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

   https://doi.org/10.1002/cpz1.14  

   Jiang, Hanjie ; Cole, Philip A. ( January 2021 , Current Protocols)

   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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2. Organocatalytic, Diastereo- and Enantioselective Synthesis of Nonsymmetric cis -Stilbene Diamines: A Platform for the Preparation of Single-Enantiomer cis -Imidazolines for Protein–Protein Inhibition

   https://doi.org/10.1021/jo501003r  

   Vara, Brandon A. ; Mayasundari, Anand ; Tellis, John C. ; Danneman, Michael W. ; Arredondo, Vanessa ; Davis, Tyler A. ; Min, Jaeki ; Finch, Kristin ; Guy, R. Kiplin ; Johnston, Jeffrey N. ( August 2014 , The Journal of Organic Chemistry)
3. Highly Efficient Computation of the Basal k on using Direct Simulation of Protein–Protein Association with Flexible Molecular Models

   https://doi.org/10.1021/acs.jpcb.5b10747  

   Saglam, Ali S. ; Chong, Lillian T. ( December 2015 , The Journal of Physical Chemistry B)
4. Structural and Biochemical Analysis of the Hordeum vulgare L. Hv GR-RBP1 Protein, a Glycine-Rich RNA-Binding Protein Involved in the Regulation of Barley Plant Development and Stress Response

   https://doi.org/10.1021/bi5007223  

   Tripet, Brian P. ; Mason, Katelyn E. ; Eilers, Brian J. ; Burns, Jennifer ; Powell, Paul ; Fischer, Andreas M. ; Copié, Valérie ( December 2014 , Biochemistry)
5. Probing protein aggregation at buried interfaces: distinguishing between adsorbed protein monomers, dimers, and a monomer–dimer mixture in situ

   https://doi.org/10.1039/d1sc04300e  

   Lu, Tieyi ; Guo, Wen ; Datar, Prathamesh M. ; Xin, Yue ; Marsh, E. Neil ; Chen, Zhan ( January 2022 , Chemical Science)

   Protein adsorption on surfaces greatly impacts many applications such as biomedical materials, anti-biofouling coatings, bio-separation membranes, biosensors, antibody protein drugs etc. For example, protein drug adsorption on the widely used lubricant silicone oil surface may induce protein aggregation and thus affect the protein drug efficacy. It is therefore important to investigate the molecular behavior of proteins at the silicone oil/solution interface. Such an interfacial study is challenging because the targeted interface is buried. By using sum frequency generation vibrational spectroscopy (SFG) with Hamiltonian local mode approximation method analysis, we studied protein adsorption at the silicone oil/protein solution interface in situ in real time, using bovine serum albumin (BSA) as a model. The results showed that the interface was mainly covered by BSA dimers. The deduced BSA dimer orientation on the silicone oil surface from the SFG study can be explained by the surface distribution of certain amino acids. To confirm the BSA dimer adsorption, we treated adsorbed BSA dimer molecules with dithiothreitol (DTT) to dissociate these dimers. SFG studies on adsorbed BSA after the DTT treatment indicated that the silicone oil surface is covered by BSA dimers and BSA monomers in an approximate 6 : 4 ratio. That is to say, about 25% of the adsorbed BSA dimers were converted to monomers after the DTT treatment. Extensive research has been reported in the literature to determine adsorbed protein dimer formation using ex situ experiments, e.g. , by washing off the adsorbed proteins from the surface then analyzing the washed-off proteins, which may induce substantial errors in the washing process. Dimerization is a crucial initial step for protein aggregation. This research developed a new methodology to investigate protein aggregation at a solid/liquid (or liquid/liquid) interface in situ in real time using BSA dimer as an example, which will greatly impact many research fields and applications involving interfacial biological molecules. 

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