scholarly journals Exploiting Protein N-Terminus for Site-Specific Bioconjugation

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3521
Author(s):  
Lucia De Rosa ◽  
Rossella Di Stasi ◽  
Alessandra Romanelli ◽  
Luca Domenico D’Andrea
Keyword(s):  
Protein Molecule ◽  
Amine Group ◽  
Reactive Site ◽  
Reaction Conditions ◽  
Selective Labeling ◽  
Site Specific ◽  
Protein Conjugates ◽  
Protein Molecules ◽  
N Terminus

Although a plethora of chemistries have been developed to selectively decorate protein molecules, novel strategies continue to be reported with the final aim of improving selectivity and mildness of the reaction conditions, preserve protein integrity, and fulfill all the increasing requirements of the modern applications of protein conjugates. The targeting of the protein N-terminal alpha-amine group appears a convenient solution to the issue, emerging as a useful and unique reactive site universally present in each protein molecule. Herein, we provide an updated overview of the methodologies developed until today to afford the selective modification of proteins through the targeting of the N-terminal alpha-amine. Chemical and enzymatic strategies enabling the selective labeling of the protein N-terminal alpha-amine group are described.

The Scope, Functions, and Dynamics of Posttranslational Protein Modifications

2019 ◽  
Vol 70 (1) ◽  
pp. 119-151 ◽  
Author(s):  
A. Harvey Millar ◽  
Joshua L. Heazlewood ◽  
Carmela Giglione ◽  
Michael J. Holdsworth ◽  
Andreas Bachmair ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Posttranslational Modification ◽  
Subcellular Distribution ◽  
Protein Function ◽  
Protein Molecule ◽  
Plant Biology ◽  
Protein Molecules ◽  
N Terminus ◽  
Functional Implications ◽  
Combined Action

Assessing posttranslational modification (PTM) patterns within protein molecules and reading their functional implications present grand challenges for plant biology. We combine four perspectives on PTMs and their roles by considering five classes of PTMs as examples of the broader context of PTMs. These include modifications of the N terminus, glycosylation, phosphorylation, oxidation, and N-terminal and protein modifiers linked to protein degradation. We consider the spatial distribution of PTMs, the subcellular distribution of modifying enzymes, and their targets throughout the cell, and we outline the complexity of compartmentation in understanding of PTM function. We also consider PTMs temporally in the context of the lifetime of a protein molecule and the need for different PTMs for assembly, localization, function, and degradation. Finally, we consider the combined action of PTMs on the same proteins, their interactions, and the challenge ahead of integrating PTMs into an understanding of protein function in plants.

scholarly journals Use of an Asparaginyl Endopeptidase for Chemo-enzymatic Peptide and Protein Labeling

2020 ◽  
Author(s):  
Simon Tang ◽  
Davide Cardella ◽  
Alexander J. Lander ◽  
Xuefei Li ◽  
Yu-Hsuan Tsai ◽  
...  
Keyword(s):  
Phenylboronic Acid ◽  
Product Formation ◽  
Protein Labeling ◽  
Recognition Sequence ◽  
Reaction Conditions ◽  
Site Specific ◽  
Asparaginyl Endopeptidase ◽  
Coupling System ◽  
Labeling System ◽  
Specific Peptide

Transpeptidases are ideal biocatalysts for site-specific peptide and protein labeling, whereas reactions that target N-terminus cysteine with commercially available reagents have become common practice. However, a versatile approach that allows bioconjugation at the terminus of choice (N or C), while avoiding the use of backbone-modified substrates (<i>e.g.</i> depsipeptide) or large excess of reagent, is highly desirable. Aiming to meet these benchmarks, we have combined the advantages of asparaginyl endopeptidase (AEP) catalysis with a N-terminal cysteine trapping reaction and created a chemo-enzymatic labeling system. In this approach, polypeptide with a Asn-Cys-Leu recognition sequence are ligated with a counterpart possessing an N-terminal Gly-Leu by AEP; the byproduct Cys-Leu is subsequently trapped by a stable and inexpensive scavenger, 2-formyl phenylboronic acid (FPBA), to yield an inert thiazolidine derivative, thereby driving the reaction forward to product formation. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP ligation/FPBA coupling system was further demonstrated by site-specific labeling the N- or C-termini of various proteins.

Site-specific N-terminus conjugation of poly(mPEG1100) methacrylates to salmon calcitonin: synthesis and preliminary biological evaluation

Soft Matter ◽  
2009 ◽  
Vol 5 (16) ◽  
pp. 3038 ◽  
Author(s):  
Claire T. Sayers ◽  
Giuseppe Mantovani ◽  
Sinead M. Ryan ◽  
Rajan K. Randev ◽  
Odin Keiper ◽  
...  
Keyword(s):  
Salmon Calcitonin ◽  
Biological Evaluation ◽  
Site Specific ◽  
N Terminus

scholarly journals Bioorthogonal protein-DNA conjugation methods for force spectroscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marie Synakewicz ◽  
Daniela Bauer ◽  
Matthias Rief ◽  
Laura S. Itzhaki
Keyword(s):  
Single Molecule ◽  
Unnatural Amino Acids ◽  
Force Spectroscopy ◽  
Diels Alder ◽  
Cysteine Residues ◽  
Single Molecule Force Spectroscopy ◽  
Site Specific ◽  
Inverse Electron Demand ◽  
Protein Conjugates ◽  
Electron Demand

Abstract Accurate and stable site-specific attachment of DNA molecules to proteins is a requirement for many single-molecule force spectroscopy techniques. The most commonly used method still relies on maleimide chemistry involving cysteine residues in the protein of interest. Studies have consequently often focused on model proteins that either have no cysteines or with a small number of cysteines that can be deleted so that cysteines can then be introduced at specific sites. However, many proteins, especially in eukaryotes, contain too many cysteine residues to be amenable to this strategy, and therefore there is tremendous need for new and broadly applicable approaches to site-specific conjugation. Here we present bioorthogonal approaches for making DNA-protein conjugates required in force spectroscopy experiments. Unnatural amino acids are introduced site-specifically and conjugated to DNA oligos bearing the respective modifications to undergo either strain-promoted azidealkyne cycloaddition (SPAAC) or inverse-electron-demand Diels-Alder (IE-DA) reactions. We furthermore show that SPAAC is compatible with a previously published peptide-based attachment approach. By expanding the available toolkit to tag-free methods based on bioorthogonal reactions, we hope to enable researchers to interrogate the mechanics of a much broader range of proteins than is currently possible.

Spectrophotometric Determination of Ephedrine Hydrochloride and Phenylephrine Hydrochloride

1982 ◽  
Vol 65 (4) ◽  
pp. 894-898
Author(s):  
Mohamed M Amer ◽  
Aly M Taha ◽  
Salwa R El-Shabouri ◽  
Pakinaz Y Khashaba
Keyword(s):  
Pharmaceutical Formulations ◽  
Eye Drops ◽  
Amine Group ◽  
Reaction Conditions ◽  
Phenylephrine Hydrochloride ◽  
Secondary Amine Group ◽  
Ephedrine Hydrochloride ◽  
Colored Product ◽  
The Relationship

Abstract A method is described for quantitative determination of the sympathomimetic amines ephedrine HCl and phenylephrine HCl. The method is based on the interaction of N-alkylvinylamine formed from the condensation of the free secondary amine group and acetaldehyde with chloranil to give a vinylaminosubstituted quinone. The colored product for ephedrine HCl and phenylephrine HCl exhibits 2 maximas at about 320 and 680 nm. All variables were studied to optimize reaction conditions. The relationship between absorbance and concentration was linear within 1-25 μg/mL under the conditions studied for both drugs at both wavelengths. The method has been applied to the analysis of some pharmaceutical formulations including tablets and eye drops with good recoveries (98.75-100.4%).

scholarly journals Water-mediated influence of a crowded environment on internal vibrations of a protein molecule

2016 ◽  
Vol 18 (6) ◽  
pp. 4881-4890 ◽  
Author(s):  
Anna Kuffel ◽  
Jan Zielkiewicz
Keyword(s):  
Protein Molecule ◽  
Protein Molecules ◽  
Single Protein ◽  
Crowded Environment ◽  
Internal Vibrations

The influence of crowding on the protein inner dynamics is examined by putting a single protein molecule close to one or two neighboring protein molecules.

scholarly journals Site-specific Fucosylation of Sialylated Polylactosamines by α1,3/4-Fucosyltransferases-V and -VI Is Defined by Amino Acids Near the N Terminus of the Catalytic Domain

2007 ◽  
Vol 282 (34) ◽  
pp. 24882-24892 ◽  
Author(s):  
Susan Shetterly ◽  
Franziska Jost ◽  
Susan R. Watson ◽  
Ronald Knegtel ◽  
Bruce A. Macher ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Site Specific ◽  
N Terminus

scholarly journals Protein Conjugation with Triazolinediones: Switching from a General Tyrosine-Selective Labeling Method to a Highly Specific Tryptophan Bioconjugation Strategy

2021 ◽  
Author(s):  
Klaas Decoene ◽  
Kamil Unal ◽  
An Staes ◽  
Kris Gevaert ◽  
Johan M. Winne ◽  
...  
Keyword(s):  
Unnatural Amino Acids ◽  
Click Reaction ◽  
Selective Labeling ◽  
Site Specific ◽  
Tyrosine Residues ◽  
Tryptophan Residues ◽  
Low Levels ◽  
Almost All ◽  
Site Selective ◽  
Labeling Method

Selective labeling of tyrosine residues in peptides and proteins can be achieved via a 'tyrosine-click' reaction with triazolinedione reagents (TAD). We have found that tryptophan residues are in fact often also labeled with this reagent. This off-target labeling is only observed at very low levels in protein bioconjugation but remains under the radar due to the low relative abundance of tryptophan compared to tyrosines in natural proteins, and because of the low availability and accessibility of their nucleophilic positions at the solvent-exposed protein surface. Moreover, because TAD-Trp adducts are known to be readily thermoreversible, it can be challenging to detect these physiologically stable but thermally labile modifications using several MS/MS techniques. We have found that fully solvent-exposed tryptophan side chains are kinetically favored over tyrosines under almost all conditions, and this selectivity can even be further enhanced by modifying the pH of the aqueous buffer to effect selective Trp-labeling. This new site-selective bioconjugation method does not rely on unnatural amino acids and has been demonstrated for peptides and for recombinant proteins. Thus, the TAD-Tyr click reaction can be turned into a highly site-specific labeling method for tryptophan.

Amino terminal sequence of type 3 streptococcal M protein extraction products

1990 ◽  
Vol 55 (10) ◽  
pp. 2562-2567
Author(s):  
Ivan Kluh ◽  
Otto Kühnemund ◽  
Manfred Pavlík ◽  
Ladislav Morávek ◽  
Jiří Havlíček
Keyword(s):  
Protein Extraction ◽  
Peptide Vaccine ◽  
Limited Proteolysis ◽  
Protein Molecule ◽  
M Protein ◽  
Terminal Sequence ◽  
Amino Terminal ◽  
Streptococcal M Protein ◽  
N Terminus ◽  
Type 3

The amino terminal hypervariable part of the M protein molecule was chosen as a basis for the preparation of a synthetic peptide vaccine against group A streptococci. As part of the mapping of various serological types the main products of extraction of type M Streptococcus pyogeneswith limited pepsin (Pep) hydrolysis at pH 5.5 and with phage-associated lysin (PAL) were sequenced. Two entirely different sequences were obtained. The sequence of PAL M3 shows the absence of the α-helical potential in the shorter N-terminal region as is characteristic of the N-terminus of the M protein molecule. The main product of limited hydrolysis Pep M3 (pH 5.5), which shows the presence of the α-helical potential from the very amino terminal residue of its chain, does not involve most likely the proper N-terminus of the M protein. Extraction with pepsin under conditions of very limited proteolysis (pH 5.8) yielded a fragment with N-terminal sequence identical with that of PAL M3 (extracted nonproteolytically).

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