Selective protein N-terminal labeling with N-hydroxysuccinimide esters

Jiang, Hanjie; D’Agostino, Gabriel D.; Cole, Philip A.; Dempsey, Daniel R.

doi:10.1016/bs.mie.2020.04.018

Cited by 16 publications

(19 citation statements)

References 94 publications

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“…Fluorescent labeling and GMP-PNP loading of KRAS for microscale thermophoresis studies. KRAS was labeled with Alexa Fluor 647 at its amino terminus following the protocol described in Jiang et al 37 Briefly, to expose the N-terminal cysteine residue, the His6 tag was removed by TEV protease. During the cleavage procedure, a 'one-pot' reaction with MESNA (Sodium 2mercaptoenthanesulfonate, Sigma Aldrich M1511) and Alexa Fluor TM 647 NHS Ester (Invitrogen cat# A20006) was initiated.…”

Section: Preparation Of Kras Proteinsmentioning

confidence: 99%

Cryo-EM structure of a RAS/RAF recruitment complex

Eck

Jeon

Park

et al. 2022

Preprint

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Cryo-EM structures of a KRAS/BRAF/MEK1/14-3-3 complex reveal KRAS bound to the flexible Ras-binding domain of BRAF, captured in two orientations. Autoinhibitory interactions are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by Ras and suggest stabilization of this pre-activation intermediate as an alternative to blocking binding of KRAS.

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Section: Preparation Of Kras Proteinsmentioning

confidence: 99%

Cryo-EM structure of a RAS/RAF recruitment complex

Eck

Jeon

Park

et al. 2022

Preprint

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“…The affinity tag will typically be cleaved with either TEV or SUMO protease, exposing an N‐terminal Cys, which should be introduced adjacent to the C‐terminus of the tag. In practice, the labeling strategy using NHS esters can be performed in two slightly different ways: a stepwise procedure (Dempsey, Jiang, Kalin, Chen, & Cole, 2018), described in Basic Protocol 1, or a one‐pot approach (Jiang, D'Agostino, Cole, & Dempsey, 2020), described in Alternate Protocol. In the stepwise procedure, cleavage of the N‐terminal affinity tag is carried out before labeling.…”

Section: Strategic Planningmentioning

confidence: 99%

N‐Terminal Protein Labeling with N‐Hydroxysuccinimide Esters and Microscale Thermophoresis Measurements of Protein‐Protein Interactions Using Labeled Protein

Jiang

Cole

2021

Current Protocols

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Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein-protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy-to-apply protocol for high-stoichiometric, site-specific labeling of a protein at its N-terminus with N-hydroxysuccinimide (NHS) esters as a means to measure protein-protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent-labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2-E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances.

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“…The versatility of this technology was further explored by labeling a 130 kDa ubiquitin carboxyl-terminal hydrolase 7 (USP7) with a readily available sulfo-cyanine5-NHS ester moiety. [73] It is noteworthy that this reaction is biocompatible. In addition, the ready availability of different commercially accessible functional NHS esters makes the whole technique versatile and cost-effective.…”

Section: Thioester Derivativesmentioning

confidence: 97%

“…The resulting conjugate then undergoes an intramolecular S-to-N acyl shift to form a native isopeptide bond, [37] resulting in the ligation of the two peptides (Figure 7A). In the N-hydroxy succinimide (NHS) ester labeling approach, [72,73] by employing a principle similar to that of NCL, the NHS ester is first pretreated with mercaptoethane sulfonate (Figure 7B). This small thiol converts the NHS ester into a chemoselective thioester, which in turn, can be reacted in situ with N-terminal cysteine, to yield a native amide bond.…”

Section: Thioester Derivativesmentioning

confidence: 99%

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Recent advances in protein modifications techniques for the targeting N‐terminal cysteine

Nicholas

Mazid

Murale³

et al. 2021

Peptide Science

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N-terminal modifications of proteins have garnered the attention of chemical biologists because of their critical roles in numerous cellular processes, including protein translocation, protein structural and metabolic stability, and the regulation of the half-life of proteins by a process known as "N-end rule pathway." In addition, other posttranslational modification processes also depend on the N-terminal signal sequences. Chemical probes are powerful tools that can be used to investigate N-terminal modifications, to gain structural and functional insights into protein modification, protein-protein interactions, protein localization, and protein dynamics. Most modifications target cysteine and lysine residues because of their high nucleophilicity. However, due to multiple occurrences of these residues in a protein at a given time, regioselective labeling can be quite difficult to accomplish. N-terminal cysteine presents itself as a unique practical option to overcome regioselectivity and site-specificity challenges. This review provides an overview of N-terminal cysteine labeling tools, including N-terminal cysteine condensation with aldehydes, cyanobenzothiazoles, cyclopropenones, and trans-thioesterification. The review also highlights the technical challenges of each of the techniques, which need to be addressed to broaden the scope of these approaches.

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Selective protein N-terminal labeling with N-hydroxysuccinimide esters

Cited by 16 publications

References 94 publications

Cryo-EM structure of a RAS/RAF recruitment complex

Cryo-EM structure of a RAS/RAF recruitment complex

N‐Terminal Protein Labeling with N‐Hydroxysuccinimide Esters and Microscale Thermophoresis Measurements of Protein‐Protein Interactions Using Labeled Protein

Recent advances in protein modifications techniques for the targeting N‐terminal cysteine

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