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Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective...
To understand the manner in which biological macromolecules interact with each other, we need not only structural information, but also details of kinetics and thermodynamics of the processes involved. This is particularly important for key proteins acting in signal transduction such as the small GTPases of the Ras superfamily. The complexity of their roles is constantly increasing since a large number...
Transcription can be regulated during initiation, elongation, and termination by an enormous variety of regulatory factors. A critical step in obtaining a mechanistic understanding of regulatory factor function is the determination of whether the regulatory factor exerts its effect through direct contact with the transcription machinery. Here I describe the application of a transcription activation-based...
This paper describes protocols for studies of structure and dynamics of DNA and protein–DNA complexes with atomic force microscopy (AFM) utilizing the surface chemistry approach. The necessary specifics for the preparation of functionalized surfaces and AFM probes with the use of silanes and silatranes, including the protocols for synthesis of silatranes are provided. The methodology of studies of...
We present a high-resolution mass spectrometric (MS) footprinting method enabling identification of contact amino acids in protein–protein complexes. The method is based on comparing surface topologies of a free protein versus its complex with the binding partner using differential accessibility of small chemical group selective modifying reagents. Subsequent MS analysis reveals the individual amino...
Since most cellular processes depend on interactions between proteins, information about protein–protein interactions (PPIs) provide valuable insights into protein function. Over the last years, quantitative affinity purification followed by mass spectrometry (q-AP-MS) has become a powerful approach to investigate PPIs in an unbiased manner. In q-AP-MS the protein of interest is biochemically enriched...
Proteins do not exert their effects in isolation of one another, but interact together in complex networks. In recent years, sophisticated methods have been developed to leverage protein–protein interaction (PPI) network structure to improve several stages of the drug discovery process. Network-based methods have been applied to predict drug targets, drug side effects, and new therapeutic indications...
Understanding of the molecular mechanisms of protein–protein interactions (PPIs) at the cell surface of living cells is fundamental to comprehend the functional meaning of a large number of cellular processes. Here we discuss how new methodological strategies derived from non-invasive fluorescence-based approaches (i.e. fluorescence resonance energy transfer, FRET) have been successfully developed...
The majority of small molecule drugs act on protein targets to exert a therapeutic function. It has become apparent in recent years that many small molecule drugs act on more than one particular target and consequently, approaches which profile drugs to uncover their target binding spectrum have become increasingly important. Classical yeast two-hybrid systems have mainly been used to discover and...
Negative protein–protein interaction datasets are needed for training and evaluation of interaction prediction methods, as well as validation of high-throughput interaction discovery experiments. In large-scale two-hybrid assays, the direct interaction of a large number of protein pairs is systematically probed. We present a simple method to harness two-hybrid data to obtain negative protein–protein...
High-throughput technologies such as affinity purification and yeast two-hybrid (Y2H) screening have been applied to explore protein–protein interactions (PPIs) in the model organism Saccharomyces cerevisiae. The “Cross-and-Capture” system is an alternative approach for an assessment of PPIs using two arrayed collections of differentially tagged yeast strains. In its current implementation, Cross-and-Capture...
The combined application of different biophysical techniques – analytical ultracentrifugation, light scattering and fluorescence-based assays – to study the ligand-linked self-association and assembly properties of the cell division protein FtsZ from Escherichia coli is described. These reactions are thought to be important for the formation of the dynamic division ring that drives bacterial cytokinesis...
A set of phage display sorting strategies and validation methodologies are presented that are capable of producing high performance synthetic antibodies (sABs) with customized properties. Exquisite control of antigen and conditions during the phage display selection process can yield sABs that: (1) recognize conformational states, (2) target specific regions of the surface of a protein, (3) induce...
Intrinsically disordered proteins (IDPs) are proteins that lack stable higher order structures for the entire protein molecule or a significant portion of it. The discovery of IDPs evolved as an antithesis to the conventional structure–function paradigm wherein a higher order structure dictates protein function. Over the last decade, a number of proteins with functionally relevant unstructured regions...
In this article, we describe our methods and protocols using collision-induced dissociative chemical crosslinking-tandem mass spectrometry (CID-CXL-MS/MS) analysis and the practical considerations when implementing these reagents and methodology for protein crosslinking studies. The synthesis of our novel chemical crosslinkers is described as well as their use for effectively labeling protein and...
Protein–protein interactions orchestrate virtually all cellular processes, therefore, their exhaustive exploration is essential for the comprehensive understanding of cellular networks. A reliable identification of interfacial residues is vital not only to infer the function of individual proteins and their assembly into biological complexes, but also to elucidate the molecular and physicochemical...
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