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2´-deoxy-2´-iodoadenosine-5´-triphosphate; nucleotide analogs; motor proteins; X-ray crystallography
Abstract:
We have synthesized 2'-deoxy-2'-iodoadenosine-5'-triphosphate (2'-IATP), a heavy-atom analog of adenosine-5'-triphosphate. This compound was made for X-ray structural studies to target the nucleotide site of ATP binding proteins. It was diffused successfully into crystals of the microtubule-based motor proteins ncd (non-claret disjunctional protein from Drosophila melanogaster) and kinesin. With ncd, the nucleotide binding site was 70% occupied and the crystals were able to diffract X-rays to 2.5 A. The iodo-analog provided a useful isomorphous derivative with overall phasing power 1.89 in the range of 25.0-2.5 A. With kinesin, 2'-IATP co-crystallized with the protein. The crystals diffracted to at least 2.8 A with a phasing power of 1.73 in the range of 20.0-5.0 A. The analog was also found to be a substrate for all of the enzymes tested, including creatine kinase, pyruvate kinase, hexokinase, and myosin, with values of Km and Vmax that were within a factor of 10 of those for ATP. The analog supported muscle contraction, relaxing fibers, and producing active tension with values not statistically different from those obtained with ATP. These results all suggest that this analog should be useful for providing a heavy-atom derivative for crystals of enzymes that bind ATP.
