Solid phase proteomics: Dramatic reinforcement of very weak protein–protein interactions

2007 ◽  
Vol 849 (1-2) ◽  
pp. 243-250 ◽  
Author(s):  
Manuel Fuentes ◽  
Cesar Mateo ◽  
Benevides C.C. Pessela ◽  
Pilar Batalla ◽  
Roberto Fernandez-Lafuente ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Solid Phase ◽  
Protein Protein Interactions

scholarly journals A High-Throughput Solid-Phase Microplate Protein-Binding Assay to Investigate Interactions between Myofilament Proteins

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Brandon J. Biesiadecki ◽  
J.-P. Jin
Keyword(s):  
Protein Binding ◽  
Protein Interactions ◽  
Protein Modification ◽  
Binding Assay ◽  
Solid Phase ◽  
Regulatory Protein ◽  
Protein Isoforms ◽  
Protein Protein Interactions ◽  
Protein Binding Assay ◽  
Phase Protein

To understand the structure-function relationship of muscle-regulatory-protein isoforms, mutations, and posttranslational modifications, it is necessary to probe functional effects at the level of the protein-protein interaction. Traditional methodologies assessing such protein-protein interactions are laborious and require significant amounts of purified protein, while many current methodologies require costly and specialized equipment or modification of the proteins, which may affect their interaction. To address these issues, we developed a novel method of microplate-based solid-phase protein-binding assay over the recent years. This method assesses specific protein-protein interactions at physiological conditions, utilizes relatively small amounts of protein, is free of protein modification, and does not require specialized instrumentation. Here we present detailed methodology for the solid-phase protein-binding assay with examples that we have successfully applied to quantify interactions of myofilament-regulatory proteins. We further provide considerations for optimization of the assay conditions and its broader application in studies of other protein-protein interactions.

scholarly journals Implementing solid-phase-enhanced sample preparation for Co-Immunoprecipitation with Mass Spectrometry for C. elegans

2021 ◽  
Author(s):  
Guelkiz Baytek ◽  
Oliver Popp ◽  
Philipp Mertins ◽  
Baris Tursun
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Preparation Method ◽  
Solid Phase ◽  
Protein Protein Interactions ◽  
Sample Preparation Method ◽  
C Elegans

Studying protein-protein interactions in vivo can reveal key molecular mechanisms of biological processes. Co-Immunoprecipitation followed by Mass Spectrometry (CoIP-MS) allows detection of protein-protein interactions in high-throughput. The nematode Caenorhabditis elegans (C. elegans) is a powerful genetic model organism for in vivo studies. Yet, its rigid cuticle and complex tissues require optimization for protein biochemistry applications to ensure robustness and reproducibility of experimental outcomes. Therefore, we optimized CoIP-MS application to C. elegans protein lysates by combining a native CoIP procedure with an efficient sample preparation method called single-pot, solid-phase-enhanced, sample preparation method (SP3). Our results based on the subunits of the conserved chromatin remodeler FACT demonstrate that our SP3-integrated CoIP-MS procedure for C. elegans samples is highly accurate and robust. Moreover, in a previous study (Baytek et al. 2021), we extended our technique to studying the chromodomain factor MRG-1 (MRG15 in human), which resulted in unprecedented findings.

scholarly journals Application of Post Solid-Phase Oxime Ligation to Fine-Tune Peptide–Protein Interactions

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2807
Author(s):  
Xue Zhi Zhao ◽  
Fa Liu ◽  
Terrence R. Burke
Keyword(s):  
Protein Interactions ◽  
Solid Phase ◽  
Hot Spot ◽  
Susceptibility Gene ◽  
Protein Tyrosine Phosphatases ◽  
Structural Diversity ◽  
Binding Energies ◽  
Protein Protein Interactions ◽  
Interacting Surfaces ◽  
Natural Amino Acids

Protein–protein interactions (PPIs) represent an extremely attractive class of potential new targets for therapeutic intervention; however, the shallow extended character of many PPIs can render developing inhibitors against them as exceptionally difficult. Yet this problem can be made tractable by taking advantage of the fact that large interacting surfaces are often characterized by confined “hot spot” regions, where interactions contribute disproportionately to overall binding energies. Peptides afford valuable starting points for developing PPI inhibitors because of their high degrees of functional diversity and conformational adaptability. Unfortunately, contacts afforded by the 20 natural amino acids may be suboptimal and inefficient for accessing both canonical binding interactions and transient “cryptic” binding pockets. Oxime ligation represents a class of biocompatible “click” chemistry that allows the structural diversity of libraries of aldehydes to be rapidly evaluated within the context of a parent oxime-containing peptide platform. Importantly, oxime ligation represents a form of post solid-phase diversification, which provides a facile and empirical means of identifying unanticipated protein–peptide interactions that may substantially increase binding affinities and selectivity. The current review will focus on the authors’ use of peptide ligation to optimize PPI antagonists directed against several targets, including tumor susceptibility gene 101 (Tsg101), protein tyrosine phosphatases (PTPases) and the polo-like kinase 1 (Plk1). This should provide insights that can be broadly directed against an almost unlimited range of physiologically important PPIs.

scholarly journals Insights into the molecular mechanisms of action of bioportides: a strategy to target protein-protein interactions

2015 ◽  
Vol 17 ◽  
Author(s):  
John Howl ◽  
Sarah Jones
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Solid Phase ◽  
Cell Penetrating Peptides ◽  
Dominant Negative ◽  
Protein Protein Interactions ◽  
Structural Modifications ◽  
Anti Cancer ◽  
The Many

Cell-penetrating peptides (CPPs) are reliable vehicles for the target-selective intracellular delivery of therapeutic agents. The identification and application of numerous intrinsically bioactive CPPs, now designated as bioportides, is further endorsement of the tremendous clinical potential of CPP technologies. The refinement of proteomimetic bioportides, particularly sequences that mimic cationic α-helical domains involved in protein-protein interactions (PPIs), provides tremendous opportunities to modulate this emergent drug modality in a clinical setting. Thus, a number of CPP-based constructs are currently undergoing clinical trials as human therapeutics, with a particular focus upon anti-cancer agents. A well-characterised array of synthetic modifications, compatible with modern solid-phase synthesis, can be utilised to improve the biophysical and pharmacological properties of bioportides and so achieve cell-and tissue-selective targeting in vivo. Moreover, considering the recent successful development of stapled α-helical peptides as anti-cancer agents, we hypothesise that similar structural modifications are applicable to the design of bioportides that more effectively modulate the many interactomes known to underlie human diseases. Thus, we propose that stapled-helical bioportides could satisfy all of the clinical requirements for metabolically stable, intrinsically cell-permeable agents capable of regulating discrete PPIs by a dominant negative mode of action with minimal toxicity.

scholarly journals Parallel solid-phase synthesis of diaryltriazoles

2012 ◽  
Vol 8 ◽  
pp. 1027-1036 ◽  
Author(s):  
Matthias Wrobel ◽  
Jeffrey Aubé ◽  
Burkhard König
Keyword(s):  
Protein Interactions ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Alpha Helix ◽  
Protein Protein Interactions ◽  
Phase Synthesis ◽  
Wang Resin ◽  
Polymer Bead ◽  
Synthesis Protocol ◽  
Parallel Fashion

A series of substituted diaryltriazoles was prepared by a solid-phase-synthesis protocol using a modified Wang resin. The copper(I)- or ruthenium(II)-catalyzed 1,3-cycloaddition on the polymer bead allowed a rapid synthesis of the target compounds in a parallel fashion with in many cases good to excellent yields. Substituted diaryltriazoles resemble a molecular structure similar to established terphenyl-alpha-helix peptide mimics and have therefore the potential to act as selective inhibitors for protein–protein interactions.

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