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A series of antipeptide antibodies designed to recognize specific sequences of the gap junction protein connexin 43 (Cx43) were developed and characterized immunochemically and immunohistologically. These antibodies bound to gap junctions and, on Western blots, to 43-kDa (often resolved as a doublet) and 41-kDa proteins in samples from heart, leptomeningeal cells, and brain. Relatively little of the...
The characterization of protein-protein interactions provides the foundation for further studies concerning protein complex function and regulation. Since the advent of the yeast two-hybrid assay, many additional genetic systems based upon the principle of protein fragment complementation have been designed. One such system, the split-ubiquitin membrane yeast two-hybrid system (MbYTH), is able to...
Molecular dynamics simulations have become a popular and powerful technique to study lipids and membrane proteins. We present some general questions and issues that should be considered prior to embarking on molecular dynamics simulation studies of membrane proteins and review common simulation methods. We suggest a practical approach to setting up and running simulations of membrane proteins, and...
The use of detergents for the structural study of membrane proteins is discussed with an emphasis on practical issues relating to membrane solubilization, protein aggregation, detergent purity and detergent quantitation. Detergents are useful reagents as mimics of lipid bilayers because of their self-assembling properties, but as a result, they have complex properties in solution. It can be difficult...
We survey computational approaches that tackle membrane protein structure and function prediction. While describing the main ideas that have led to the development of the most relevant and novel methods, we also discuss pitfalls, provide practical hints and highlight the challenges that remain. The methods covered include: sequence alignment, motif search, functional residue identification, transmembrane...
Structural and functional information of membrane proteins at ever-increasing resolution is being obtained by electron crystallography. While a large amount of work on the development of methods for electron microscopy and image processing has resulted in tremendous advances in terms of speed of data collection and resolution, general guidelines for crystallization are first starting to emerge. Yet...
Size exclusion chromatography is an established technique for the determination of hydrodynamic volumes of proteins or protein complexes. When applied to membrane proteins, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate measurements for the protein. In a detergent series, in which the detergents...
This paper describes the use of Atomic Force Microscopy (AFM) to investigate the plasma membrane of Xenopus laevis oocyte. Different protocols of sample preparation to perform an AFM investigation of both external and intracellular sides of the oocyte plasma membrane are presented and discussed. Reproducible AFM images allowed visualization and dimensional characterization of protein complexes differently...
Aptamers are chemical antibodies that bind to their targets with high affinity and specificity. These short stretches of nucleic acids are identified using a repetitive in vitro selection and partitioning technology called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since the emergence of this technology, many modifications and variations have been introduced to enable the selection...
Biochemical and biophysical analysis on integral membrane proteins often requires monodisperse and stable protein samples. Here we describe a method to characterize protein thermostability by measuring its melting temperature in detergent using analytical size-exclusion chromatography. This quantitative method can be used to screen for compounds and conditions that stabilize the protein. With this...
A challenging requirement for X-ray crystallography or NMR structure determination of membrane proteins (MPs), in contrast to soluble proteins, is the necessary use of amphiphiles to mimic the hydrophobic environment of membranes. A number of new detergents, lipids and non-detergent-like amphiphiles have been developed that stabilize MPs, and these have contributed to increased success in MP structural...
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing...
Crystallographic studies of membrane proteins have been steadily increasing despite their unique physical properties that hinder crystal formation. Co-crystallization with antibody fragments has emerged as a promising solution to obtain diffraction quality crystals. Antibody binding to the target membrane protein can yield a homogenous population of the protein. Interantibody interactions can also...
Binding of ligands, ranging from proteins to ions, to membrane proteins is associated with absorption or release of heat that can be detected by isothermal titration calorimetry (ITC). Such measurements not only provide binding affinities but also afford direct access to thermodynamic parameters of binding – enthalpy, entropy and heat capacity. These parameters can be interpreted in a structural context,...
Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure...
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