Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mRNA coding for rat intestinal calcium-binding protein, a vitamin D3-induced protein (Mr 7500), has been partially purified from growing rat duodenum. Double-stranded DNA synthesized from the purified mRNA preparation was inserted into the PstI site of pBR322, using the oligo(dG-dC) tailing procedure. Clones containing DNA complementary to vitamin D-dependent calcium-binding protein mRNA were selected by differential colony hybridization with [32P] cDNA synthesized from enriched or low vitamin D-dependent calcium-binding protein mRNA preparations. Plasmid DNAs from the selected clones were each verified by both a solution hybrid-arrest assay and a filter hybrid-selection assay. Four recombinant clones showed identical endonuclease restriction maps and contained inserts ranging from 250 to 380 base pairs.
 ...
PMID:Synthesis, molecular cloning, and restriction analysis of DNA complementary to vitamin D-dependent calcium-binding protein mRNA from rat duodenum. 629 2

A gene coding for the Nereis sarcoplasmic calcium-binding protein (NSCP) was synthesized and expressed in Escherichia coli. The sequence of the gene was derived from the protein sequence by reverse translation. It possesses a number of unique, regularly spaced, restriction endonuclease cleavage sites to facilitate future site-directed mutagenesis. For the cloning strategy the gene sequence was divided into four parts. Three parts were cloned by ligation of hybridized oligomers and one part by inverse PCR. The protein was expressed as a fusion protein with the bacterial chloramphenicol acetyl-transferase (CAT), which could be easily purified by affinity chromatography. At the junction of the CAT and NSCP moieties a recognition site for the proteolytic enzyme factor Xa was built in. However, the distance between the moieties appeared to be crucial to warrant cleavage. A kinetic analysis showed that NSCP prepared from the sandworm and the one expressed by E. coli behaved in the same way. This system provides a basis for site-specific mutagenesis studies, in order to elucidate the molecular mechanism of cation binding and concomitant conformational changes.
 ...
PMID:Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase. 814 89

In this article we focus on presenting a broad range of examples illustrating low-energy transitions via hinge-bending motions. The examples are divided according to the type of hinge-bending involved; namely, motions involving fragments of the protein chains, hinge-bending motions involving protein domains, and hinge-bending motions between the covalently unconnected subunits. We further make a distinction between allosterically and nonallosterically regulated proteins. These transitions are discussed within the general framework of folding and binding funnels. We propose that the conformers manifesting such swiveling motions are not the outcome of "induced fit" binding mechanism; instead, molecules exist in an ensemble of conformations that are in equilibrium in solution. These ensembles, which populate the bottoms of the funnels, a priori contain both the "open" and the "closed" conformational isomers. Furthermore, we argue that there are no fundamental differences among the physical principles behind the folding and binding funnels. Hence, there is no basic difference between funnels depicting ensembles of conformers of single molecules with fragment, or domain motions, as compared to subunits in multimeric quaternary structures, also showing such conformational transitions. The difference relates only to the size and complexity of the system. The larger the system, the more complex its corresponding fused funnel(s). In particular, funnels associated with allosterically regulated proteins are expected to be more complicated, because allostery is frequently involved with movements between subunits, and consequently is often observed in multichain and multimolecular complexes. This review centers on the critical role played by flexibility and conformational fluctuations in enzyme activity. Internal motions that extend over different time scales and with different amplitudes are known to be essential for the catalytic cycle. The conformational change observed in enzyme-substrate complexes as compared to the unbound enzyme state, and in particular the hinge-bending motions observed in enzymes with two domains, have a substantial effect on the enzymatic catalytic activity. The examples we review span the lipolytic enzymes that are particularly interesting, owing to their activation at the water-oil interface; an allosterically controlled dehydrogenase (lactate dehydrogenase); a DNA methyltransferase, with a covalently-bound intermediate; large-scale flexible loop motions in a glycolytic enzyme (TIM); domain motion in PGK, an enzyme which is essential in most cells, both for ATP generation in aerobes and for fermentation in anaerobes; adenylate kinase, showing large conformational changes, owing to their need to shield their catalytic centers from water; a calcium-binding protein (calmodulin), involved in a wide range of cellular calcium-dependent signaling; diphtheria toxin, whose large domain motion has been shown to yield "domain swapping;" the hexameric glutamate dehydrogenase, which has been studied both in a thermophile and in a mesophile; an allosteric enzyme, showing subunit motion between the R and the T states (aspartate transcarbamoylase), and the historically well-studied lac repressor. Nonallosteric subunit transitions are also addressed, with some examples (aspartate receptor and BamHI endonuclease). Hence, using this enzyme-catalysis-centered discussion, we address energy funnel landscapes of large-scale conformational transitions, rather than the faster, quasi-harmonic, thermal fluctuations.
 ...
PMID:Folding funnels and conformational transitions via hinge-bending motions. 1059 56