Annual 2005 - Hauptman Woodward Institute - University at Buffalo

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Pioneers of Science High School Educational Outreach: Western New York has a long, proud and rich history of producing leaders in many branches of science. To salute some of these individuals and to focus local and national attention on the high caliber of scientific work that is thriving here, Hauptman-Woodward hosts our Pioneers of Science Conference. The event is made up of two separate programs: one, a morning lecture and series of workshops for area high school science students and the second, a dinner and awards presentation gala. The objective of the conference is to inspire high school students through the achievements and accomplishments of leading Western New York scientists. They have the opportunity to meet in small workshop sessions led by each of the honored scientists. The students also will learn of current and future research and career opportunities. The target audience for this conference is science-inclined high school students currently attending Erie County public and private schools. Approximately 200 students participate in this program. A New Public Science-Math High School: HWI is participating in the development of a new high school that will be part of the Buffalo Public School system where the concentration for the students will be focused primarily on the sciences and mathematics. The goal is for the school to open in the fall of 2006. Overall, HWI is strategically moving into the next 50 years. We are in a new home, growing our HWI family with new scientists, technicians and support staff and we are continually expanding the boundaries into new arenas of science. The Science at the Hauptman-Woodward Medical Research Institute Structural biology is the study of the shapes of molecules, such as proteins, that exist in the body. Each protein has a distinct shape and function and is very often involved in one or more diseases. Knowing the threedimensional structure of these proteins enables scientists to understand how they work and can allow them to develop new medications to treat disease. At Hauptman-Woodward, our scientists specialize in structural biology and the use of x-ray crystallography. What is the Process? The structural biology process is often complicated and time consuming, but each step is critical to understanding disease and to developing new ways to treat, prevent, and possibly cure them. There are six distinct steps that take place in the structural biology process at HWI. 1. Protein production and purification: In this stage, genes implicated in diseases are identified, isolated, cloned, and introduced into protein expression systems. Finally, these proteins are over-expressed in various systems to determine the highest level of protein production, and purified using high-pressure liquid chromatography to achieve greater than 99 percent purity. Breast cancer drug target 2. Crystal growth: Once a purified protein is obtained, crystals are produced, using high-throughput crystallization, a technique created by our scientists that performs 1536 experiments at a time using a robotic system, and which records the results in a database for future access. 3. 4. X-ray diffraction: After crystals of adequate diffraction quality are obtained, they are mounted on a diffractometer or brought to a synchrotron facility where a unique diffraction pattern is produced. Analysis of diffraction data: A computer analysis of the diffraction data is performed that allows the location of atoms in the molecules to be determined. An electron density map is then created and viewed, using state-of-the-art computer graphics. Methods developed by Dr. Hauptman are used at this stage. AIDS-related research 5. Analysis of molecular structure and function: Scientists are then able to view the three-dimensional structure that has been determined to understand how structure guides biological function. Pioneer of Science honoree Edith Flanigen explains her research to local high school students at the Pioneers of Science student symposium. Structure-Based Drug Design: By studying the structure and function of molecules we can design new medications that are more selective and effective in the fight against diseases. The AIDS drugs that have proved to be so successful in extending patients' lives were designed using the three-dimensional Protein essential for cell proliferation and growth structure of the AIDS protease. 12 13 6.
What Are The Tools Used to Get the Job Done? There are a wealth of laboratory resources available to HWI scientists and graduate students. Among them are: The Molecular Biology Lab - Important disease-related genes are identified and isolated. The gene that codes for the protein of interest is cloned and introduced into an appropriate cell line. The molecular biology laboratory is the starting point for most structural biology projects. These projects involve the study of protein molecules, most of which are present in the body only in minute quantities. The instructions for making proteins are found within the genes, the hereditary material that is composed of deoxyribonucleic acid (DNA), contained in the chromosomes present in each cell, and passed from one generation to the next. The main objective of the molecular biology lab is to manipulate the DNA that codes for a protein of interest in ways that make it possible to produce the large quantities of protein required for structural studies. The Protein Production & Purification Lab – Cells are cultured under conditions that support large-scale production (expression) of the desired protein. The cells are then disrupted, and techniques such as electrophoresis and column chromatography are used to separate the target protein from other (contaminating) proteins on the basis of properties such as charge and size. Many molecular biologists and biochemists can use nanogram quantities of protein for their experiments. In order to carry out successful structure determinations, however, crystallographers require a million times more protein. HWI’s protein production and purification laboratory has been designed for the purpose of producing proteins on a 1-100 mg scale and to achieve 99 percent purity for crystallization and subsequent structural studies. The Diffraction Lab – Single crystals are exposed to X-rays to create a diffraction pattern that is recorded electronically by an area detector. In most cases, diffraction data are also measured using the high intensity X-ray beams available from synchrotron radiation sources at Cornell University and the Brookhaven and Argonne National Laboratories. The Crystal Growth Lab – Here the purified protein sample is combined with crystallization solutions to grow high quality single crystals. Automated, high-throughput screening techniques are applied to micro samples in order to identify suitable growth conditions. The high-throughput crystallization laboratory at HWI houses state-of-the-art facilities for screening conditions suitable to grow the high-quality single crystals required for molecular structure determination by X-ray diffraction. As currently configured, the lab has the capacity to evaluate as many as 200 new samples each month. A large number of experiments (1536) can be set up within minutes after a protein sample is received, thereby reducing the chance for degradation. The approach used for screening growth conditions is to incubate, under paraffin oil, small aqueous aliquots of the sample protein with chemical mixtures (called ‘cocktails’) that are designed to induce a state of supersaturation. The protein molecules will then be driven out of solution and, under proper conditions, crystals will form. The high-throughput experiments are set up using Robbins Scientific Tango pipetting robots. Two custom-made photomicrographic reader tables, that can accommodate as many as 28 crystallization plates each, are used to document the results with a digital camera. Digital images are recorded and the plates are stored in temperature-controlled incubators. The crystallization lab also contains all the equipment needed to optimize crystal size and quality once the initial conditions have been found. The instruments available for preparing and characterizing macromolecular solutions and crystallization cocktails include pH meters, electronic balances, centrifuges, refractometers, a spectrophotometer, a viscometer, and an osmometer. Two DynaPro temperature-controlled dynamic light scattering instruments are available to make measurements of solution homogeneity. These measurements are useful for predicting the likelihood that crystals will form from a solution. The Computational Lab – The diffraction data are analyzed to find the atomic positions using software including the program SnB written at HWI. With the help of three-dimensional computer graphics, electronic models of the protein structure are then constructed. Finally, color graphics are used to depict the overall molecular architecture, as well as the locations of chemically and biologically important regions. The crystal growth lab uses high-throughput screening techniques to identify suitable growth conditions. Eric Drake, research associate, works on one of the many steps involved in protein purification. Angela Lauricella, research associate, is pictured (bottom right) working in the crystal growth lab. 14 15
Page 2 and 3: President’s Message The Human Fro
Page 4 and 5: HWI Focuses on Crystallography A ne
Page 6 and 7: Future Growth Requires Physical Gro
Page 10 and 11: What is Being Done in Research and
Page 12 and 13: HAUPTMAN-WOODWARD MEDICAL RESEARCH
Page 14 and 15: Recent Publications • Cody, V., L

Annual 2005 - Hauptman Woodward Institute - University at Buffalo

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