Resumen de La tripsina como modelo de difracción de rayos X a alta resolución en proteínas

J.A. Cuesta-Seijo, Santiago García Granda

• español

  En la última década se ha hecho posible obtener datos de difracción de rayos X hasta resolución atómica (

• English

  In the last decade has been made possible to obtain X-ray diffraction data to atomic resolution (<1.2¿) of proteins (more than 1000 atoms in the molecule). The atomic resolution has interest in the accurate determination of atomic positions, but it also gives dinamic information from the study of individual anisotropic displacement factors and more information on the conformational freedom. Besides it allows for the experimental observation of the positions of Hydrogen atoms, often playing a key role in the catalytic mechanisms.

  Traditionaly lisozyme has been used as the proteic model for diffraction studies at high resolution, partialy by inertia due to its paradigmatic character in protein diffraction. Trypsin shows also useful properties for these estudies. Recently, crystallization conditions allow the obtention of big size and high quality crystals in a fast, easy and reproducible way have been determined. That crystallization is possible using commercial trypsin without previous purification, simplifiying the procedure, and achiving higher resolution than lisozyme. Moreover, trypsin has many organic, inorganic and proteic inhibitors, showing also one active centre accesible in crystaline form, to medium size peptides, by diffusion. In that way, it becomes one ideal model for the study of protein-inhibitor interactions and for protein interactions in general.

  Bovine Trypsin allows the easy, fast and reproducible obtention of protein crystals able to diffract to atomic resolution. Our laboratory has grown crystals able to diffract to the limit of 1.20 ¿ using a Cu sealed tube as X-ray source. That crystals include native Trypsin and Trypsin inhibited with a reversible inhibitor. Synchrotron radiation data collected at ESRF(bm14) with a bending magnet as X-ray source show a signal/noise of about 5 to 6 in the last shell of resolution shell (to 1.02¿) using a exposure time of a few seconds per image, which suggest a diffraction limit quite below than 0.90¿, therefore being suitables for X-ray diffraction studies of proteins at very high resolution. Similar results have been obtained with trypsins of other species, Rypniewski (2001); Leiros (2001). The resulting models show a high structural order besides the atomic resolution. This fact itself justifies the adoption of Trypsin as a model for X ray diffaction studies of proteins to atomic resolution. Crystals of Trypsin have in addition chanels of solvent reaching the active center making it accesible to inhibitors and/or substrates by diffusion. That allows to extend the studies to the linkage of inhibitors (organic or inorganic) and herefore the use of Trypsin as a model for studies of protein-inhibitor interactions.