beta-aggregation. This observation may have relevance to the molecular events which lead to the accumulation of abnormal proteineous structures in Alzheimer's disease.

Amino Acid Sequence↗

PubMed1993

Circular dichroism and Fourier-transform infrared spectroscopic studies on T-cell epitopic peptide fragments of influenza virus hemagglutinin.

Epitopic peptides representing the C-terminal (HA1) region of cleaved hemagglutinin of influenza virus from different serotypes were synthesized. Circular dichroism and Fourier-transform infrared spectroscopic data showed that peptides HS2 and HS3 have a predominantly alpha-helical conformation in trifluoroethanol. Recently a component band appearing between 1640 and 1635 cm-1 in the amide I region of the Fourier-transform infrared spectra of polypeptides has been correlated with strongly H-bonded beta-turns (Ref. 8). Using this assignment, HS1 was found to contain less alpha-helix but have tendency to adopt beta-turn(s). Interestingly, fragment HS2 with the highest alpha-helix content proved to be the poorest T-cell epitope among serotypes HS1-HS3.

Amino Acid Sequence↗

PubMed1993

Prolyl oligopeptidase catalysis. Reactions with thiono substrates reveal substrate-induced conformational change to be the rate-limiting step.

Prolyl oligopeptidase, a member of the new family of serine proteases, exhibits significant mechanistic differences compared with the enzymes of the chymotrypsin and subtilisin families. Our kinetic study using the thiono substrate, benzyloxycarbonyl-Gly-Pro[CS-NH]-2-naphthylamide suggests that the putative oxyanion binding site is important in prolyl oligopeptidase catalysis, although to a lesser extent than in the chymotrypsin- and subtilisin-catalyzed reactions. By using another thiono substrate, benzyloxycarbonyl-Gly[CS-NH]Pro-2-naphthylamide, it is demonstrated that the distant S2P2 hydrogen bond (formed between the S2 subsite and P2 peptide residue) makes a greater contribution to catalysis than does stabilization by the oxyanion binding site involved directly in the bond cleavage. In contrast to the reactions catalyzed by chymotrypsin and subtilisin, no kinetic deuterium isotope effect is apparent in the acylation of prolyl oligopeptidase measured either with the specific benzyloxycarbonyl-Gly-Pro-2-naphthylamide, or with the very poor substrate, benzyloxycarbonyl-Gly-Pro[CS-NH]-2-naphthylamide. This indicates that the rate-limiting conformational change is induced by the substrate.

Animals↗

PubMed1993

ATP induces a conformational change of the 90-kDa heat shock protein (hsp90).

The 90-kDa heat shock protein (hsp90) is a well conserved, abundant cytosolic protein believed to be a "chaperone" of most steroid receptors. We have recently demonstrated that hsp90 has an ATP-binding site and autophosphorylating activity (Csermely, P., and Kahn, C. R. (1991) J. Biol. Chem. 266, 4943-4950). Circular dichroism analysis of highly purified hsp90 from rat liver shows that ATP induces an increase of beta-pleated sheet content of hsp90. Vanadate, molybdate, and heat treatment at 56 degrees C induce a similar change in the circular dichroism spectrum. Fourier transformed infrared spectroscopy reveals an ATP-induced increase in the interchain interactions of the 90-kDa heat shock protein due to an increase in its beta-pleated sheet content. In further studies we found that ATP: 1) decreases the tryptophan fluorescence of hsp90 by 11.6 +/- 1.9%; 2) increases the hydrophobic character of the protein as determined by its distribution between an aqueous phase and phenyl-Sepharose; and 3) renders hsp90 less susceptible to tryptic digestion. Our results suggest that hsp90 undergoes an "open-->closed" conformational change after the addition of ATP, analogous in many respects to the similar changes of the DnaK protein, the immunoglobulin heavy chain binding protein (BiP/GRP78), and hsp70. The ATP-induced conformational change of hsp90 may be important in regulating its association with steroid receptors and other cellular proteins.

Adenosine Triphosphate↗

PubMed1993

Characterization of beta-turns in cyclic hexapeptides in solution by Fourier transform IR spectroscopy.

The beta-turn represents a structural element frequently encountered in globular proteins. However, in spite of various theoretical and experimental studies the ir signature bands of pure beta-turns are still not established beyond doubt. Although considerable information exists now on the ir spectra of alpha-helical and beta-sheet structures, the lack of knowledge concerning turn structures in general, and that of beta-turns in particular, presents a major uncertainty in the estimation of global protein secondary structures from ir spectroscopic data. To obtain more specific information about the characteristic amide bands in beta-turns, we report herein an ir spectroscopic analysis of a series of five cyclic pseudo-hexapeptides known to form beta-turns from previous CD and nmr studies [A. Perczel, M. Hollósi, B. M. Foxman, and G. D. Fasman (1991) Journal of the American Chemical Society, Volume 113, pp. 9772-9784]. We show here that in these cyclic peptides the amide groups involved in beta-turns that comprise a ten-membered hydrogen-bonded ring (and represent the first H-bond pair in a beta-sheet), give rise to characteristic amide I bands in the range 1638-1646 cm-1, with the exact position depending on the solvent and the nature of the side-chain substituents.

Amino Acid Sequence↗

PubMed1993

Ca(2+)-induced conformational transitions of phosphorylated peptides.

CD spectroscopic studies on protected peptides containing lysine and serine, or phosphoserine, and on serine-containing fragments of the neurofilament protein midsized subunit, both in the unphosphorylated and phosphorylated form, are reported. The introduction of the phosphoryl group was not found to have a significant spectral effect in aqueous solution. In trifluoroethanol (TFE), spectral shifts toward unordered (type U) spectra or the appearance of distorted spectra likely reflect the adoption of aperiodic polypeptide conformations due to salt bridge(s) between negatively charged phosphoserine and positive lysine side-chain groups. A turn-stabilizing effect of phosphorylation was also observed. CD-monitored titration experiments in TFE revealed a high conformational sensitivity of phosphopeptides toward Ca2+ ions. The appearance of the unordered spectra or spectral shifts were the sign of a bulk disordering effect of Ca2+ ions. Spectra with specific spectroscopic features reflect the formation of Ca2+ complexes and the adoption of ordered unique backbone conformations. When ordered structures were obtained on addition of Ca2+ ions, the observed CD curves showed a resemblance to the spectrum of beta-pleated sheets. This may originate from chain extension and the formation of beta-pleated sheet segments fixed by Ca2+ bridges between PO3H-1 groups of adjacent peptide chains. The data clearly show that the effect of the Ca2+ ions is highly specific: the sequence, chain length, presence and distribution of charged side-chain groups, degree and site of phosphorylation, and environmental factors appear to be determining in the process of chain extension or beta-sheet formation.

Amino Acid Sequence↗

PubMed1993

Synthesis and conformational analysis of N-glycopeptides. II. CD, molecular dynamics, and NMR spectroscopic studies on linear N-glycopeptides.

The comprehensive structural analysis reported herein of eight N-glycopeptides, in three different solvents, is based on quantitative CD experiments, homonuclear nuclear Overhauser effect measurements, and molecular dynamics (MD) calculations. Although several orientations of the two amide planes attached to the carbohydrate pyranose ring are possible, according to NOE, CD data, and MD simulations, of all of the glycopeptide models, regardless of the type of the carrier peptide, only one dominant conformer population was found. This conformer is characterized by a nearly trans orientation of the CH and NH hydrogens of both acetamido groups. This finding is in perfect agreement with x-ray crystallographic data on the solid state conformation of the 1-N-acetyl- and 1-N-(beta-aspartyl)-2-acetamido-2-deoxy-beta-D-glucopyranosyla min e. The precise identification of this dominant conformer of N-glycopeptides in solution was the major question addressed herein by the structural analyses. A "CD additivity" experiment was carried out using an equimolar solution of Boc-Pro-Asp-NHCH3 and 1-N-acetyl-3,4,6- tri-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranosylamine at ambient temperature in acetonitrile. The CD spectrum obtained from the equimolar solution of the above two molecules (the "spectroscopic sum") was identical with the CD curve obtained from the algebraic summation of the individually recorded CD spectra of the peptide and the carbohydrate moiety ("mathematical sum"). The global picture of the CD spectral analyses of the eight parent peptides with the eight N-glycopeptides revealed that in trifluoroethanol and acetonitrile, the side-chain modification of the Asn models (natural N-glycopeptide analogues) by N-glycosylation has a significant effect on the conformation of the carrier peptide, resulting in a decrease in the original type I beta-turn content. Simultaneously, the type II beta-turn conformational percentage increased to approximately 20%. Such a conformational ratio change seems to be larger than the expected errors arising from the CD analyses, and agrees with the results of MD calculations. N-glycosylation of Asn residues causes perturbations, not only through the covalent bond, but also through specific hydrogen bonds between the backbone and side chain atoms. CD spectroscopy, augmented by efficient CD curve deconvolution techniques, has proved to be a useful tool for studying multicomponent conformer mixtures of small linear peptides in solution and changes of conformational equilibria caused by N-glycosylation.

Amino Acid Sequence↗

PubMed1993

New Gaba-containing analogues of human growth hormone releasing hormone (1-30)-amide: II. Detailed in vivo biological examinations.

Analogues of human growth hormone-releasing hormone-(1-30)-amide [GH-RH(1-30)-amide] were tested for their ability to stimulate GH release in vivo by injecting the peptides intravenously (iv), subcutaneously (sc), and intramuscularly (im). The analogues involved derivatization with Nle27 and Gaba substituents at the C-terminus with or without D-amino acid(s) in the peptide chain. The potency of the analogues was compared to that of GH-RH(1-29)-amid testing their ability to release GH at 5, 15 and 30 min after the administration. In iv test the potency of the analogues was 1.2-2 times higher than that of the GH-RH(1-29)-amide, and no significant differences were detected between the potencies of the analogues with or without D-amino acid. In the sc test the analogue with D-Ala2, Nle27, and Gaba30 substitutions expressed 8.0-51.7 times higher potency than the GH-RH(1-29)-amide, however, the analogue with similar modifications but with L-Ala2 showed the same low potency (1.2-2.1) as in the iv test. Results from the im experiments were similar to those of SC test. The most potent analogues were those which had D-Ala2, Nle27, and Gaba30 substitutions with Gly15 or Leu15. Circular dichroism (CD) spectra of the analogues showed that Leu in position 15 increased the stability of the predominant alpha-helix conformation, which improved the absorption of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

The evaluation of type I and type II beta-turn mixtures. Circular dichroism, NMR and molecular dynamics studies.

Circular dichroism (CD) and 1H-(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 beta-turn models. In the crystal, 1 and 3 adopt an ideal type I beta-turn, while 2 is characterized by a semifolded backbone geometry incorporating a cis Boc-Pro tert-amide bond. The predominance of a beta-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 beta-turn conformation according to heteronuclear NOE measurement. Positive 1H-(1H)NOEs were detected between the H alpha(Pro)/NH(Ser), H alpha(Ser)/NH(Ser) and NH(NHCH3)/HN(Ser) protons in the trans Boc-Pro rotamer form of 2 at -20 degrees 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) beta-turn structure, one of the main conformations of models 1-5 is close to the type II beta-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 beta-turn conformation. These acyclic models are the first carefully investigated examples of -L-L- triamide systems, containing a significant amount of a type II beta-turn, as well as the type I beta-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.

Amino Acid Sequence↗