Crystallization of GABAA Receptor

LoVerde Crystallization of GABAA Receptor Marilena LoVerde Energy Research Undergraduate Fellowship University of California, Berkeley Stanford Linear Accelerator Center Stanford, CA 94309 18 August 2000 Prepared in partial fulfillment of the requirements of the Office of Science, Energy research Undergraduate Laboratory Fellowship Program under the direction of Dr Aina Cohen and Dr Paul Ellis in the protein crystallography division of Stanford Synchrotron Radiation Laboratory Structural Molecular Biology Group at Stanford Linear Accelerator Center Participant: _ Signature Research Advisors: _ Signature _ Signature LoVerde Table of Contents Abstract Introduction Materials and Methods Results Discussion and Conclusion Acknowledgements Works Cited and Bibliography Figures and Tables 8 9 LoVerde Abstract Crystallization of GABAA Receptor Marilena LoVerde (University of California at Berkeley, Berkeley, California 94703) Aina Cohen and Paul Ellis (Stanford Linear Accelerator Center, Stanford, California 94025) X-ray diffraction is the most reliable and informative method for determining three-dimensional molecular structure and electron density This process requires crystals of sufficient quality for successful solution of the crystal structure Crystallization is often the most laborious step in the X-ray diffraction process Crystallization conditions for the GABAA receptor were sought by the Vapor Diffusion Method using a total of 192 different solutions No protein crystals have been grown to date, however the screens will be periodically inspected for possible crystal growth If crystals are obtained, ideal crystallization conditions will be researched by Dr.’s Paul Ellis and Aina Cohen Crystals of sperm whale myoglobin and egg white lysozyme were grown under known conditions for the use of the Stanford Synchrotron Radiation Laboratory Protein Crystallography Group In addition an instructional web page for diffraction experiments was developed This page includes instructions for use of beamline hardware and data collection software LoVerde Introduction The goal of this research is to obtain crystallization conditions for the GABA (aminobutyric acid) receptor Crystallization is the first step in the process of solving a macromolecular structure by diffraction analysis GABA is the major inhibitory neurotransmitter in the brain Determination of the GABA receptor structure will further medical research on structure based pharmaceuticals that alter neurotransmission In order to understand molecular function, physical structure as well as composition must be understood Crystallography is a method by which macromolecules can be investigated to obtain information such as three dimensional shape and electron density Crystallography with synchrotron radiation is the process whereby a crystalline sample is investigated with the intense radiation emitted from high energy electrons circling in a storage ring At Stanford Synchrotron Radiation Laboratory, SSRL, electrons circle the ring at 3GeV Synchrotron radiation is advantageous because of the intensity of the X-rays provided and the ability to select the wavelength most appropriate for the study When visible light hits a macroscopic object the light is reflected Our eyes focus these reflections to recreate an image of the original object The individual atoms of molecules are not visible to the human eye because they are to small to reflect visible light X-rays are small enough to interact with molecules However, there is no way to focus reflected X-rays to produce a visible image so it is the diffracted X-rays that are studied Diffraction occurs when an electromagnetic wave front encounters the boundary of an object and interferes with itself A crystalline protein sample will diffract X-rays because the waves travel different path lengths as they are reflected off lattice planes LoVerde If the path difference is an integer multiple of wavelengths the wave amplitudes will add and a maximum occurs If the path length difference is a multiple of half wavelengths destructive interference occurs; a minimum is present The maxima of the diffraction pattern can be located using Bragg’s law (figure 1): n = 2dsin where  is the wavelength of light, d is the spacing between adjacent lattice planes, and  the angle of the incident reflection Proteins are large molecules composed of amino acids bonded together in a polypeptide chain The polypeptide is folded and its shape maintained by hydrogen bonds Proteins not naturally form crystals Small molecule crystals often form if the molecule is heated in solution and abruptly cooled Proteins are delicate molecules and such extreme conditions will cause them to denature Initially conditions for protein crystallization are sought through trial and error Once any crystals are produced the conditions are refined to produce the best quality crystal The quality of a crystal is defined by its shape, mosaicity, resolution limit, and size (Helliwell, 1985) A more symmetric crystal grants better statistical data accuracy Mosaicity refers to the regularity of the crystal; a crystal with high mosaicity would have groups of unit cells that were angularly misaligned from the rest of the crystal Low mosaicity is desirable The resolution limit of a crystal is the largest angle at which a diffraction spot can be experimentally observed Resolution limit is determined by the order of the crystal structure; a highly ordered crystal will have a high resolution limit A crystal with a high resolution limit would have a large lattice plane spacing and diffract at large angles A large crystal will produce a stronger diffraction pattern LoVerde Protein crystallization requires the protein to precipitate out of solution at slow enough rates to form a well ordered crystalline solid At present there is no reliable method for determining ideal crystallization conditions other than trial and error The solutions used in the screening process often precipitates inorganic crystals Therefore crystals obtained must be tested for protein crystal content Unlike inorganic crystals, protein crystals contain large amounts of solvent Commercial indicators are available that will penetrate protein crystals via the solvent channels altering the crystal color Protein crystals are more delicate than inorganic crystals therefore fragility is also a representative characteristic of a protein crystal An inorganic crystal would be rigid and difficult to break when prodded with an acupuncture needle, whereas a protein crystal would be penetrable and dismember The most reliable method for determining crystal content is to look at the diffraction pattern A protein crystal contains larger unit cells and is more disordered than an inorganic crystal The diffraction pattern for an inorganic crystal appears more regular than that of a protein crystal Materials and Methods Initially the solution contained 5M Argimine, 0.1M tris, 01M EDTA, 5% Cholic acid, 3mM Cystine, The protein solution was first purified by draining the solution through a 10 kDa concentrator centrifuged at 4700rpm The drained buffer was replaced with tris to reduce the concentration of Arginine to 015M The protein was then concentrated to 8.5 mg/mL and sterilized using 22 m acetate sterilizing filters Wizard I, Wizard II, Cryo I and Cryo II screen sets bought from Emerald Biosystems were used Crystallization screen were set up according to the vapor diffusion Hanging Drop Method illustrated in figure Seven hundred microliters of each solvent or mother liquor was LoVerde put in a 1.5 x 1.8 cm well One microliter of solvent and one microliter of protein solution (~8.5 mg/mL) were placed on a coverslip placed on the well and sealed with oil All screens were prepared at 22C on July 24, 2000 Cryo I, Wizard I, and Wizard II screens were prepared at 4C on July 27, 2000 Cryo II 4C screen was prepared on August 1, 2000 Screens were checked for crystals on July 31, 2000 All screen containing possible crystals were tested with 2L IzIt? brand indicator and checked four hours later A previously grown lysozyme crystal was also tested with the IzIt? solution to represent a positive for protein crystal content Crystals that were initially darker than the IzIt? solution were tested by physically prodding the sample with an acupuncture needle Results Solutions containing possible crystals are listed in tables 1-3 Images of drops representative of all results are pictured in figure four with IzIt? solution After four hours the lysozyme crystal turned royal blue and the surrounding solution did not appear to contain any IzIt? None of the crystals grown illustrated a dramatic color change when tested with IzIt? brand indicator All crystals remained lighter than the surrounding liquid Crystals in drops six, seven, and eight of figure were readily penetrated by the needle, and their high viscosity and lack of rigidity suggested a gel like composition Large crystals such as that in drop two of figure were jabbed and exhibited no flexibility nor fragility Some drops like number three include a wispy precipitate Drops such as number four contain small translucent objects, too small to observe confidently under the microscope LoVerde Discussion and Conclusion Of all drops with possible crystalline precipitate analyzed with IzIt?, none exhibited the color change signifying the presence of a protein crystal Confidence in this result is heightened with the comparison to the lysozme IzIt? interaction and with the physical rigidity or extreme malleability of the crystals Thus far no protein crystals have been grown The crystals demonstrating extreme rigidity are positively not protein and most likely salts Crystals in drops six, seven, and eight appear to be a gel precipitate Results in drop four are either small inorganic crystals or evidence of phase separation Drop three is protein precipitate Drop six evaporated due to a poorly sealed well Despite the absence of crystal growth at present, all screens will remain intact for further inspection As crystal growth is often a long process all trays will be analyzed every month for an indefinite period Acknowledgements I thank the Department of Energy’s Energy Research Undergraduate Laboratory Fellowships program for granting me the opportunity to partake in professional laboratory research Special thanks to my mentors Dr.’s Paul Ellis and Aina Cohen for their granting me their time and knowledge I would also like to thank Peter Kuhn, Mike Soltis, Tom Rabedeau, Scot McPhilips, Amanda Prado, and Tim McPhilips for their continual assistance In addition I would like to thank the entire Protein Crystallography Group for their help My coworker Amy Wu, was instrumental in the overall success of my experience LoVerde Works Cited and Bibliography Glusker, J., Trueblood, K (1985) Crystal Stucture Analysis Oxford University Press, NY Helliwell, J (1992) Macromoleculr Crystallography with Synchrotron Radiation Cambridge University Press Great Britain Lawrence Livermore National Laboratory Crystallography 101 http://www-structure.llnl.gov/Xray/101index.html Figures and Tables Figure Bragg’s Law Figure The Hanging Drop Method LoVerde LoVerde 10 Figure Drops observed on 31 July 2000, 50x ... crystals of sufficient quality for successful solution of the crystal structure Crystallization is often the most laborious step in the X-ray diffraction process Crystallization conditions for the GABAA. .. for use of beamline hardware and data collection software LoVerde Introduction The goal of this research is to obtain crystallization conditions for the GABA (aminobutyric acid) receptor Crystallization. .. and Bibliography Figures and Tables 8 9 LoVerde Abstract Crystallization of GABAA Receptor Marilena LoVerde (University of California at Berkeley, Berkeley, California 94703) Aina Cohen and Paul