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Title: Track: High Throughput Protein Science Thermal and mechanical stability of highly-luminescent protein NanoLuc in presence and absence of chaperones; Award Winners/Abstracts from the 36th Annual Symposium of The Protein Society, July 7 – 10, 2022
Award ID(s):
1817556
NSF-PAR ID:
10475461
Author(s) / Creator(s):
; ;
Publisher / Repository:
Wiley
Date Published:
Journal Name:
Protein Science
Volume:
32
Issue:
S1
ISSN:
0961-8368
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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  2. 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 withN‐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. © 2021 Wiley Periodicals LLC.

    This article was corrected on 18 July 2022. See the end of the full text for details.

    Basic Protocol 1: Labeling a protein of interest at its N‐terminus with NHS esters through stepwise reaction

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    Basic Protocol 2: Purifying the N‐terminal fluorescent‐labeled protein and determining its concentration and labeling efficiency

    Basic Protocol 3: Using MST to determine the binding affinity of an N‐terminal fluorescent‐labeled protein to a binding partner.

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