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Molecular Cell Sep 2021CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display...
CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.
Topics: CRISPR-Cas Systems; Epitopes; Gene Editing; Humans; Mutagenesis; Peptide Library; Protein Binding; RNA, Guide, CRISPR-Cas Systems
PubMed: 34390675
DOI: 10.1016/j.molcel.2021.07.027 -
Methods in Enzymology 2006Glycolipids are found on all eukaryotic cells. Their expression varies among tissues, with the highest density found in the brain, where glycolipids are the most... (Review)
Review
Glycolipids are found on all eukaryotic cells. Their expression varies among tissues, with the highest density found in the brain, where glycolipids are the most abundant of all glycoconjugate classes. In addition to playing roles in membrane structure, glycolipids also act as cell surface recognition molecules, mediating cell-cell interactions, as well as binding certain pathogens and toxins. Because of their amphipathic nature, underivatized glycolipids are amenable to immobilization on hydrophobic surfaces, where they can be probed with lectins, antibodies, pathogens, toxins, and intact cells to reveal their binding specificities and affinities. Three particularly useful methods to probe specific glycolipid-mediated recognition events are microwell adsorption (ELISA), thin layer chromatography overlay, and surface plasmon resonance (SPR) spectroscopy.
Topics: Animals; Glycolipids; Humans; Protein Binding; Proteins
PubMed: 17132507
DOI: 10.1016/S0076-6879(06)17015-9 -
Identification of inhibitors of biological interactions involving intrinsically disordered proteins.International Journal of Molecular... Apr 2015Protein-protein interactions involving disordered partners have unique features and represent prominent targets in drug discovery processes. Intrinsically Disordered... (Review)
Review
Protein-protein interactions involving disordered partners have unique features and represent prominent targets in drug discovery processes. Intrinsically Disordered Proteins (IDPs) are involved in cellular regulation, signaling and control: they bind to multiple partners and these high-specificity/low-affinity interactions play crucial roles in many human diseases. Disordered regions, terminal tails and flexible linkers are particularly abundant in DNA-binding proteins and play crucial roles in the affinity and specificity of DNA recognizing processes. Protein complexes involving IDPs are short-lived and typically involve short amino acid stretches bearing few "hot spots", thus the identification of molecules able to modulate them can produce important lead compounds: in this scenario peptides and/or peptidomimetics, deriving from structure-based, combinatorial or protein dissection approaches, can play a key role as hit compounds. Here, we propose a panoramic review of the structural features of IDPs and how they regulate molecular recognition mechanisms focusing attention on recently reported drug-design strategies in the field of IDPs.
Topics: DNA-Binding Proteins; Drug Discovery; Humans; Intrinsically Disordered Proteins; Pharmaceutical Preparations; Protein Binding
PubMed: 25849651
DOI: 10.3390/ijms16047394 -
Biomolecules Apr 2021The binding affinity of small molecules to receptor proteins is essential to drug discovery and drug repositioning. Chemical methods are often time-consuming and costly,...
The binding affinity of small molecules to receptor proteins is essential to drug discovery and drug repositioning. Chemical methods are often time-consuming and costly, and models for calculating the binding affinity are imperative. In this study, we propose a novel deep learning method, namely CSConv2d, for protein-ligand interactions' prediction. The proposed method is improved by a DEEPScreen model using 2-D structural representations of compounds as input. Furthermore, a channel and spatial attention mechanism (CS) is added in feature abstractions. Data experiments conducted on ChEMBLv23 datasets show that CSConv2d performs better than the original DEEPScreen model in predicting protein-ligand binding affinity, as well as some state-of-the-art DTIs (drug-target interactions) prediction methods including DeepConv-DTI, CPI-Prediction, CPI-Prediction+CS, DeepGS and DeepGS+CS. In practice, the docking results of protein (PDB ID: 5ceo) and ligand (Chemical ID: 50D) and a series of kinase inhibitors are operated to verify the robustness.
Topics: Amino Acid Sequence; Deep Learning; Drug Discovery; Drug Repositioning; Forecasting; Ligands; Neural Networks, Computer; Protein Binding; Proteins
PubMed: 33925310
DOI: 10.3390/biom11050643 -
Expert Reviews in Molecular Medicine Jul 2012Protein-protein interactions (PPIs) control the assembly of multi-protein complexes and, thus, these contacts have enormous potential as drug targets. However, the field... (Review)
Review
Protein-protein interactions (PPIs) control the assembly of multi-protein complexes and, thus, these contacts have enormous potential as drug targets. However, the field has produced a mix of both exciting success stories and frustrating challenges. Here, we review known examples and explore how the physical features of a PPI, such as its affinity, hotspots, off-rates, buried surface area and topology, might influence the chances of success in finding inhibitors. This analysis suggests that concise, tight binding PPIs are most amenable to inhibition. However, it is also clear that emerging technical methods are expanding the repertoire of 'druggable' protein contacts and increasing the odds against difficult targets. In particular, natural product-like compound libraries, high throughput screens specifically designed for PPIs and approaches that favour discovery of allosteric inhibitors appear to be attractive routes. The first group of PPI inhibitors has entered clinical trials, further motivating the need to understand the challenges and opportunities in pursuing these types of targets.
Topics: Allosteric Regulation; Animals; Humans; Protein Binding; Proteins; Surface Properties
PubMed: 22831787
DOI: 10.1017/erm.2012.10 -
Annual Review of Pharmacology and... Jan 2018The adhesion G protein-coupled receptors (aGPCRs) are an evolutionarily ancient family of receptors that play key roles in many different physiological processes. These... (Review)
Review
The adhesion G protein-coupled receptors (aGPCRs) are an evolutionarily ancient family of receptors that play key roles in many different physiological processes. These receptors are notable for their exceptionally long ectodomains, which span several hundred to several thousand amino acids and contain various adhesion-related domains, as well as a GPCR autoproteolysis-inducing (GAIN) domain. The GAIN domain is conserved throughout almost the entire family and undergoes autoproteolysis to cleave the receptors into two noncovalently-associated protomers. Recent studies have revealed that the signaling activity of aGPCRs is largely determined by changes in the interactions among these protomers. We review recent advances in understanding aGPCR activation mechanisms and discuss the physiological roles and pharmacological properties of aGPCRs, with an eye toward the potential utility of these receptors as drug targets.
Topics: Animals; Drug Delivery Systems; Humans; Protein Binding; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 28968187
DOI: 10.1146/annurev-pharmtox-010617-052933 -
Molecules (Basel, Switzerland) Aug 2021The recognition of specific DNA sequences in processes such as transcription is associated with a cooperative binding of proteins. Some transcription regulation...
The recognition of specific DNA sequences in processes such as transcription is associated with a cooperative binding of proteins. Some transcription regulation mechanisms involve additional proteins that can influence the binding cooperativity by acting as corepressors or coactivators. In a conditional cooperativity mechanism, the same protein can induce binding cooperativity at one concentration and inhibit it at another. Here, we use calorimetric (ITC) and spectroscopic (UV, CD) experiments to show that such conditional cooperativity can also be achieved by the small DNA-directed oligopeptides distamycin and netropsin. Using a global thermodynamic analysis of the observed binding and (un)folding processes, we calculate the phase diagrams for this system, which show that distamycin binding cooperativity is more pronounced at lower temperatures and can be first induced and then reduced by increasing the netropsin or/and Na+ ion concentration. A molecular interpretation of this phenomenon is suggested.
Topics: DNA; Distamycins; Netropsin; Oligopeptides; Protein Binding; Sodium; Thermodynamics; Transcription, Genetic
PubMed: 34500619
DOI: 10.3390/molecules26175188 -
British Journal of Clinical Pharmacology Dec 2017
Topics: Female; Humans; Pregnancy; Protein Binding; Serum Albumin
PubMed: 28983934
DOI: 10.1111/bcp.13432 -
Experimental Biology and Medicine... Jun 2021The chaperone heat shock protein 70 (Hsp70) and its network of co-chaperones serve as a central hub of cellular protein quality control mechanisms. Domain organization... (Review)
Review
The chaperone heat shock protein 70 (Hsp70) and its network of co-chaperones serve as a central hub of cellular protein quality control mechanisms. Domain organization in Hsp70 dictates ATPase activity, ATP dependent allosteric regulation, client/substrate binding and release, and interactions with co-chaperones. The protein quality control activities of Hsp70 are classified as foldase, holdase, and disaggregase activities. Co-chaperones directly assisting protein refolding included J domain proteins and nucleotide exchange factors. However, co-chaperones can also be grouped and explored based on which domain of Hsp70 they interact. Here we discuss how the network of cytosolic co-chaperones for Hsp70 contributes to the functions of Hsp70 while closely looking at their structural features. Comparison of domain organization and the structures of co-chaperones enables greater understanding of the interactions, mechanisms of action, and roles played in protein quality control.
Topics: Cytosol; HSP70 Heat-Shock Proteins; Humans; Molecular Chaperones; Protein Binding; Protein Folding
PubMed: 33730888
DOI: 10.1177/1535370221999812 -
Journal of the Royal Society, Interface Feb 2013Interactions between proteins are orchestrated in a precise and time-dependent manner, underlying cellular function. The binding affinity, defined as the strength of... (Review)
Review
Interactions between proteins are orchestrated in a precise and time-dependent manner, underlying cellular function. The binding affinity, defined as the strength of these interactions, is translated into physico-chemical terms in the dissociation constant (K(d)), the latter being an experimental measure that determines whether an interaction will be formed in solution or not. Predicting binding affinity from structural models has been a matter of active research for more than 40 years because of its fundamental role in drug development. However, all available approaches are incapable of predicting the binding affinity of protein-protein complexes from coordinates alone. Here, we examine both theoretical and experimental limitations that complicate the derivation of structure-affinity relationships. Most work so far has concentrated on binary interactions. Systems of increased complexity are far from being understood. The main physico-chemical measure that relates to binding affinity is the buried surface area, but it does not hold for flexible complexes. For the latter, there must be a significant entropic contribution that will have to be approximated in the future. We foresee that any theoretical modelling of these interactions will have to follow an integrative approach considering the biology, chemistry and physics that underlie protein-protein recognition.
Topics: Allosteric Regulation; Calorimetry; Crystallography; Fluorescence; Models, Molecular; Multiprotein Complexes; Protein Binding; Protein Interaction Mapping; Structure-Activity Relationship; Surface Plasmon Resonance
PubMed: 23235262
DOI: 10.1098/rsif.2012.0835