In Situ Quenching of Trialkylphosphine Reducing Agents Using Water-Soluble PEG-Azides Improves Maleimide Conjugation to Proteins

Kantner, Terrence; Alkhawaja, Bayan; Watts, Andrew G.

doi:10.1021/acsomega.7b01094

Cited by 19 publications

(16 citation statements)

References 21 publications

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“…Unmodified and fluorophore-labeled peptides could not be separated, resulting in labeling reagents contaminated with up to 70% non-fluorescent peptides . For the current work, we optimized labeling by adapting a protocol published by Watts and co-workers . This method quenches the reducing agent (i.e., TCEP) before the addition of excess reactive fluorophore.…”

Section: Resultsmentioning

confidence: 99%

Orthogonal Versatile Interacting Peptide Tags for Imaging Cellular Proteins

et al. 2023

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Genetic tags are transformative tools for investigating the function, localization, and interactions of cellular proteins. Most studies today are reliant on selective labeling of more than one protein to obtain comprehensive information on a protein’s behavior in situ. Some proteins can be analyzed by fusion to a protein tag, such as green fluorescent protein, HaloTag, or SNAP-Tag. Other proteins benefit from labeling via small peptide tags, such as the recently reported versatile interacting peptide (VIP) tags. VIP tags enable observations of protein localization and trafficking with bright fluorophores or nanoparticles. Here, we expand the VIP toolkit by presenting two new tags: TinyVIPER and PunyVIPER. These two tags were designed for use with MiniVIPER for labeling up to three distinct proteins at once in cells. Labeling is mediated by the formation of a high-affinity, biocompatible heterodimeric coiled coil. Each tag was validated by fluorescence microscopy, including observation of transferrin receptor 1 trafficking in live cells. We verified that labeling via each tag is highly specific for one- or two-color imaging. Last, the self-sorting tags were used for simultaneous labeling of three protein targets (i.e., TOMM20, histone 2B, and actin) in fixed cells, highlighting their utility for multicolor microscopy. MiniVIPER, TinyVIPER, and PunyVIPER are small and robust peptide tags for selective labeling of cellular proteins.

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Section: Resultsmentioning

confidence: 99%

Orthogonal Versatile Interacting Peptide Tags for Imaging Cellular Proteins

et al. 2023

View full text Add to dashboard Cite

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“…Unmodified and fluorophore-labeled peptides could not be separated, resulting in labeling reagents contaminated with up to 70% non-fluorescent peptides 17 . For the current work, we optimized labeling by adapting a protocol published by Watts and coworkers 40 . This method quenches excess reducing agent (i.e., TCEP) before the addition of excess reactive fluorophore.…”

Section: Resultsmentioning

confidence: 99%

A set of orthogonal versatile interacting peptide tags for imaging cellular proteins

Suyama

Devlin

Macias-Contreras

et al. 2022

Preprint

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Genetic tags are transformative tools for investigating the function, localization, and interactions of cellular proteins. Most studies today are reliant on selective labeling of more than one protein to obtain comprehensive information on a protein's behavior in situ. Some proteins can be analyzed by fusion to protein tag, such as green fluorescent protein, HaloTag, or SNAP-Tag. Other proteins benefit from labeling via small peptide tags, such as the recently reported versatile interacting peptide (VIP) tags. VIP tags enable observations of protein localization and trafficking with bright fluorophores or nanoparticles. Here we expand the VIP toolkit by presenting two new tags: TinyVIPER and PunyVIPER. These two tags were designed for use with MiniVIPER for labeling up to three distinct proteins at once in living cells. Labeling is mediated by the formation of a high affinity, biocompatible heterodimeric coiled coil. Each tag was validated by fluorescence microscopy, including observation of transferrin receptor 1 trafficking in live cells. We verified that labeling via each tag is highly specific, with no cross-reactivity between the three VIP tags under cellular conditions. Lastly, the self-sorting tags were used for simultaneous labeling of three protein targets (i.e., TOMM20, histone 2B, and actin), highlighting their utility for multicolor microscopy. MiniVIPER, TinyVIPER, and PunyVIPER are small and robust peptide tags for selective labeling of cellular proteins.

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“…Cysteine residues of purified proteins (25uM in the total volume of 50ul In PBS) were reduced with 50mM Tris(−2-carboethyl)phosphine (TCEP) at 40-fold molar excess for 30 minutes. Excess TCEP was removed with ZebaTM Spin desalting columns 7kDa MWCO (ThermoFisher) as it may interfere with the Maleimide reaction 41 . The proteins were then labeled with 25-fold molar excess monoreactive maleimide-Dibenzocyclooctyne (DBCO) (Sigma Aldrich) in Phosphate Buffered Saline (PBS) pH 7.4 overnight at room temperature.…”

Section: Methodsmentioning

confidence: 99%

iMAXFRET (Information Maximized FRET) for multipoint single-molecule structural analysis

Joshi,

de Lannoy,

Howarth

et al. 2023

Preprint

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Understanding the structure of biomolecules is vital for deciphering their characteristics and roles in biological systems. While current structural analysis techniques like nuclear magnetic resonance and X-ray crystallography excel in many aspects, they fall short in capturing comprehensive single-molecule information. To address this limitation and to better capture the heterogeneity and dynamic range of biomolecular reactions, there is a need for single-molecule structural analysis tools. To achieve this, we introduce iMAX FRET, a one-pot FRET-based single-molecule method integrated with geometrical 3D reconstruction, offering comprehensiveab initiostructural analysis. Through the stochastic exchange of fluorescent weak binders, iMAX FRET allows simultaneous assessment of multiple spatial coordinates on a biomolecule within a few minutes of time to generate distinct FRET fingerprints for 3D structural profiling. We demonstrate a mathematical approach forde novostructural prediction using iMAX data, opening avenues for native biomolecule analysis. Furthermore, this method facilitates the investigation of conformational changes in individual molecules, illuminating single-molecule structural dynamics. Our technique has the potential to emerge as a powerful approach to advance our understanding of biomolecular structures.

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In Situ Quenching of Trialkylphosphine Reducing Agents Using Water-Soluble PEG-Azides Improves Maleimide Conjugation to Proteins

Cited by 19 publications

References 21 publications

Orthogonal Versatile Interacting Peptide Tags for Imaging Cellular Proteins

Orthogonal Versatile Interacting Peptide Tags for Imaging Cellular Proteins

A set of orthogonal versatile interacting peptide tags for imaging cellular proteins

iMAXFRET (Information Maximized FRET) for multipoint single-molecule structural analysis

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