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Title: Optimization of Protein–Protein Interaction Measurements for Drug Discovery Using AFM Force Spectroscopy
Award ID(s):
1826135
NSF-PAR ID:
10111723
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
IEEE Transactions on Nanotechnology
Volume:
18
ISSN:
1536-125X
Page Range / eLocation ID:
509 to 517
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Intracellular protein gradients serve a variety of functions, such as the establishment of cell polarity or to provide positional information for gene expression in developing embryos. Given that cell size in a population can vary considerably, for the protein gradients to work properly they often have to be scaled to the size of the cell. Here, we examine a model of protein gradient formation within a cell that relies on cytoplasmic diffusion and cortical transport of proteins toward a cell pole. We show that the shape of the protein gradient is determined solely by the cell geometry. Furthermore, we show that the length scale over which the protein concentration in the gradient varies is determined by the linear dimensions of the cell, independent of the diffusion constant or the transport speed. This gradient provides scale-invariant positional information within a cell, which can be used for assembly of intracellular structures whose size is scaled to the linear dimensions of the cell, such as the cytokinetic ring and actin cables in budding yeast cells.

     
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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

    Alternate Protocol: Labeling a protein of interest at its N‐terminus with NHS esters through a one‐pot reaction

    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.

    Basic Protocol 4: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay

     
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  3. null (Ed.)