Synthesis of Polypeptides with High-Fidelity Terminal Functionalities under NCA Monomer-Starved Conditions

Controlled polypeptide synthesis via α-amino acid N-carboxylic anhydride (NCA) polymerization using conventional primary amine initiators encounters two major obstacles: (i) normal amine mechanism (NAM) and activated monomer mechanism (AMM) coexist due to amine basicity and nucleophilicity and (ii) NCA is notoriously sensitive towards moisture and heat and unstable upon storage. We serendipitously discover that N-phenoxycarbonyl-functionalized α-amino acid (NPCA), a latent NCA precursor, could be polymerized solely based on NAM with high initiating efficiency by using primary amine hydrochloride as an initiator. The polymerization affords well-defined polypeptides with narrow polydispersity and high-fidelity terminal functionalities, as revealed by the clean set of MALDI-TOF MS patterns. We further demonstrate successful syntheses of random and block copolypeptides, even under open-vessel conditions. Overall, the integration of moisture-insensitive and air-tolerant NPCA precursors with stable primary amine hydrochloride initiators represents a general strategy for controlled synthesis of high-fidelity polypeptides with sophisticated functions.

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Detection of Structural and Conformational Changes in ALS-Causing Mutant Profilin1 With Hydrogen/Deuterium Exchange Mass Spectrometry and Bioinformatics Techniques

10.21203/rs.3.rs-217926/v1 ◽

2021 ◽

Author(s):

Ahmad Shahir Sadr ◽

zahra abdollahpour ◽

Atousa Aliahmadi ◽

Changiz Eslahchi ◽

Mina Nekouei ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Amino Acid ◽

Conformational Changes ◽

Docking Simulation ◽

Hydrogen Deuterium Exchange ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Hydrogen Deuterium ◽

Tof Ms

Abstract The hydrogen/deuterium exchange (HDX) is a reliable method to survey the dynamic behavior of proteins and epitope mapping. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a quantifying tool to assay for HDX in the protein of interest. We combined HDX-MALDI-TOF MS and molecular docking/MD simulation to identify accessible amino acids and analyze their contribution in the structural changes of profilin1 (PFN1). The molecular docking/MD simulations are computational tools for enabling the analysis of the type of amino acids that may be involved via HDX identified under the lowest binding energy condition. Glycine to Valine amino acid (G117V) substitution mutation is linked to amyotrophic lateral sclerosis (ALS). This mutation is found to be in the actin-binding site of PFN1 and prevents the dimerization/polymerization of actin and invokes a pathologic toxicity that leads to ALS. In this study, we sought to understand the PFN1 protein dynamic behavior using purified wild type and mutant PFN1 proteins. The data obtained from HDX-MALDI-TOF MS for PFN1WT and PFN1G117V at various time intervals, from seconds to hours, revealed multiple peaks corresponding to molecular weights from monomers to multimers. PFN1/Benzaldehyde complexes identified 20 accessible amino acids to HDX that participate in the docking simulation in the surface of WT and mutant PFN1. Consistent results from HDX-MALDI-TOF MS and docking simulation predict candidate amino acid(s) involved in the dimerization/polymerization of PFNG117V. This information may shed critical light on the structural and conformational changes with details of amino acid epitopes for mutant PFN1s’ dimerization, oligomerization, and aggregation.

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Isolation and amino acid sequencing by MALDI-TOF-MS/MS of a novel antimicrobial anionic peptide from the skin secretion of Osteocephalus taurinus (Anura, Hylidae)

Journal of the Brazilian Chemical Society ◽

10.1590/s0103-50532012001200002 ◽

2012 ◽

Vol 23 (12) ◽

pp. 2133-2136 ◽

Cited By ~ 4

Author(s):

Túlio O. G. Costa ◽

Richardson A. Almeida ◽

Jorge T. Melo ◽

Hector H. F. Koolen ◽

Felipe M. A. da Silva ◽

...

Keyword(s):

Amino Acid ◽

Skin Secretion ◽

Amino Acid Sequencing ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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Molecular characterization and phylogenetic analysis of a novel glutenin gene (Dy10.1t) fromAegilops tauschii

Genome ◽

10.1139/g06-032 ◽

2006 ◽

Vol 49 (7) ◽

pp. 735-745 ◽

Cited By ~ 25

Author(s):

Yanzhen Zhang ◽

Qiaoyun Li ◽

Yueming Yan ◽

Jigang Zheng ◽

Xueli An ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Amino Acid ◽

Aegilops Tauschii ◽

Pcr Primers ◽

Duplication Event ◽

Nucleotide Polymorphisms ◽

Coding Region ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

A novel y-type high molecular mass glutenin subunit (HMM-GS) possessing a mobility that is slightly slower than that of the subunit Dy10 obtained by SDS–PAGE, named Dy10.1t, in the wild wheat Aegilops tauschii was identified by 1- and 2-dimensional gel electrophoresis, capillary electrophoresis, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI–TOF–MS). The gene encoding the HMM subunit Dy10.1twas amplified with allele-specific PCR primers, and the amplified products were cloned and sequenced. The coding domain of the Dy10.1tsubunit gene consisted of 1980 bp encoding a protein of 658 residues with an Mrsof 68 611 Da, which was similar to the Mrsdetermined by MALDI–TOF–MS. The deduced amino acid sequence indicated that Dy10.1tsubunit displayed a greater similarity to the Dy12 subunit, differing by only 8 amino acid substitutions. Six coding region single-nucleotide polymorphisms were discovered in the Dy10.1tgene by multiple alignments (1 per 330 bp), 1 in the N-terminal domain and the others in the central repeats. Five of them resulted in residue substitutions, whereas 3 created enzyme site changes. The homology and neighbour-joining trees constructed from code domain sequences of 20 x- and y-type glutenin genes from different Triticum species separated into 2 halves, which corresponded to the x-type and y-type HMM glutenin alleles. Phylogenetic analysis revealed that the Glu-1 gene duplication event probably occurred at about 16.83 million years ago, whereas the divergence times of A, B, and D genomes within x-type and y-type halves were before 7.047 and 10.54 million years ago, respectively.Key words: HMW glutenin genes, MALDI-TOF-MS, AS-PCR, cSNP, phylogenetic analysis, Aegilops tauschii.

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