TEMP DEPEND OF AVERAGE & SITE-SPECIFIC RADIATION DAMAGE IN PROTEIN CRYSTALS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We will try to address two questions: 1. Is there any difference in damage versus dose to protein crystals when data is collected at T=100 K (nitrogen gas) and T=20 K (helium gas)? Several experiments have seen little or no effect, but the effects may be too small to be reliably quantified using standard methods. Postdoctoral Associate (now Assistant Professor) Jan Kmetko developed a data collection and analysis protocol that should give the accuracy to reliably resolve differences. In this part of the experiment, we will dose crystals and collect data in a narrow angular wedge (5 degrees), which will not be sufficient for a full structural refinement, out to doses of 20 MGray or more. 2. Does site-specific radiation damage depend on temperature at temperatures well below water's glass transition temperature? There is a belief among those currently studying radiation damage to protein crystals that the observation of site-specific damage (e.g., breakage of disulfide bonds) at relatively low doses is evidence for secondary radiation damage. However, we believe (based on old ESR experiments and basic considerations) that this damage occurs via electron transfer, not by thermal diffusion of atomic and molecular radicals, and therefore is primary damage. Even if site specific damage is due entirely to electron motion, this motion may occur via tunneling or thermal hopping. By comparing site-specific damage to protein crystals at T=100 K and T=20 K, we hope to be able to nail down the nature of site-specific damage. These experiments will involve taking a series of data sets (each with enough information to evalate the status of disulfide bonds) from the same spot on the same crystal , and watching the growth of site specific damage with total absorbed dose. Comparison of data collected at T=100 K and T=20 K will reveal any differences in the rate and extent of site specific damage.