scholarly journals Cell-Binding Assays for Determining the Affinity of Protein–Protein Interactions

2016 ◽  
pp. 21-44 ◽  
Author(s):  
S.A. Hunter ◽  
J.R. Cochran
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Binding Assays ◽  
Cell Binding

scholarly journals Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins

2014 ◽  
Vol 112 (1) ◽  
pp. 112-117 ◽  
Author(s):  
Gurkan Guntas ◽  
Ryan A. Hallett ◽  
Seth P. Zimmerman ◽  
Tishan Williams ◽  
Hayretin Yumerefendi ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Protein Design ◽  
Protein Interactions ◽  
Mammalian Cell Culture ◽  
Large Change ◽  
Small Gtpase ◽  
Protein Protein Interactions ◽  
Binding Assays ◽  
Light Stimulation ◽  
Inducible Protein

The discovery of light-inducible protein–protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.

scholarly journals Biological insights from SMA-extracted proteins

2021 ◽  
Author(s):  
Lucas Unger ◽  
Alejandro Ronco-Campaña ◽  
Philip Kitchen ◽  
Roslyn M. Bill ◽  
Alice J. Rothnie
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
Protein Interactions ◽  
Divalent Cations ◽  
Structural Data ◽  
Protein Protein Interactions ◽  
Binding Assays ◽  
Real Time Measurement ◽  
Low Concentrations ◽  
Binding Pockets

In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein–lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein–protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.

Application of AlphaLISA in bioanalysis

2020 ◽  
pp. 16-37
Author(s):  
Stanislav Cherepushkin
Keyword(s):  
Drug Development ◽  
Ligand Binding ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
High Sensitivity ◽  
Medical Diagnostics ◽  
Protein Protein Interactions ◽  
Binding Assays ◽  
Post Translational Modifications

The use and development of biotherapeutics increases and the need for accurate, sensitive and robust bioanalytical methods is also increasing. ELISA and other ligand-binding assays are the most widely used methods for the quantification of macromolecules in complex biological samples. One of the alternatives to ELISA is AlphaLISA — a versatile chemiluminescent ligand binding assay using a homogeneous no-wash protocol. AlphaLISA assays are suited for automation and exhibit high sensitivity, high throughput and wide analytical range. Since the early 2000s, this method has been used in science, medicine, and drug development for wide variety of applications, including the quantification of analytes, immunogenicity, protein-protein interactions, enzyme activity, post-translational modifications and epigenetics. In this review, we describe the principles of the AlphaLISA assay and its application in bioanalytical studies (pharmacokinetics and immunogenicity) and high-throughput screening in drug development, medical diagnostics and pathogens detection.

scholarly journals Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nitesh Shashikanth ◽  
Meridith A. Kisting ◽  
Deborah E. Leckband
Keyword(s):  
Protein Interactions ◽  
Intercellular Junctions ◽  
Binding Kinetics ◽  
Protein Protein Interactions ◽  
Binding Assays ◽  
Kinetic Measurements ◽  
Adhesion Protein ◽  
Membrane Bound ◽  
And Function ◽  
The Impact

Abstract The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions.

AR Suppresses Transcription of the α Glycoprotein Hormone Subunit Gene Through Protein-Protein Interactions with cJun and Activation Transcription Factor 2

2001 ◽  
Vol 15 (9) ◽  
pp. 1496-1504
Author(s):  
Joan S. Jorgensen ◽  
John H. Nilson
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Transcriptional Activity ◽  
Protein Protein Interactions ◽  
Binding Assays ◽  
Reporter Construct ◽  
Glutathione S Transferase ◽  
Glycoprotein Hormone ◽  
Bzip Proteins ◽  
Bzip Family

Abstract Previously, we reported that the AR directly suppressed transcription of the α glycoprotein hormone subunit (αGSU) gene in a ligand-dependent fashion while ER had no effect. Mutagenesis studies of the αGSU promoter indicated that two elements were required for AR-mediated suppression: the α basal element and tandem cAMP response elements (CREs). Because several members of the bZip family of transcriptional proteins can bind the CREs, we used several functional assays to determine whether AR interacts selectively with cJun, activation transcription factor 2 (ATF2), or CRE binding protein (CREB). When tested by cotransfection with AR, cJun and ATF2 specifically rescued androgen-mediated suppression of theα GSU-reporter construct in a gonadotrope-derived cell line. In contrast, cotransfected CREB displayed no activity in this rescue assay. In fact, overexpression of CREB alone diminished activity of theα GSU promoter, suggesting that the transcriptional activity normally conferred by the tandem CREs in gonadotropes requires their occupancy by cJun/ATF2 heterodimers. Binding assays carried out with a glutathione-S-transferase-AR fusion protein indicated that the receptor itself also displayed a clear preference for binding cJun and ATF2. Furthermore, we ruled out the possibility that AR suppressed activity of the αGSU promoter by reducing synthesis of these bZip proteins. Additional experiments suggested that phosphorylation of AR or histone acetylation are unlikely requirements for AR suppression of αGSU promoter activity. Thus, our data suggest that AR suppresses activity of the αGSU promoter through direct protein-protein interactions with cJun and ATF2.

Protein-protein interactions of the p53 family with the Bcl-2 family in hepatocellular carcinoma

2011 ◽  
Vol 49 (08) ◽  
Author(s):  
LC König ◽  
M Meinhard ◽  
C Sandig ◽  
MH Bender ◽  
A Lovas ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
P53 Family

Partial Purification of a Specific Plasminogen Activator from Porcine Parotid Glands

1974 ◽  
Vol 31 (03) ◽  
pp. 403-414 ◽  
Author(s):  
Terence Cartwright
Keyword(s):  
Nucleic Acid ◽  
Salivary Glands ◽  
Plasminogen Activator ◽  
Protein Interactions ◽  
Partial Purification ◽  
Protein Protein Interactions ◽  
Parotid Glands ◽  
Two Stage ◽  
Preliminary Characterization ◽  
Specific Inhibitors

SummaryA method is described for the extraction with buffers of near physiological pH of a plasminogen activator from porcine salivary glands. Substantial purification of the activator was achieved although this was to some extent complicated by concomitant extraction of nucleic acid from the glands. Preliminary characterization experiments using specific inhibitors suggested that the activator functioned by a similar mechanism to that proposed for urokinase, but with some important kinetic differences in two-stage assay systems. The lack of reactivity of the pig gland enzyme in these systems might be related to the tendency to protein-protein interactions observed with this material.

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