Molecular biological techniques, protein

Toxin Any toxic substance that can be produced by an animal, plant, or microbe. Some toxins can also be produced by molecular biological techniques (protein toxins) or by chemical synthesis (low-molecular-weight toxins). Chemical agents, such as soman, sarin, VX, cyanide, and mustard agents, typically man-made for weaponization, are not included in this discussion except for comparison. 

A major requirement for structural studies is the availability of large quantities of pure, functional protein. Essentially all proteins are present in only very small quantities in native cells or tissues, so molecular biology techniques... 

The first two volumes in the series New Comprehensive Biochemistry appeared in 1981. Volume 1 dealt with membrane structure and Volume 2 with membrane transport. The editors of the last volume (the present editor being one of them) tried to provide an overview of the state of the art of the research in that field. Most of the chapters dealt with kinetic approaches aiming to understand the mechanism of the various types of transport of ions and metabolites across biological membranes. Although these methods have not lost their significance, the development of molecular biological techniques and their application in this field has given to the area of membrane transport such a new dimension that the appearance of a volume in the series New Comprehensive Biochemistry devoted to molecular aspects of membrane proteins is warranted. 

Due to the ready accessibility of SH2 domains by molecular biology techniques, numerous experimentally determined 3D structures of SH2 domains derived by X-ray crystallography as well as heteronuclear multidimensional NMR spectroscopy are known today. The current version of the protein structure database, accessible to the scientific community by, e.g., the Internet (http //www.rcsb.org/pdb/) contains around 80 entries of SH2 domain structures and complexes thereof. Today, the SH2 domain structures of Hck [62], Src [63-66], Abl [67], Grb2 [68-71], Syp [72], PLCy [73], Fyn [74], SAP [75], Lck [76,77], the C- and N-terminal SH2 domain ofp85a [78-80], and of the tandem SH2 domains Syk [81,82], ZAP70 [83,84], and SHP-2 [85] are determined. All SH2 domains display a conserved 3D structure as can be expected from multiple sequence alignments (Fig. 4). The common structural fold consists of a central three-stranded antiparallel ft sheet that is occasionally extended by one to three additional short strands (Fig. 5). This central ft sheet forms the spine of the domain which is flanked on both sides by regular a helices [49, 50,60]. 

Unstable proteins may be modified by the molecular biological technique of site-directed mutagenesis to remove the site of instability— for instance, an oxidizable cysteine. Such techniques are appropriate for commercial production of proteins, but may of course alter natural functioning parameters. Increased thermostability can be one modification, although it is not easy to predict mutations that will improve that parameter. Thermostable proteins originating from thermophilic bacteria do not need structural modification and, if expressed in large amounts, can be purified satisfactorily in one step by simply heat-treating the extract at 70°C for 30 min, which denatures virtually all the host proteins (e.g., see Oka et al., 1989). 

The amino acid sequences of numerous transport proteins have been elucidated during the last 10 years. As with conventional enzymes, molecular biological techniques now make it possible to modify individual amino acid residues in the proteins and thus to probe the roles these residues play in catalysis of facilitated diffusion or active transport. 

Ache and coworkers demonstrated that both cyclic nucleotides and inositol phosphates mediate the transduction of environmental chemical signals by the olfactory neurons of P. argus.62 65 Both biochemical and molecular biological techniques have shown that the receptor cells contain various G-protein subunits that would be necessary for signal detection by G-protein-associated chemoreceptors.48 49 66-69 In combination with electrophysiological studies,... 

---