Template-assembled Synthetic Proteins Research Articles

Characterization of a Selective Antagonist of Neuropeptide Y at the Y2 Receptor: SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF A Y2 ANTAGONIST

Neuropeptide Y (NPY) is a potent inhibitor of neurotransmitter release through the Y2 receptor subtype. Specific antagonists for the Y2 receptors have not yet been described. Based on the concept of template-assembled synthetic proteins we have used a cyclic template molecule containing two beta-turn mimetics for covalent attachment of four COOH-terminal fragments RQRYNH2 (NPY 33-36), termed T4-[NPY(33-36)]4. This structurally defined template-assembled synthetic protein has been tested for binding using SK-N-MC and LN319 cell lines that express the Y1 and Y2 receptor, respectively. T4-[NPY(33-36)]4 binds to the Y2 receptor with high affinity (IC50 = 67.2 nM) and has poor binding to the Y1 receptor. This peptidomimetic tested on LN319 cells at concentrations up to 10 microM shows no inhibitory effect on forskolin-stimulated cAMP levels (IC50 for NPY = 2.5 nM). Furthermore, we used confocal microscopy to examine the NPY-induced increase in intracellular calcium in single LN319 cells. Preincubation of the cells with T4-[NPY(33-36)]4 shifted to the right the dose-response curves for intracellular mobilization of calcium induced by NPY at concentrations ranging from 0.1 nM to 10 microM. Finally, we assessed the competitive antagonistic properties of T4-[NPY(33-36)]4 at presynaptic peptidergic Y2 receptors modulating noradrenaline release. the compound T4-[NPY(33-36)]4 caused a marked shift to the right of the concentration-response curve of NPY 13-36, a Y2-selective fragment, yielding a pA2 value of 8.48. Thus, to our best knowledge, T4-[NPY(33-36)]4 represents the first potent and selective Y2 antagonist.

Non-native architectures in protein design and mimicry.

Protein design aims to mimic some of the structural and functional properties of native proteins. The complexity of the folding mechanism, i.e. the pathway by which a linear polypeptide chain finds its unique 3D-structure, represents one of the most intriguing hurdles in this rapidly growing field. In order to bypass this well-known protein-folding problem, some years ago we proposed the construction of non-native chain architectures with a high propensity for folding. According to this concept, termed TASP (template-assembled synthetic proteins), topological templates are used as a built-in device for directing covalently attached peptide blocks to a predetermined packing arrangement, resulting in branched chain architectures. Recent progress in the synthetic methodology for assembling peptides now allows us to access the full potential of the TASP concept. In this article, we discuss the state of the art of template-based protein de novo design, with special emphasis on progress in peptide synthesis and template design and show that some fundamental questions in protein assembly, structure and function can be approached by designing protein mimetics of reduced structural and functional complexity.

Redesigning Protein Function: Towards Synthetic Vaccines and MHC Mimetics

Template Assembled Synthetic Proteins (TASP) approach has been applied to mimic 3D-conformations of the hen egg white lysozyme antigen (HEL) and the major histocompatibility complex (MHC) and some of their biological properties.

Protein de novo Design

The ultimate goal in protein de novo design is the construction of artificial proteins exhibiting tailor-made structural and functional properties. To create native-like macromolecules in copying nature's way has proven to be difficult because the mechanism of folding in its complexity has yet to be unraveled. In order to bypass the well-known folding problem, we have developed the concept of template assembled synthetic proteins (TASP); this meanwhile widely accepted strategy uses topological templates as 'built-in' devices for the induction of well-defined folding topologies. Progress in synthetic strategies, e.g., chemoselective ligation methods and orthogonal protection techniques open the way for the design of more complex TASP molecules featuring functional properties such as membrane channels, vaccines, catalysts, receptors, or ligands.

Template Assembled Synthetic Proteins (TASP) as Functional Mimetics of Proteins

A review with 26 refs. Methodologies for the synthesis of functional TASP compds. are given. [on SciFinder (R)]

Protein de novo design: Macromolecules with tailormade properties

AbstractIn the absence of a detailed knowledge of the folding mechanism of natural proteins, the general design of polypeptide sequences with a high propensity to fold into a predetermined three dimensional structure still appears to be out of reach. The concept of Template Assembled Synthetic Proteins (TASP) represents a means by which one can nonetheless construct macromolecules with native‐like structural features, with the result that systems with tailormade structural and functional properties are now becoming attainable.

Template assisted protein de novo design

Abstract

A specific template-assembled peptidic agonist for the angiotensin II receptor subtype 2 (AT2) and its effect on inferior olivary neurones.

We synthesized a molecule composed of two angiotensin II 4-8 pentapeptide fragments attached to a carrier molecule (TA), according to the template-assembled synthetic proteins concept. This molecule was investigated for receptor binding on angiotensin type-1 and type-2 receptors (AT1 and AT2) and its biological activity was determined by iontophoretic experiments on neurones of the inferior olive (ION) that express only AT2 receptors. TA binds exclusively to the AT2 receptor and mediates an agonistic angiotensin-II effect on the ION. TA is the first agonist available to study the direct stimulation of AT2 receptors.

Templates in protein de novo design

The de novo design of polypeptide sequences with a three-dimensional structure necessary for many biological functions is limited by the complex folding process, or ‘protein folding problem’. This problem can be bypassed through constructing protein-like molecules with a ‘built-in’ device for intramolecular folding, that is, proteins of non-natural chain architecture (template-assembled synthetic proteins, TASP). Topological templates have become a versatile tool for inducing and stabilizing secondary structures (protein loops, β-turns, α-helices, β-sheets) and are widely adopted design elements for the construction of protein-like molecules, exhibiting interesting structural and functional properties.

Preparation of the very acid-sensitive Fmoc-Lys(Mtt)-OH. Application in the synthesis of side-chain to side-chain cyclic peptides and oligolysine cores suitable for the solid-phase assembly of MAPs and TASPs.

N alpha-9-Fluorenylmethoxycarbonyl-N epsilon-4=methyltrityl-lysine, [Fmoc-Lys(Mtt)-OH], was prepared in two steps from lysine, in 42% overall yield. The N epsilon-Mtt function can be quantitatively removed upon treatment with 1% TFA in dichloromethane or with a 1:2:7 mixture of acetic acid/trifluoroethanol/dichloromethane for 30 min and 1 h at room temperature, respectively. Under these conditions, groups of the tert-butyl type and peptide ester bonds to TFA-labile resins, such as the 2-chlorodiphenylmethyl- and the Wang-resin, remained intact. The utility of the new derivative in peptide synthesis has been exemplified with the synthesis of a cyclic cholecystokinin analog. As an example of further application, five types of lysine cores suitable for the solid-phase synthesis of one, two or three epitopes containing antigenic peptides or template-assembled synthetic proteins have been synthesized on Merrifield, Wang and 2-chlorodiphenylmethyl resin.

Chemoselective ligation of multifunctional peptides to topological templates via thioether formation for TASP synthesis

Covalent attachment of multifunctional peptides to a topological template containing four maleimide functions was achieved via thioether formation; the resulting TASP (Template Assembled Synthetic Protein) molecules are obtained in high yield and purity.

Protein design as a challenge for peptide chemists.

All efforts to turn the ultimate goal in protein de novo design into reality-the construction of new macromolecules with predetermined three-dimensional structure and well-defined functionality-failed because the mechanism of folding has still to be unravelled. In the present review, various attempts to apply synthetic tools for inducing native-like structural features in peptides in order to bypass the folding problem are described. Besides well-established methods for the nucleation and stabilization of secondary structures, e.g. alpha-helices, beta-sheets and beta-turns, topological templates as 'built-in' folding devices have more recently become the key elements for the induction of protein-like folding units (template-assembled synthetic proteins, TASP). Progress in the synthetic strategy and structural characterization of this new type of macromolecules opens the way for the design of functional TASP molecules.

Synthesis of antiparallel 4?-helix bundle TASP by chemoselective ligation

The use of chemoselective ligation methods and orthogonal protection techniques allows access to Template-Assembled Synthetic Protein (TASP) molecules exhibiting a large variety of packing topologies. This is demonstrated for the synthesis of an antiparallel 4α-helical bundle TASP by condensing amphiphilic peptide blocks, containing aldehyde functions at the C- or N-terminus, to a selectively addressable topological template via oxime bond formation. The resulting antiparallel 4α-helix TASP is obtained in high yield and shows a template-induced helical conformation.

Convenient total synthesis of a 4-helix template-assembled synthetic protein (TASP) molecule by chemoselective ligation

The chemoselective ligation approach to the preparation of complex peptides has been used in a simple, convenient synthesis of a template-assembled synthetic protein (TASP) molecule. Reaction of a readily prepared synthetic pro-helical peptide-αCOSH, in unprotected form, with a synthetic (BrAc) 4 template molecule, also in unprotected form, proceeded rapidly in aqueous solution to give a uniform product in high yield. The resulting 4-helix TASP was simply purified to homogeneity, and the covalent structure was defined by ion-spray mass spectrometry [obs MW 6647.1±2.8 D; calc 6645.6 (monoisotopic), 6649.9 (average isotope composition)]. The conformation of the resulting macromolecule was determined by circular dichroism (CD) and was highly helical

Template-assembled synthetic proteins designed to adopt a globular, four-helix bundle conformation form ionic channels in lipid bilayers

Template-assembled synthetic proteins (TASPs) designed to adopt globular, four-helix bundle structures form ion channels in lipid bilayers. The rationale behind this work is that a bundle of amphiphilic alpha-helices may constitute a functional pore-forming motif in a lipid environment. TASPs designated T4(4alpha11), T4(4alpha15), and T4(4alpha18) contain four identical, amphiphilic, helical modules of 11, 15, or 18 residues, respectively; secondary structure modules are attached to the lysine epsilon-amino groups of a cyclic template, Ac-CysLysAlaLysProGlyLysAlaLysCys-NH2, which directs the intramolecular folding. T4(4alpha15) and T4(4alpha18) form cation selective channels with single channel conductances in 500 mM NaCl of 10 and 50 pS for T4(4alpha15) and 9 and 25 pS for T4(4alpha18). In contrast, T4(4alpha11) does not produce discrete conductance events. Notably, channel activity is observed only for molecules that display well-defined alpha-helical structure in solution; K(alpha15), Which contains a single peptide module attached to the epsilon-amino group of Ac-Lys-NH2, does not elicit single channels and displays low alpha-helical content. By contrast, K(alpha18) displays spectral features associated with alpha-helical structure and forms channels with primary conductances of 3 and 9 pS. The occurrence of multiple conductances suggests that molecules aggregate and form heterogeneous conductive oligomers. The minimum length of helical modules required to span a lipid bilayer is established by investigating the channel activity of T4(4alpha15) in bilayers of increasing width. Taken together, results suggest that the relative orientation of amphiphilic segments depends on the hydrophobicity of surrounding media; accordingly, TASP molecules may form ionic channels through a reorientation of template-assembled helical modules to expose charged residues to a central hydrophilic pore.

Template‐Assembled Synthetic Proteins (TASP). Cyclic Templates with Incorporated Turn‐Inducing Mimics

AbstractThe 8‐amino‐5,6,7,8‐tetrahydronaphth‐2‐oic acid (1), 8‐(aminomethyl)‐5,6,7,8‐tetrahydronaphth‐2‐oic acid (2), and 8‐(aminomethyl)naphth‐2‐oic acid (3) were synthesized in their protected forms as turn‐inducing dipeptide mimics. Two of them (2 and 3) were incorporated into a novel type of cyclic, peptide‐based structures (see 21 and 34–36) designed as templates for the synthesis of TASP molecules.

The TASP concept : Mimetics of peptide ligands, protein surfaces and folding units

The TASP (Template Assembled Synthetic Protein) concept in protein de novo design has been introduced for the construction of protein-like molecules exhibiting tailor-made functional properties. After establishing some synthetic and conformational foundations in TASP strategy, we describe a 4α - helix bundle TASP molecule mimicking immunological properties of human MHC-I. Furthermore, we focus on structural motifs (e.g. topological templates) disposing functional groups in defined spatial positions as candidates to mimic structural and conformational features of peptides and proteins for molecular recognition studies.

Synthetic peptide and template‐assembled synthetic protein models of the hen egg white lysozyme 87–97 helix: Importance of a protein‐like framework for conformational stability in a short peptide sequence

In the native structure of hen egg white lysozyme (HEL), the amino acid sequence 87-97 (HEL 87-97) forms an amphiphilic helix, with hydrophilic residues in the sequence directed toward the solvent. A synthetic version of the HEL 87-97 sequence (with the cysteine corresponding to position 94 of HEL replaced by alanine) displays conformational features in solution typical of an unordered structure as judged by CD. However, various modifications in the sequence result in increased helix-forming potential of the HEL 87-97 analogues. Further stabilization of the helical conformation in the most helical analogue of the HEL 87-97 sequence is obtained when 4 copies of this peptide sequence are coupled on a peptide carrier molecule following the template-assembled synthetic protein (TASP) approach [M. Mutter and S. Vuilleumier (1989) Angew. Chem. Int. Ed. Engl., Vol. 28, pp. 535-554 "A Chemical Approach to Protein Design-Template-Assembled Synthetic Proteins (TASP)." This suggests that long-range interactions of the peptide with its environment contribute to conformational stability in short peptide sequences. TASP molecules may prove useful for the study of the factors that determine secondary structure formation in short peptides by providing a protein-like framework.

Template assembled synthetic proteins: Condensation of a multifunctional peptide to a topological template via chemoselective ligation

A chemoselective ligation via oxime bond formation is used for the chemical synthesis of template assembled peptides according to the TASP (Template Assembled Synthetic Proteins) approach. Aminooxyacetylation of the multifunctional partial sequence Lys- Arg- Asp- Ser of lactoferrin and subsequent condensation in aqueous solution with a topological template containing four selectively addressable aldehyde functions as attachment sites gives readily access to the TASP molecule.

Total chemical synthesis, characterization, and immunological properties of an MHC class I model using the TASP concept for protein de novo design.

The design, total chemical synthesis, and immunological properties of a four-alpha-helix bundle template-assembled synthetic protein (TASP) mimicking some of the structural features of the major histocompatibility complex (MHC) class I is described. In a first approach, the native sequence 58-74 of the alpha 1 heavy chain domain of HLA-A2 was modeled in order to increase helix stability and amphiphilicity of the 17-mer peptide, preserving the residues for potential T-cell receptor (TcR) binding properties. According to the TASP concept, these helical segments were covalently attached to a cyclic template molecule designed for the induction of a four-helix-bundle topology of the assembled peptide blocks. After extensive HPLC purification, stepwise solid-phase synthesis resulted in a TASP molecule of high chemical purity as demonstrated by analytical HPLC, mass spectrometry, and amino acid analysis. CD spectroscopic investigations are consistent with the onset of a partial alpha-helical conformation in aqueous buffer as well as in TFE. Antibodies raised directly against this four-alpha-helix bundle TASP molecule (without prior conjugation to a carrier molecule) were detected by ELISA. Flow cytometry studies showed that these antibodies recognize the native MHC class I molecule on the surface of HLA-A2-positive cells. The results indicate that the TASP approach represents a versatile tool for mimicking conformational epitopes.