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

Abstract

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.

Enzymatic analysis of WWP2 E3 ubiquitin ligase using protein microarrays identifies autophagy-related substrates

“…To characterize further the interaction between autophagy receptors and WWP2, we turned to microscale thermophoresis (MST) to determine the affinity of NDP52 and OPTN binding to WWP2 ( 56 ). MST measures the thermophoresis of molecules, which can correlate with the molecule size, charge, conformation, and hydration shell ( 57 ).…”

“…As autophagy receptors are relatively large in molecular weight (NDP52, ∼52 kDa; OPTN, ∼66 kDa), MST provides relatively robust signals in their association with the ∼100 kDa WWP2 protein compared to fluorescence anisotropy. For this purpose, we site-specifically labeled the WWP2 N-terminus with a fluorescent probe (Cy5 NHS ester) as described previously ( 56 , 58 ) and varied the concentration of the autophagy receptor proteins. Using this approach, the K D of WWP2 binding to NDP52 was found to be 19 μM and for OPTN, 39 μM ( Fig.…”

“…As described previously ( 56 ), MST was used to evaluate the binding affinity K D between WWP2 and autophagy receptors NDP52 and OPTN. In brief, the N-terminal Cy5-labeled WWP2 at a final concentration of 10 nM was used in the MST assays, with a buffer containing 25 mM Hepes pH 7.8, 250 mM NaCl, 1 mM TCEP, 0.5 mg/ml BSA, and 0.05% Tween-20.…”

Utilizing a Baculovirus/Insect Cell Expression System and Expressed Protein Ligation (EPL) for Protein Semisynthesis

“…Although site‐specific installation of PTMs into proteins of interest is the focus of these protocols, users can also employ EPL to stoichiometrically label proteins for biophysical studies. A recent expansion of the EPL approach for N‐terminal fluorescence labeling of proteins has involved pretreatment of commercial fluorophore N ‐hydroxysuccinimide (NHS) esters with MESNA to form a thioester that can be selectively linked to the N‐terminal Cys‐containing proteins of interest (Dempsey, Jiang, Kalin, Chen, & Cole, 2018; see also Jiang & Cole, 2021). Without the need for peptide synthesis, this approach provides non‐chemists with an attractive method for easy N‐terminal fluorescent labeling of proteins.…”

Exploiting Protein N-Terminus for Site-Specific Bioconjugation

“…Therefore, such a thiol group could be exploited for pursuing second site-specific labeling using a thiol reactive probe, for instance, through the thiol-maleimide chemistry [64,66]. Conveniently, thioester probes can be obtained from commercially available NHS-ester derivatives by treatment with thiols [65,67]. N-terminal cysteine can also be directly tagged using 2-cyanobenzothiazole (CBT) through a watercompatible condensation reaction [34] that mimics the last reaction step in the synthesis of D-luciferin in firefly [68] (Figure 2b).…”