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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. Size-Dependent Relationships between Protein Stability and Thermal Unfolding Temperature Have Important Implications for Analysis of Protein Energetics and High-Throughput Assays of Protein–Ligand Interactions

   https://doi.org/10.1021/acs.jpcb.7b05684  

   Watson, Matthew D. ; Monroe, Jeremy ; Raleigh, Daniel P. ( January 2018 , The Journal of Physical Chemistry B)
3. The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense

   https://doi.org/10.1128/mSystems.00817-20  

   Gerhardt, Edileusa C. ; Parize, Erick ; Gravina, Fernanda ; Pontes, Flávia L. ; Santos, Adrian R. ; Araújo, Gillize A. ; Goedert, Ana C. ; Urbanski, Alysson H. ; Steffens, Maria B. ; Chubatsu, Leda S. ; et al ( December 2020 , mSystems)

   Porto, Carla (Ed.)

   ABSTRACT The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status. IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense . The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors. 

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4. Alternative complexes formed by the Escherichia coli clamp loader accessory protein HolC (x) with replication protein HolD (ψ) and repair protein YoaA

   https://doi.org/10.1016/j.dnarep.2020.103006  

   Sutera, Vincent A. ; Weeks, Savannah J. ; Dudenhausen, Elizabeth E. ; Baggett, Helen B. ; Shaw, McKay C. ; Brand, Kirsten A. ; Glass, David J. ; Bloom, Linda B. ; Lovett, Susan T. ( April 2021 , DNA Repair)

   null (Ed.)
5. Effects of SARS‐CoV‐2 mutations on protein structures and intraviral protein–protein interactions

   https://doi.org/10.1002/jmv.26597  

   Wu, Siqi ; Tian, Chang ; Liu, Panpan ; Guo, Dongjie ; Zheng, Wei ; Huang, Xiaoqiang ; Zhang, Yang ; Liu, Lijun ( April 2021 , Journal of Medical Virology)

   null (Ed.)