scholarly journals Water-soluble allyl sulfones for dual site-specific labelling of proteins and cyclic peptides

2016 ◽  
Vol 7 (5) ◽  
pp. 3234-3239 ◽  
Author(s):  
Tao Wang ◽  
Andreas Riegger ◽  
Markus Lamla ◽  
Sebastian Wiese ◽  
Patrick Oeckl ◽  
...  
Keyword(s):  
Cyclic Peptides ◽  
Specific Protein ◽  
Water Soluble ◽  
Site Specific ◽  
Protein Modifications ◽  
Dual Site ◽  
Specific Labelling

Allyl sulfones as efficient disulfide rebridging agents for site-specific protein modifications with up to two additional functionalities in water.

scholarly journals Site-specific protein modifications through pyrroline-carboxy-lysine residues

2011 ◽  
Vol 108 (26) ◽  
pp. 10437-10442 ◽  
Author(s):  
W. Ou ◽  
T. Uno ◽  
H.-P. Chiu ◽  
J. Grunewald ◽  
S. E. Cellitti ◽  
...  
Keyword(s):  
Specific Protein ◽  
Site Specific ◽  
Protein Modifications ◽  
Lysine Residues

scholarly journals Transition metal catalyzed site-selective cysteine diversification of proteins

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Muhammad Jbara
Keyword(s):  
Transition Metal ◽  
Aqueous Media ◽  
Basic Research ◽  
Specific Protein ◽  
Water Soluble ◽  
Cysteine Residues ◽  
Site Specific ◽  
Organometallic Reagents ◽  
Native Proteins ◽  
Metal Catalyzed

AbstractSite-specific protein conjugation is a critical step in the generation of unique protein analogs for a range of basic research and therapeutic developments. Protein transformations must target a precise residue in the presence of a plethora of functional groups to obtain a well-characterized homogeneous product. Competing reactive residues on natural proteins render rapid and selective conjugation a challenging task. Organometallic reagents have recently emerged as a powerful strategy to achieve site-specific labeling of a diverse set of biopolymers, due to advances in water-soluble ligand design, high reaction rate, and selectivity. The thiophilic nature of various transition metals, especially soft metals, makes cysteine an ideal target for these reagents. The distinctive reactivity and selectivity of organometallic-based reactions, along with the unique reactivity and abundancy of cysteine within the human proteome, provide a powerful platform to modify native proteins in aqueous media. These reactions often provide the modified proteins with a stable linkage made from irreversible cross-coupling steps. Additionally, transition metal reagents have recently been applied for the decaging of cysteine residues in the context of chemical protein synthesis. Orthogonal cysteine protecting groups and functional tags are often necessary for the synthesis of challenging proteins, and organometallic reagents are powerful tools for selective, rapid, and water-compatible removal of those moieties. This review examines transition metal-based reactions of cysteine residues for the synthesis and modification of natural peptides and proteins.

scholarly journals A Simple Method to Dual Site-Specifically Label a Protein Using Tryptophan Auxotrophic Escherichia coli

2021 ◽  
Author(s):  
Ti Wu ◽  
Simpson Joseph
Keyword(s):  
Escherichia Coli ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Specific Protein ◽  
Cysteine Residue ◽  
Simple Method ◽  
Site Specific ◽  
E Coli ◽  
Expanded Genetic Code ◽  
Dual Site

Site-specifically labeling proteins with multiple dyes or molecular moieties is an important yet non-trivial task for many research, such as when using Föster resonance energy transfer (FRET) to study dynamics of protein conformational change. Many strategies have been devised, but usually done on a case-by-case basis. Expanded genetic code provided a general platform to incorporate non-canonical amino acids (ncAA), which can also enable multiple site-specific labeling, but it’s technically complicated and not suitable for some applications. Here we present a streamlined method that could enable dual site-specific protein labeling by using a tryptophan auxotroph of Escherichia coli to incorporate a naturally found tryptophan analog, 5-hydroxytryptophan into a recombinant protein. As a demonstration, we incorporated 5-hydroxytryptophan into E. coli release factor 1 (RF1), a protein known to possess two different conformations, and site-specifically attached two different fluorophores, one on 5-hydroxytryptophan and another on a cysteine residue. This method is simple, generally applicable, efficient, and can serve as an alternative way for researchers who want to install an additional labeling site in their proteins.

scholarly journals Site-Specific Protein Modifications by an Engineered Asparaginyl Endopeptidase from Viola canadensis

2021 ◽  
Vol 9 ◽  
Author(s):  
Yu Chen ◽  
Dingpeng Zhang ◽  
Xiaohong Zhang ◽  
Zhen Wang ◽  
Chuan-Fa Liu ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Specific Protein ◽  
Wild Type ◽  
Peptide Bonds ◽  
Site Specific ◽  
Protein Modifications ◽  
Cyclic Product ◽  
Binding Pockets ◽  
Key Residues ◽  
Mcf 7

Asparaginyl endopeptidases (AEPs) or legumains are Asn/Asp (Asx)-specific proteases that break peptide bonds, but also function as peptide asparaginyl ligases (PALs) that make peptide bonds. This ligase activity can be used for site-specific protein modifications in biochemical and biotechnological applications. Although AEPs are common, PALs are rare. We previously proposed ligase activity determinants (LADs) of these enzymes that could determine whether they catalyze formation or breakage of peptide bonds. LADs are key residues forming the S2 and S1′ substrate-binding pockets flanking the S1 active site. Here, we build on the LAD hypothesis with the engineering of ligases from proteases by mutating the S2 and S1′ pockets of VcAEP, an AEP from Viola canadensis. Wild type VcAEP yields <5% cyclic product from a linear substrate at pH 6.5, whereas the single mutants VcAEP-V238A (Vc1a) and VcAEP-Y168A (Vc1b) targeting the S2 and S1′ substrate-binding pockets yielded 34 and 61% cyclic products, respectively. The double mutant VcAEP-V238A/Y168A (Vc1c) targeting both the S2 and S1′ substrate-binding pockets yielded >90% cyclic products. Vc1c had cyclization efficiency of 917,759 M−1s−1, which is one of the fastest rates for ligases yet reported. Vc1c is useful for protein engineering applications, including labeling of DARPins and cell surface MCF-7, as well as producing cyclic protein sfGFP. Together, our work validates the importance of LADs for AEP ligase activity and provides valuable tools for site-specific modification of proteins and biologics.

Neurotoxicity and Neuroprotective Effects of Polyimides (PI) and Polyphenylenevinylene (PPV) against Hydrogen Peroxide (H2O2)

2011 ◽  
Vol 8 (2) ◽  
pp. 33
Author(s):  
Norfaezah Mazalan ◽  
Mazatulikhma Mat Zain ◽  
Nor Saliyana Jumali ◽  
Norhanim Mohalid ◽  
Zurina Shaameri ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Drug Delivery Systems ◽  
Neuronal Cells ◽  
Water Soluble ◽  
Brain Delivery ◽  
Specific Therapy ◽  
Neuroprotective Effects ◽  
Water Soluble Polymer ◽  
Site Specific ◽  
Novel Drugs

Recently, research and development in the field of drug delivery systems (DDS) facilitating site-specific therapy has reached significant progression. DDS based on polymer micelles, coated micro- and nanoparticles, and various prodrug systems including water-soluble polymer have been prepared and extensively studied as novel drugs designed for cancer chemotherapy and brain delivery. Since polymers are going to be used in human, this study has the interest of testing two types of polymer, polyimides (PI) and polyphenylenevinylene (PPV) on neuronal cells. The objective of this study was to determine the possible neurotoxicity and potential neuroprotective effects of PI and PPV towards SH-SY5Y neuronal cells challenged by hydrogen peroxide (H2O2) as an oxidant. Cells were pretreated with either PI or PPV for 1 hour followed by incubation for 24 hour with 100 µM of H2O2. MTS assay was used to assess cell viability. Results show that PI and PPV are not harmful within the concentration up to 10 µM and 100 µM, respectively. However, PI and PPV do not protect neuronal cells against toxicity induced by H2O2 or further up the cell death.

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

2018 ◽  
Author(s):  
Daniel D. Brauer ◽  
Emily C. Hartman ◽  
Daniel L.V. Bader ◽  
Zoe N. Merz ◽  
Danielle Tullman-Ercek ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Modification ◽  
Positive Charge ◽  
Specific Protein ◽  
High Yield ◽  
Site Specific ◽  
Protein Cage ◽  
Protein Carriers ◽  
Site Selective ◽  
Targeted Library

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

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