The insertion of azobenzene moiety in complex molecular protein or peptide systems can lead to molecular switches to be used to determine kinetics of folding/unfolding properties of secondary structures, such as -helix, -turn or -hairpin. In fact, in azobenzene, absorption of light induces a reversible trans ↔ cis isomerization, which in turns generates a strain or a structure relaxation in the chain that causes peptide folding/unfolding. In particular azobenzene may permit reversible conformational control of hairpin formation. In the present work a synthetic photochromic azobenzene amino acid derivative was incorporated as a turn element to modify the synthetic peptide [Pro7,Asn8,Thr10]CSF114 previously designed to fold as a type I -turn structure in biomimetic aqueous solution. In particular, the P-N-H fragment at positions 7-9, involved in a -hairpin, was replaced by an azobenzene amino acid derivative (synthesized ad hoc) to investigate if the electronic properties of the novel peptidomimetic analogue could induce variations in the isomerization process. The absorption spectra of the azopeptidomimetic analogue of the type I -turn structure and of the azobenzene amino acid as control were measured as a function of the irradiation time exciting into the respective first * and n* transition bands. Isomerization of the azopeptidomimetic results strongly favoured by exciting into the * transition. Moreover, conformational changes induced by the cis↔ trans azopeptidomimetic switch were investigated by NMR in different solvents.
Abstract 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.
Les coudes β etant des motifs de reconnaissance frequents entre proteines, l’objectif de ce travail a ete de developper des mimes potentiels de coudes β et de les evaluer sur le plan structural et comme inhibiteurs d’interactions proteine-proteine. Le complexe α amylase/tendamistat a constitue notre modele d’etude et nous avons developpe trois approches pour mimer le coude β du tendamistat. Dans une premiere approche, des peptides contenant des enchainements d’acides β-amines ont ete synthetises. L’analyse structurale par RMN a montre que ces peptides ne sont pas structures en coude β en solution mais ils possedent une affinite pour l’α amylase similaire a celle de l’α peptide de reference et presentent l’avantage d’etre resistants a la proteolyse. L’utilisation d’un peptide biotinyle a permis de capturer specifiquement l’α amylase dans un melange complexe de proteines, la reproductibilite des experiences devant encore etre optimisee. Dans une deuxieme approche, trois α hexapeptides cycliques ont ete synthetises pour comparer les effets conformationnels des cyclisations par liaison amide, pont disulfure et chimie « click ». Alors que le dernier peptide presente un equilibre conformationnel, un repliement en coude β est observe dans le methanol et l’eau pour le peptide cyclise par liaison amide et dans le methanol et lors de l’interaction avec l’α amylase pour l’analogue contenant un pont disulfure. Ces deux peptides sont deux fois plus affins pour la proteine que l’α peptide controle. Enfin, nous avons montre que les enchainements Dprolinoamino acide-Nmethylamino acide et Dprolinoamino acide-cyclopropylamino acide induisent le repliement en coude β de pseudotetrapeptides.
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).
