Maud Larregola

Azatryptophans as tools to study polarity requirements for folding of green fluorescent protein

Aequorea victoria green fluorescent protein and its widely used mutants enhanced green fluorescent protein and enhanced cyan fluorescent protein (ECFP) are ideal target proteins to study protein folding. The spectral signals of their chromophores are directly correlated with the folding status of the surrounding protein matrix. Previous studies revealed that tryptophan at position 57 (Trp57) plays a crucial role for the green fluorescent proteins structural and functional integrity. To precisely dissect its role in ECFP folding, we performed its substitution with the isosteric analogs 4‐azatryptophan [(4‐Aza)Trp] and 7‐azatryptophan [(7‐Aza)Trp]. Although Trp is moderately hydrophobic, these isosteric analogs are hydrophilic, which makes them an almost ideal tool to study the role of Trp57 in ECFP folding. We achieved high‐level expression of both (4‐Aza)Trp‐ECFP and (7‐Aza)Trp‐ECFP. However, great portions (70–90%) of protein samples were insoluble and did not contain a maturated chromophore. All attempts to refold the insoluble protein fractions failed. Nevertheless, low amounts of fully labeled, soluble, chromophore containing fractions with altered spectral features were also isolated and identified. The most probable reason for the high yield of misfolding is the introduction of strong hydrophilicity at position 57 which strongly interferes with productive and efficient folding of ECFP. In addition, the results support a strong correlation between translational kinetics of non‐canonical amino acids in the ribosome and in vivo folding of the related modified protein sequence. Copyright

Prolinoamino Acids as Tools to Build Bifunctionalized, Stable β-Turns in Water

Keywords: beta-turn ; bioorganic chemistry ; NMR spectroscopy ; peptidomimetics ; prolinoamino acid ; Ultraviolet Circular-Dichroism ; Side-Chain Contribution ; Asymmetric-Synthesis ; Amino-Acids ; Conformation ; Constraints ; Derivatives ; Peptides ; Ligands ; Analogs Reference EPFL-ARTICLE-149200doi:10.1002/cbic.200900572 Record created on 2010-06-03, modified on 2017-05-12

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH2 and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH2 hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β3‐hSer‐(S)‐Trp‐(S)‐β3‐hArg‐(S)‐β3‐hTyr‐NH2, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH2, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright

The Combination of Prolinoamino Acids and Cyclopropylamino Acids Leads to Fully Functionalized, Stable β‐Turns in Water

The conformational freedom of peptides is often considered a major drawback in their use as suitable therapeutic agents. Indeed, flexibility both of backbone and of amino acid side chains is associated with low selectivity, together with poor metabolic stability. However, this flexibility is also responsible for the high affinities of peptides for their biological targets, allowing them to interact in their ideal conformations upon binding. The restriction of peptide conformational space is a major approach to improving both receptor selectivity and metabolic stability. However, achievement of high binding affinities with this strategy requires a range of tools allowing fine-tuning in the control of the three-dimensional space of the peptide backbone while keeping all the crucial information contributed by amino acids’ side chains, in terms of functional groups and spatial orientation. To this end, for the past few years we have been exploring synthetic methodologies that are easy to scale up, in order to prepare natural amino acid surrogates such as proline chimeras and cyclopropyl and b-amino acid derivatives. These tools have known applications for pharmaceutical use, for probing bioactive conformations of peptides, and for stabilizing peptide secondary structure elements such as PPII helix or b-turns. 9] The b-turn motif, a recognition element often involved in receptor–ligand interactions, is a major subject of investigation in the development of synthetic mimics of peptide secondary structure. 11] We have recently validated the use of cis-3-substituted prolinoamino acids (denoted P3Xaa) in Piv-d-P c 3Xaa-lNMeYaa-NHMe sequences to stabilize short b-turns (in water) that retain the side-chain functionalities in both the i+1 and the i+2 positions of the turn. These short peptides incorporate three motifs—a heterochiral sequence, a proline scaffold, and a N-methyl group—that are known to promote strong bturn propensity. The prolinoamino acid allows the canonical staggered rotamers of the side chains in the i+1 position to be mimicked with minimal deviation. In contrast, the N-methylation restricts the conformational space of the side chain in the i+2 position to gauche(c1 608) and trans (c1 1808) orientations around the c angle, the gauche + rotamer (c1 +608) being destabilized by unfavorable interaction with the N-methyl group. Here we have studied the replacement of Nmethylamino acids by cyclopropyl amino acids (or 2,3-methanoamino acids) in order to characterize the three-dimensional space available for the side chains of these constrained amino acids when combined with proline chimeras and to analyze the influence of side chain interactions on b-turn folding. Pivd-P3Xaa-d-c3Yaa-NHMe and Piv-d-P c 3Xaa-l-c3Yaa-NHMe sequences incorporating the side chains of aromatic hTrp and cationic Lys or Arg amino acids were prepared (Scheme 1).

Glaser oxidative coupling on peptides: stabilization of β-turn structure via a 1,3-butadiyne constraint.

The Glaser-Eglinton reaction between either two C or N propargylglycine (Pra or NPra) amino acids, in the presence of copper(II), led to cyclic hexa- and octapeptides constrained by a butadiyne bridge. The on-resin cyclization conditions were analyzed and optimized. The consequences of this type of constraint on the three dimensional structure of these hexapeptides and octapeptides were analyzed in details by NMR and molecular dynamics. We show that stabilized short cyclic peptides could be readily prepared via the Glaser oxidative coupling either with a chiral (Pra), or achiral (NPra) residue. The 1,3-butadiyne cyclization, along with disulfide bridged and lactam cyclized hexapeptides expands the range of constrained peptides that will allow exploring the breathing of amino acids around a β-turn structure.

Antibody Recognition in multiple sclerosis and rett syndrome using a collection of linear and cyclic N‐glucosylated antigenic probes

Antibody detection in autoimmune disorders, such as multiple sclerosis (MS) and Rett syndrome (RTT) can be achieved more efficiently using synthetic peptides. The previously developed synthetic antigenic probe CSF114(Glc), a type I′ β‐turn N‐glucosylated peptide structure, is able to recognize antibodies in MS and RTT patients’ sera as a sign of immune system derangement. We report herein the design, synthesis, conformational analysis, and immunological evaluation of a collection of glycopeptide analogs of CSF114(Glc) to characterize the specific role of secondary structures in MS and RTT antibody recognition. Therefore, we synthesized a series of linear and cyclic short glucosylated sequences, mimicking different β‐turn conformations, which were evaluated in inhibition enzyme‐linked immunosorbent assays (ELISA). Calculated IC50 ranking analysis allowed the selection of the candidate octapeptide containing two (S)−2‐amino‐4‐pentynoic acid (L‐Pra) residues Ac‐Pra‐RRN(Glc)GHT‐Pra‐NH2, with an IC50 in the nanomolar range. This peptide was adequately modified for solid‐phase ELISA (SP‐ELISA) and surface plasmon resonance (SPR) experiments. Pra‐RRN(Glc)GHT‐Pra‐NH2 peptide was modified with an alkyl chain linked to the N‐terminus, favoring immobilization on solid phase in SP‐ELISA and differentiating IgG antibody recognition between patients and healthy blood donors with a high specificity. However, this peptide displayed a loss in IgM specificity and sensitivity. Moreover, an analog was obtained after modification of the octapeptide candidate Ac‐Pra‐RRN(Glc)GHT‐Pra‐NH2 to favor immobilization on SPR sensor chips. SPR technology allowed us to determine its affinity (KD = 16.4 nM), 2.3 times lower than the affinity of the original glucopeptide CSF114(Glc) (KD = 7.1 nM).

Epitope mapping of anti-myelin oligodendrocyte glycoprotein (MOG) antibodies in a mouse model of multiple sclerosis: Microwave-assisted synthesis of the peptide antigens and ELISA screening

The role of pathologic auto‐antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35–55). In this scenario, we analyzed the anti‐MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1–117). To assess the presence of a B‐cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1–117 sequence of MOG, including MOG(35–55). For this purpose, we cloned, expressed in Escherichia coli and on‐column refolded MOG(1–117), and we applied an optimized microwave‐assisted solid‐phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid‐phase immunoenzymatic assay testing both naïve and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35–55), we hypothesize the presence of both linear and conformational epitopes on MOG(35–55) sequence. Copyright

Role of Lipoylation of the Immunodominant Epitope of Pyruvate Dehydrogenase Complex: Toward a Peptide-Based Diagnostic Assay for Primary Biliary Cirrhosis.

Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.

Structure–Activity Relationship Studies, SPR Affinity Characterization, and Conformational Analysis of Peptides That Mimic the HNK-1 Carbohydrate Epitope

The design of molecules that mimic biologically relevant glycans is a significant goal for understanding important biological processes and may lead to new therapeutic and diagnostic agents. In this study we focused our attention on the trisaccharide human natural killer cell‐1 (HNK‐1), considered the antigenic determinant of myelin‐associated glycoprotein and the target of clinically relevant auto‐antibodies in autoimmune neurological disorders such as IgM monoclonal gammopathy and demyelinating polyneuropathy. We describe a structure–activity relationship study based on surface plasmon resonance binding affinities aimed at the optimization of a peptide that mimics the HNK‐1 minimal epitope. We developed a cyclic heptapeptide that shows an affinity of 1.09×10−7 m for a commercial anti‐HNK1 mouse monoclonal antibody. Detailed conformational analysis gave possible explanations for the good affinity displayed by this novel analogue, which was subsequently used as an immunological probe. However, preliminary screening indicates that patients′ sera do not specifically recognize this peptide, showing that murine monoclonal antibodies cannot be used as a guide to select immunological probes for the detection of clinically relevant human auto‐antibodies.