9317981 Feigelson This proposal is for the development of a multi-station, computer controlled apparatus for the growth of high quality, single crystals of biological macromolecules (proteins) suitable for use in structure determinations by x- ray diffraction. The difficulties in growing suitable crystals 1 has been one of the major factors limiting the number of protein structures which have been completely determined 2 . The determination of the molecular structure of proteins has become an important segment of modern biology and pharmacology and its potential has been barely tapped 2 . The development of the proposed apparatus is directed toward the growth of crystals with a high degree of internal perfection which will facilitate the process of structure determination. In order to growth the highest quality crystals, it is necessary to separately control the nucleation and growth phases during crystallization. Previous work in our laboratory has demonstrated that the nucleation of crystals in protein solutions can be controlled by controlling the temperature in a small region of the solution. The subsequent growth was controlled by changing the ambient temperature around the growth cell. This technique, Thermonucleation. has been used to nucleate and grow crystals of both inorganic and protein materials 3,4 . Thermonucleation relies on the independent control of the temperatures in both the nucleating region and the bulk of the solution. Its use is limited to those proteins which have some temperature dependence of solubility within the temperature range of approximately 0 to 35 C and thus is applicable to a large number of proteins. The intent of the proposed program is to develop a user friendly, computer controlled, multi-station apparatus to grow protein crystals using the principle of Thermonucleation. The growth module will be downsized from the research instrument previously developed and will use thermoclectric heating and cooling . The new design will emphasize ease of crystal harvesting and changing of protein solutions. The actual growth cell will be of a size consistent with the small solution volumes used in protein crystallization. Temperature sensing will be built into each growth module. The module will also incorporate a nucleation detection system which will allow switching from a computer controlled nucleation program to the appropriate growth program. Nucleation detection will be based on static light scattering techniques 5 . The development of the apparatus will begin with the design, construction and testing of the growth module. Concurrent with this, the nucleation detector will be developed. These two units will be integrated into a single module. At this point, interfacing with the computer and computer program development will begin. When the single module has been thoroughly tested under computer control using a number of crystallizing systems multiple unit apparatus will be constructed and tested. Results at each stage of the apparatus development will be used to optimize the final apparatus. The goal of the proposed program will be to develop this technology into a user friendly, semi-automated apparatus which will allow biochemical worker to reproducibly grow high quality single crystals of proteins for x-ray structure determinations with a minimum of operator interaction.
