Molecular dynamics conformational search of six cyclic peptides used in the template assembled synthetic protein approach for protein de novo design.

Six cyclic peptides, designed to act as topological templates in the TASP (template assembled synthetic protein) approach in protein de novo design, were investigated employing a 100-ps, 900-K molecular dynamics conformational search. The peptides are composed of two Lys-X-Lys (X = Gly, Ala) tripeptides connected at its N- and C-terminal end by a Pro-Gly motif and a cystine bridge (I), two Pro-Gly units (II), naphthalene derivatives (III), and tetrahydronaphthalene derivatives of different stereochemistry (IV-VI). The molecular dynamics conformational search established that template I had beta-sheet like geometry. Templates II-VI showed different preferential geometries, among them, e.g., distinct preferences for type V turns in Pro-Gly containing peptides and close spatial arrangement of hydrophobic naphthalene moieties. The orientation of the lysine side chains within preferential geometries of the individual templates is analyzed and a tentative evaluation for their potential to stabilize TASP molecules of 4-helix bundle topology is given.     

Novel strategy for the synthesis of template-assembled analogues of rat relaxin.

The 'template-assembled synthetic protein' (TASP) concept provides a simple and elegant approach for the preparation of analogues that retain key structural elements. We have synthesized TASP molecules containing the putative active site of relaxin, a peptide that has similar structural features to insulin but a markedly different biological role. Two types of chemoselective thiol ligation strategies (thioether and thiazolidine) were used and compared. The synthetic pendant peptides contain an essential region for bioactivity that is located in the alpha-helical region of the relaxin B-chain. Depending on whether the thioether or the thiazolidine chemistry was used to attach the peptides to the template, the reacting amino acid was placed either at the C-terminus or N-terminus, respectively, thus allowing the choice of orientation relative to the carrier molecule. The template molecule consists of a decapeptide with two proline-glycine turns and four evenly spaced lysine residues that were functionalized with the appropriate chemical moiety. This allowed reaction with the appropriately derivatized peptides in solution. To improve the template ligation step using the thioether approach, a pendant peptide C-terminal cysteamine residue was used to reduce potential steric hindrance during conjugation. The design of the peptides as well as the synthetic strategy resulted in the acquisition of mimetics showing weak non-competitive and weak competitive antagonist properties.     

Solution structure of regioselectively addressable functionalized templates: An NMR and restrained molecular dynamics investigation

Three cyclic peptides that are Regioselectively Addressable Functionalized Templates (RAFT) for use in protein de novo design have been investigated using a combination of nmr, restrained molecular dynamics, and CD spectroscopy. These peptides contain up to four selectively addressable sites (orthogonally protected lysine side chains) or have selectively addressable faces. The results show a common stable conformation for templates of this kind based on two type II β-turns and an associated β-sheet structure. A preferential orientation for the side chains is also demonstrated. The significance of these findings is discussed in the context of applications of RAFT that rely on their conformational rigidity and ability to present functionalities in a defined spatial arrangement. © 1996 John Wiley & Sons, Inc.     

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

Summary 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 4-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 4-helix TASP is obtained in high yield and shows a template-induced helical conformation.     

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.     

Chemoselectively addressable HCan building blocks in peptide synthesis: L-homocanaline derivatives

(S-2-amino-5-(aminooxy)pentanoic acid (L -homocanaline, HCan), a structural analogue of lysine, contains a reactive alkyloxyamine side chain and is therefore considered to react chemoselectively with carbonyl compounds by forming a kinetically stable oxime bond. The chemical synthesis of L -homocanaline starting from protected glutamic acid derivatives is described. Two orthogonally protected homocanaline derivatives were synthesized and their use in standard SPPS procedures was exemplified for the synthesis of a chemoselectively addressable cyclic peptide for use in TASP design. Moreover, the wide range of applications of this unique building block was demonstrated for the chemoselective ligation of an unprotected disaccharide to a HCan containing model peptide resulting in a chimeric glycopeptide structure. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Total chemical synthesis of a 27 kDa TASP protein derived from the MscL ion channel of M. tuberculosis by ketoxime-forming ligation

A 27-kDa TASP protein, T(5)Msc(103-151), that was derived from the cytoplasmic domain (amino acid residues 103-151) of the MscL ion channel of M. tuberculosis was synthesized by ketoxime-forming chemoselective ligation between a template molecule carrying five pyruvic acid groups, and linear channel peptides carrying one aminooxyacetic acid group. Ketoxime-forming ligation provided for highly efficient assembly of this large totally synthetic protein construct with yields >90% with modest excess (1.5x) of the aminooxy peptide. Formation of the desired TASP molecule was confirmed by SDS-PAGE analysis and MALDI mass spectrometry. The effect of template attachment on the structure of the peptides constituting the TASP was assessed by circular dichroism spectroscopy. Attachment of the peptides to the topological template induces predominantly helical secondary structure, whereas an analogous peptide that did not bear an aminooxy group, MscL(103-151), does not exhibit significant secondary structure at pH 7 and is found to be monomeric in concentrations up to 65 microM. This observation can be explained by entropic destabilization of the unfolded state of T(5)Msc(103-151) due to the attachment to the template and the resulting loss of degrees of freedom. Pyruvic acid-based ketoxime-forming chemoselective ligation may thus prove to be a useful tool for the assembly of large, non-native protein constructs and their biophysical study.     

The construction of new proteins: V. A template-assembled synthetic protein (TASP) containing both a 4-helix bundle and beta-barrel-like structure

The construction of a template-assembled synthetic protein (TASP) designed to contain both a 4-helix bundle and a beta-barrel as two folding "domains" is described. For the de novo design of proteins, amphiphilic helices (alpha) and beta-sheets (beta) are covalently attached to a template peptide (T) carrying functional side chains suitably oriented to promote intramolecular folding of the secondary structure blocks into a characteristic packing arrangement, i.e., T8-(4 alpha)(4 beta). The design of this new macromolecule was assisted by computer modeling, which suggested a low-energy conformation with tight hydrophobic packing of the secondary structure subunits. Solid-phase synthesis of the "two-domain" TASP molecule was achieved using orthogonal protection techniques. The solution properties as well as circular dichroism (CD) and infrared spectroscopy (IR) data under various experimental conditions are consistent with the folded conformation suggested by modeling.     

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. α-helices, β-sheets and β-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.     

Protein design and folding: template trapping of self-assembled helical bundles.

An experimental system is described, permitting a detailed and systematic analysis of the factors governing self-assembly of amphipathic helices, e.g. to a four-helical bundle, a subject of major relevance for tertiary structure formation, protein folding and design. Following the Template Assembled Synthetic Proteins (TASP) approach, helices of different packing potential are competitively assembled in solution with a preformed two-helix TASP molecule, and after equilibration are covalently attached ('template trapping') via chemoselective thioether formation. The quantitative analysis of the individual TASP molecules by high performance liquid chromatography (HPLC) and electrospray mass spectrometry (ES-MS) allows the delineation of the role of complementary packing in helix bundle formation. The procedure established represents a general tool for the experimental verification of modern concepts in molecular recognition.     

Template-dependent polymerization across discontinuous templates by the heterodimeric primase from the hyperthermophilic archaeon Sulfolobus solfataricus

The eukaryotic-like primase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsoPriSL) exhibits a range of activities including template-dependent de novo primer synthesis, primer extension and template-independent terminal nucleotidyl transfer using either rNTPs or dNTPs. Remarkably, the enzyme is able to synthesize products far longer than templates in vitro. Here we show that the long products resulted from template-dependent polymerization across discontinuous templates (PADT) by SsoPriSL. PADT was initiated through either primer synthesis or terminal transfer, and occurred efficiently on templates containing contiguous dCs. Template switching took place when the 3'-end of a growing strand synthesized on one template annealed to another template directly or following the terminal addition of nucleotides, and was subsequently extended on the new template. The key to PADT was the ability of SsoPriSL to promote strand annealing. SsoPriSL catalyzed PADT with either dNTPs or rNTPs as the substrates but preferred the latter. The enzyme remained active in PADT but became inefficient in primer synthesis in vitro when temperature was raised from 55°C to 70°C. Our results suggest that SsoPriSL is capable of bridging noncomplementary DNA ends and, therefore, may serve a role in double-strand DNA break repair in Archaea.     

Role of small molecules in regulation of D-serine deaminase synthesis.

Cyclic AMP is required for optimal synthesis of D-serine deaminase synthesis from dsdO+ templates and for optimal hyperinducible synthesis from low constitutive dsdO templates both in vitro and in vivo. Neither D-serine, cyclic AMP, nor dsdC activator has an effect on expression of a high constitutive dsdO template. The synthesis of the dsdC activator itself in vitro is independent of cyclic AMP. Guanosine tetraphosphate does not have a significant effect on in vitro D-serine deaminase synthesis from dsdO+ or dsdO templates. A previously described class of dsdO mutants showing partial catabolite sensitivity of constitutive D-serine deaminase synthesis proved to be low dsdO types. They all contain a low constitutive dsdC mutation; the two effects are additive with regard to level of constitutivity, but only that portion of synthesis attributable to the dsdC mutation is cyclic AMP dependent.     

Synthesis of multitopic neoglycopeptides displaying recognition and detection motifs

We describe herein the synthesis of cyclic decapeptide template displaying clustered carbohydrate recognition motifs and detection agent on spatially separated domains. Such multitopic labeled neoglycopeptides represent attractive tools for binding assays with carbohydrate binding proteins in glycomic research.     

Carbopeptides: chemoselective ligation of peptide aldehydes to an aminooxy-functionalized D-galactose template.

Multifunctional, topological template molecules such as linear and cyclic peptides have been used for the attachment of peptide strands to form novel protein models of, for example, 4-alpha-helix bundles. The concept of carbohydrates as templates for de novo design of potential protein models has been previously described and these novel chimeric compounds were termed carbopeptides. Here, a second generation strategy in which carbopeptides are synthesized by chemoselective ligation of a peptide aldehyde to an aminooxy-functionalized alpha-D-galactopyranoside is described. This template was prepared by per-O-acylation of methyl alpha-D-galactopyranoside with N,N-Boc2-aminooxyacetic acid to form a tetra-functionalized template, followed by treatment with TFA-CH2Cl2 to release the aminooxy functionality. The peptide aldehydes Fmoc-Ser-Gly-Gly-H and H-Ala-Leu-Ala-Lys-Leu-Gly-Gly-H were synthesized by a BAL strategy. Four identical copies of peptide aldehyde were smoothly attached to the template by chemoselective ligation to form a 2.1 and a 2.9 kDa carbopeptide, respectively.     

Versatile synthesis of Boc protected hydrazinoacetic acid and its application to the chemoselective ligation of TASP molecules

This paper describes a convenient synthesis of protected hydrazine derivatives, i.e. 1,2-bis-Boc-hydrazinoacetic acid, and its application for hydrazone ligation techniques in convergent template assembled synthetic protein (TASP) synthesis.     

Assembly of binding loops on aromatic templates as VCAM‐1 mimetics

The design and synthesis of cyclic mimetics of VCAM‐1 protein that reproduce the integrin‐binding domain are presented. The unprotected peptide precursor 37 – 43 , Thr‐Gln‐Ile‐Asp‐Ser‐Pro‐Leu, was grafted onto functional templates of type naphthalene, biphenyl and benzyl through the chemoselective formation of C‐ and N‐terminal oximes resulting in a mixture of four isomeric forms due to syn–anti isomerism of the oxime bonds. Some isomers could be monitored by HPLC and identified by NMR. The molecule containing a naphthalene‐derived template was found to inhibit the VCAM‐1/VLA‐4 interaction more efficiently than previously reported for sulfur‐bridged cyclic peptides containing similar sequences. The finding confirms the importance of incorporating conformational constraints between the terminal ends of the peptide loop 37 – 43 in the design of synthetic inhibitors of the VCAM‐1/integrin interaction. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

DNA-dependent RNA polymerase III from cauliflower. Characterization and template specificity

Class III DNA-dependent RNA polymerase (EC 2.7.7.6) was highly purified from cauliflower (Brassica oleracea, var. bortytis) by using polyethyleneimine precipitation. The specific activity of the enzyme was comparable to that reported for mammalian enzymes. Glycerol gradient sedimentation analysis indicated that the sedimantation coefficient (23 S) was slightly higher than that of enzyme II from cauliflower. The class III enzyme was inhibited by alpha-amanitin at high concentrations (50% inhibition at 200 microgram/ml). The Km value for nucleoside triphosphate was determined. Template specificities for single synthetic polymers showed that the enzyme read pyrimidine homopolymers as templates and preferred poly(dT) to poly(dC). The enzyme transcribed both strands of homopolymer pairs of poly(dI). poly(dC) and poly(dA).poly(dT). The synthetic polyribonucleotides were not effectively read. Competition experiments with these synthetic polymers indicated that the enzyme had different binding specificities which were not the same as their template specificities. The different binding affinities and template specificites for synthetic templates of the three classes of enzyme suggest that the enzyme can discriminate among different template sequences.     

Solid-phase total synthesis and antimicrobial activities of loloatins A-D

The first total synthesis of the decapeptide antibiotics loloatins A-D (1-4), originally isolated from the marine bacterial isolate MK-PNG-276A, possibly in the genus Bacillus, was accomplished by solid-phase peptide synthesis (SPPS), followed by 'head-to-tail' cyclization of the activated linear precursors, without protection of nucleophilic side-chain functions, on a safety-catch resin. The synthetic peptides were equally active as the natural products isolated from the bacterial source and found to possess similar bacterial selectivity as other members in the family of amphipathic antimicrobial cyclic decapeptides.     

A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix‐assisted laser desorption/ionisation mass spectrometry

Short cyclic peptides have a great interest in therapeutic, diagnostic and affinity chromatography applications. The screening of ‘one‐bead‐one‐peptide’ combinatorial libraries combined with mass spectrometry (MS) is an excellent tool to find peptides with affinity for any target protein. The fragmentation patterns of cyclic peptides are quite more complex than those of their linear counterparts, and the elucidation of the resulting tandem mass spectra is rather more difficult. Here, we propose a simple protocol for combinatorial cyclic libraries synthesis and ring opening before MS analysis. In this strategy, 4‐hydroxymethylbenzoic acid, which forms a benzyl ester with the first amino acid, was used as the linker. A glycolamidic ester group was incorporated after the combinatorial positions by adding glycolic acid. The library synthesis protocol consisted in the following: (i) incorporation of Fmoc‐Asp[2‐phenylisopropyl (OPp)]‐OH to Ala‐Gly‐oxymethylbenzamide‐ChemMatrix, (ii) synthesis of the combinatorial library, (iii) assembly of a glycolic acid, (iv) couple of an Ala residue in the N‐terminal, (v) removal of OPp, (vi) peptide cyclisation through side chain Asp and N‐Ala amino terminus and (vii) removal of side chain protecting groups. In order to simultaneously open the ring and release each peptide, benzyl and glycolamidic esters were cleaved with ammonia. Peptide sequences could be deduced from the tandem mass spectra of each single bead evaluated. The strategy herein proposed is suitable for the preparation of one‐bead‐one‐cyclic depsipeptide libraries that can be easily open for its sequencing by matrix‐assisted laser desorption/ionisation MS. It employs techniques and reagents frequently used in a broad range of laboratories without special expertise in organic synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.