For solution of the macromolecular phase problem, the most commonly used methods (multiple isomorphous replacement (MIR) and that of multiple wavelength anomalous dispersion (MAD)) still involve the incorporation of heavy atoms into protein crystals.Full Description
For solution of the macromolecular phase problem, the most commonly used methods (multiple isomorphous replacement (MIR) and that of multiple wavelength anomalous dispersion (MAD)) still involve the incorporation of heavy atoms into protein crystals. After crystallization, finding such derivatives is the second major bottle neck in the determination of the 3D structure of bio-macromolecules.
Most labelling procedures focus on the protein itself in a “trial and error” fashion. Halogenated ATP and GTP analogs however, provide an alternative method that allows rational incorporation of heavy atoms into a large number of physiologically relevant enzymes:
- In the human genome alone, estimated 5,000 to 10,000 proteins interact with ATP or GTP (protein- and nucleotide-kinases, motor proteins, chaperones… or the superfamily of GTPases, respectively).
- The incorporation of iodine or bromine allows MIR or MAD phasing for proteins with molecular weights of at least up to 50 kDa. Importantly, for MIR experiments, such derivatives are likely to be isomorphous with the native crystal.
- The kinetics of binding of 2′-halogenated ATP analogs to most enzymes so far investigated (including myosin, creatine kinase, hexokinase, pyruvate kinase and UMP/CMP kinase) are similar to those of non-substituted ATP.
- The complexes of 2′-halogenated GTP analogs with the GTPases p21ras or Rab5 have dissociation constants well suitable for crystallization.
- Several proteins including kinesin, non-claret disjunctional protein and TMP kinase have been crystallized with 2′-halogenated analogs.
- Gruen et al. (1999) 2′-Halo-ATP and -GTP analogues: Rational phasing tools for protein crystallography. Protein Sci. 8:2524.
- Gruen et al. (1999) Synthesis of 2′-iodo- and 2′-bromo-ATP and GTP analogues as potential phasing tools for X-ray crystallography. Nucleos. Nucleot. 18 (1):137.
- Naber et al. (1995) A novel adenosine-triphosphate analog with a heavy-atom to target the nucleotide-binding site of proteins. Protein Sci. 4:1824.