Bioactive Conformation of Luteinizing Hormone-Releasing Hormone: Evidence from a Conformationally Constrained Analog

Freidinger, Roger; Veber, Daniel F.; Perlow, Debra S.; Brooks, J. R.; Saperstein, Richard

doi:10.1126/science.7001627

Cited by 308 publications

(169 citation statements)

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“…Over the past 5 years there has been an escalating need for synthetic peptides in a wide variety of applications, including the development of synthetic peptide vaccines (1)(2)(3)(4)(5), the detailed study of antigen-antibody interactions (6)(7)(8)(9)(10)(11), the preparation of optimal analogs of biologically active peptides (12)(13)(14), the optimization of peptide antigens of clinical diagnostic utility (15)(16)(17), the mapping of protein products of brain-specific genes (18,19), and the study of protein conformational parameters (20). In the majority of these studies, the limiting factor has been the availability and cost of the desired peptides.…”

mentioning

confidence: 99%

General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids.

Houghten¹

1985

Proc. Natl. Acad. Sci. U.S.A.

1,532

747

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A novel yet simple method is described that facilitates the synthesis of large numbers of peptides to the extent that the synthesis process need no longer be the limiting factor in many studies involving peptides. By using the methods described, 10-20 mg of 248 different 13-residue peptides representing single amino acid variants of a segment of the hemagglutinin protein (HAI) have been prepared and characterized in less than 4 weeks. Through examination of the binding of these analogs to monoclonal antibodies raised against residues 75-110 of HAl, it was found that a single amino acid, aspartic acid at position 101, is of unique importance to the interaction. Two other residues, aspartic acid-104 and alanine-106, were found to play a lesser but significant role in the binding interaction. Other single positional residue variations appear to be of little or no importance.Over the past 5 years there has been an escalating need for synthetic peptides in a wide variety of applications, including the development of synthetic peptide vaccines (1-5), the detailed study of antigen-antibody interactions (6-11), the preparation of optimal analogs of biologically active peptides (12)(13)(14), the optimization of peptide antigens of clinical diagnostic utility (15-17), the mapping of protein products of brain-specific genes (18,19), and the study of protein conformational parameters (20). In the majority of these studies, the limiting factor has been the availability and cost of the desired peptides. Clearly, these studies would be greatly facilitated if synthetic methods were available that would permit the synthesis of larger numbers of peptides in a more cost efficient manner and in a shorter period of time per peptide. While it is currently possible to produce large numbers ofpeptides (i.e., hundreds) using available methods, the effort is so time-consuming and expensive that to actually carry out this number of syntheses is impractical, even in especially well-equipped laboratories. The first part of this paper describes a general method that can be utilized for the rapid (2-6 weeks) synthesis of large numbers of peptides (>100) in quantities that satisfy most needs (>10 mg).It is known from recent studies that antibodies raised against synthetic peptides frequently react with sites in the parent protein (1,2,11). These findings allow more detailed understanding of immunological interactions. Thus, instead of defining the reactivity of a protein antigen at the whole protein or subunit level, antigen-antibody interactions can be localized within the antigen to a short sequence of immunological importance (7,11). The next step is the refinement of our understanding of the antigenic sites of proteins or peptides at the level of individual amino acids. To illustrate the utility of the synthesis procedures presented here, the second part of this paper presents a detailed study of peptide antigen-antibody interactions. In the example presented, 248 individual peptides were synthesized and used in a study of one part...

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mentioning

confidence: 99%

General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids.

Houghten¹

1985

Proc. Natl. Acad. Sci. U.S.A.

1,532

747

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“…This approach has previously provided insight into the conformation-activity relationships of several naturally occurring linear and cyclic peptide hormones, including the enkephalins (13), luteinizing hormone-releasing hormone (14), somatostatin (15), oxytocin (16)(17)(18), vasopressin (18,19), and bradykinin (20). Based on our earlier reports of the exceptional in vitro (11) and in vivo (21) biological properties of [Nle4,DPhe7]-a-MSH on both normal and transformed melanophores, the evaluation of possible secondary structures (22) in a-MSH, the known conformational effect of introducing D-amino acids into peptides (23,24), and examination of three-dimensional molecular models of this stereoisomeric analogue, we concluded that a ,B-turn or other peptide chain-reversal region within the central active site (His-Phe-Arg-Trp) of a-MSH might be functionally related to its biologically active conformation.…”

mentioning

confidence: 99%

[half-Cys4,half-Cys10]-alpha-Melanocyte-stimulating hormone: a cyclic alpha-melanotropin exhibiting superagonist biological activity.

Sawyer¹,

Hruby²,

Darman³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

158

112

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a-Melanocyte-stimulating hormone (ai-melanotropin; a-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Ser-MetGlu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) that reversibly darkens amphibian skins by stimulating melanosome (pigment granule) dispersion within melanophores. By using a number of in vitro melanocyte assays, we have examined the conformational requirements for a-MSH activity. Synthesis of [half-Cys4,half-Cys 0.l-a MSH, a cyclic, conformationally restricted, "isosteric" analogue of a-MSH, provided a melanotropin with a potency > 10,000 times that of the native hormone in stimulating frog (Rana pipen) skin darkening. The cyclic analogue also showed substantially prolonged activity relative to the native hormone. Cys'l0-a-MSH was =30 times more potent than a-MSH in stimulating lizard (Anolis carolinensis) skin melanophores in vitro. By using a cell-free Cloudman S-91 mouse melanoma plasma membrane preparation, we found the cyclic analogue to be "'3 times as potent as the native hormone in stimulating adenylate cyclase activity. These results provide insight into the conformational requirements for biological activity of a-MSH, and the comparative conformational requirements of a-MSH at a number of pigment cell receptors.Peptide hormones and their analogues provide useful molecular probes for investigating the chemical-physical basis ofbiological information transfer at specific target tissues. a-Melanocytestimulating hormone (a-MSH; a-melanotropin) is a linear tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-GlyLys-Pro-Val-NH2, that is synthesized and secreted by the pars intermedia of the vertebrate pituitary (1). The hormone may have important physiological roles in the control of vertebrate pigment cell melanogenesis (2), neural functioning related to learning and behavior (3, 4), and fetal development (4).a-Melanotropin structure-function relationships have been studied by analysis of the biological activities of a number of structurally or stereochemically modified a-MSH analogues and fragments in amphibian melanophores (5-8) and, more recently, on mammalian melanoma cells (9-11). The amino acid residues believed to be important in the expression of melanotropic activity of a-MSH have been systematically examined and, based on these studies, a-MSH apparently contains two message sequences, (Glu)-His-Phe-Arg-Trp and Gly-Lys-ProVal-NH2. Each ofthese sequences can independently stimulate melanosome dispersion in amphibian melanophores in vitro (7). It has been reported (12) that a-MSH, like other so-called sychnologically organized hormones [e.g., corticotropin (ACTH), f3-lipotropin, cholecystokinin] is a conformationally flexible molecule in aqueous solution. Perhaps because of the postulated inherent conformational flexibility of this peptide, the threedimensional topochemical requirements of a-MSH, which may be important for its biologically active conformation § at the melanotropin receptor, have not been experimentally examined.The various conformational requirements in a peptide hormone that ...

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“…This observation led to the suggestion that -turns may serve as sites for molecular recognition. 3,4 Moreover, turns in peptides and proteins have been frequently suggested to be the bioactive conformations [5][6][7][8][9][10][11] involved in receptor binding, immune recognition, posttranslational modifications, and other processes. 12 Since turns are the shortest relatively stable and ordered structural elements, it has been proposed that they act as nucleation sites in the process of protein folding.…”

Section: Introductionmentioning

confidence: 99%

Optical spectroscopic elucidation of β‐turns in disulfide bridged cyclic tetrapeptides

2006

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Vibrational circular dichroism (VCD) spectroscopic features of type II β‐turns were characterized previously, but, criteria for differentiation between β‐turn types had not been established yet. Model tetrapeptides, cyclized through a disulfide bridge, were designed on the basis of previous experimental results and the observed incidence of amino acid residues in the i + 1 and i + 2 positions in β‐turns, to determine the features of VCD spectra of type I and II β‐turns. The results were correlated with electronic circular dichroism (ECD) spectra and VCD spectra calculated from conformational data obtained by molecular dynamics (MD) simulations. All cyclic tetrapeptides yielded VCD signals with a higher frequency negative and a lower frequency positive couplet with negative lobes overlapping. MD simulations confirmed the conformational homogeneity of these peptides in solution. Comparison with ECD spectroscopy, MD, and quantum chemical calculation results suggested that the low frequency component of VCD spectra originating from the tertiary amide vibrations could be used to distinguish between types of β‐turn structures. On the basis of this observation, VCD spectroscopic features of type II and VIII β‐turns and ECD spectroscopic properties of a type VIII β‐turn were suggested. The need for independent experimental as well as theoretical investigations to obtain decisive conformational information was recognized. © 2006 Wiley Periodicals, Inc. Biopolymers 85: 1–11, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Bioactive Conformation of Luteinizing Hormone-Releasing Hormone: Evidence from a Conformationally Constrained Analog

Cited by 308 publications

References 19 publications

General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids.

General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids.

[half-Cys4,half-Cys10]-alpha-Melanocyte-stimulating hormone: a cyclic alpha-melanotropin exhibiting superagonist biological activity.

Optical spectroscopic elucidation of β‐turns in disulfide bridged cyclic tetrapeptides

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