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Biocrystallography course: from gene to drug 8 - 11 September 2003. Trieste, Italy
The course will be held in the H3 building of the Trieste University, Lecture Hall B3, Via A.Valerio (Comprensorio Universitario di Piazzale Europa), Trieste
Monday 8 Sept. : Large-scale protein production for structural biology.
High-throughput protein expression and purification plays a pivotal role in structural genomics. In fact, crystallographic-quality protein production, on the scale required to generate tens to hundreds of different proteins per day, will probably be the greatest obstacles for the conversion of protein structure determination to a high-throughput format. High-throughput efforts in structural biology place unique restrictions on protein expression and purification. These requirements are today addressed by employing different eterologous expression systems (E. coli, Yeast, Baculovirus, Plants) and either large or small N-terminal expression tags and by the use of particular affinity purification tags. Protein crystal growth requires stringent protein purity either in terms of homogeneity and conformation, lacking denatured species and other structural microheterogeneities that adversely affect crystal growth. To this purpose, prior to the performance of crystallization trials, the purity and homogeneity of protein samples must be confirmed. SDS-PAGE analyses and matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometry can be used for purity assessment with dynamic light scattering measurements used to verify sample dispersity and the degree of aggregation. Systematic studies will lay the groundwork for the assembly of comprehensive information databases that can then be used to refine the procedures necessary for efficient genome-scale protein expression and purification efforts. This part of the course will be organized in a day with the aim of giving a theoretical and applied overview of the most common recombinant protein expression systems (E. coli, Baculovirus, Yeast, Plants and Mammalians).
8:15- 8:45 - Registration
8:45- 8:55 - Welcome Address and Opening Remarks
8:55- 9:00 - Introduction to 1st day: Large-scale protein production for structural biology (Gianluca Tell)
9:00-10:00 - Bacterial expression and E. coli as an expression host (Gunter Stier)
10:00-10:30 - Coffee break
10:30-11:30 - Foreign gene expression in Yeast and Baculovirus (Sergey Fedosov)
11:30-12:30 - The Plant expression system (Stefano Marchetti)
12:30-14:00 - Lunch
14:00-15:00 - Stable Gene expression in mammalian cell lines (Antonio Leonardi)
15:00-16:00 - Protein purification I (Jan-Christer Janson)
16:00-16:30 - Coffee break
16:30-17:30 - Protein purification II (Jan-Christer Janson)
17:30-18:30 - Quality assessment on protein samples by Mass Spectrometry analysis (Andrea Scaloni)
18:30-19:00 - Open discussion
19.00 - Welcome party
Tuesday 9 Sept. : Crystallization of the protein and crystallographic data collection.
Since the pioneering work on the structure on heme-proteins by the Nobel Laureates M. Perutz and J. Kendrew, X-ray crystallography has become a very important technique for understanding protein properties at the molecular level. The capability of this technique for the elucidation of the structure of biopolymers and their complexes is now very large as shown by the recent X-ray structural determination of the 30S and 50S ribosomal sub-units at atomic resolution. The accuracy of protein structure determination by synchrotron radiation is some time similar to that achieved for small molecules, and fine details can be discerned without bond distance restraints. Intense, tuneable synchrotron X-ray beams, cryo-crystallography and sensitive area detectors have all contributed to this tremendous advancement in technology. Hence, individual protein structures can be studied at atomic level in multiple forms via multiple binding of inhibitors and substrate analogues: single and double bond distances can be resolved and confirmed by the location of hydrogen atoms. The interactions of enzymes with substrates and inhibitors have notable applications in the fields of drug discovery and pollution control. Methods are continuously being developed to analyse new protein structure and their relationships to those already existing However, one of the most important factors limiting the rate at which protein structures are determined by X-ray crystallography is the difficulty of obtaining high-quality crystals. This part of the course will be organized with the aim of giving a theoretical and applied overview of several aspects of X-ray crystallography starting from the crystallization techniques and diffraction data collection to structure solution techniques. A visit to the X-ray diffraction beam line at the Elettra Synchrotron is planned.
8:55- 9:00 - Introduction to 2nd day: Crystallization of the protein and crystallographic data collection (Silvano Geremia)
9:00-10:00 - Crystallization of biological macromolecules (Adriana Zagari)
10:00-10:30 - Coffee break
10:30-11:30 - Crystal growth and crystal improvement strategies (Naomi Chayen)
11:30-12:30 - X-ray sources and data diffraction from protein crystals (Alberto Cassetta)
12:30-14:00 - Lunch
14:00-15:00 - Fundamentals of macromolecule structure determination I (Louise Johnson)
15:00-16:00 - Fundamentals of macromolecule structure determination II (Louise Johnson)
16:00-16:30 - Open discussion
16:30-19:00 - Visit to X-ray diffraction beam line at the Elettra Synchrotron
19.00 - Osmizza tour
Wednesday 10 Sept. : New biological knowledge derived by structural biology
It is well know that the knowledge of gene information cannot be directly converted into the knowledge of the actually working molecular machine (protein). As a result, after the genomics era, a new area of research has appeared which is called "proteomics". The "proteomics" is a science dealing with cataloguing of proteins based on a combination of several methods: 2D- electrophoresis, mass-spectrometric analysis of molecular mass, structural characterization etc. and subsequent analysis of the results obtained by use of bioinformatical methods. Biocrystallography has a key role in this scenario. Furthermore, the structure–function relationship of biological macromolecules must be studied at the molecular level in order to gain a full understanding of biological processes. X-ray diffraction from protein crystals is the principal source of data for determination of large structures to atomic resolution. The recent successes of structure determination for a number of different biological systems indicate the versatility and potential for biocrystallography above and beyond static structure determination. This part of the course will be organized with the aim of giving an overview of several aspects of the structural biology
8:55- 9:00 - Introduction to 3rd day: New biological knowledge derived by structural biology (Silvano Geremia)
9:00-10:00 - Overview of structural biology (Louise Johnson)
10:00-10:30 - Coffee break
10:30-11:30 - Structural biology of cell cycle regulatory protein (Andrea Musacchio)
11:30-12:30 - Structural biology of cytoskeleton (Kristina Djinovic)
12:30-14:00 - Lunch
14:00-15:00 - Structural studies of proteins of medical and biotechnological interest (Doriano Lamba)
15:00-16:00 - Advantages of 3rd and 4th generation synchrotron light in the structure determination of metallo-proteins (Stefano Mangani)
16:00-16:30 - Coffee break
16:30-17:30 - Structural Basis of T cell-mediated immune response (Massimo Degano)
17:30-18:30 - The liver "basic" Fatty Acid-Binding Proteins (Hugo L. Monaco)
18:30-19:00 - Open discussion
19.00 - Social dinner
Thursday 11 Sept. : Computational methods
The recently published draft sequences of the complete human genome, contains an estimated 26,000 - 38,000 genes encoding a comparable number of proteins. To date, only a small fraction of known proteins, and even fewer of the number hypothesized based on genome sequencing data, have had their structures solved by crystallographic methods or by nuclear magnetic resonance (NMR) spectroscopy. These methods cannot keep pace with the demand to know the structures of all proteins, especially those postulated to be involved in medical maladies. The effort to map the structural features of proteins, and the structural basis of protein-protein, protein-ligand, and protein-drug interactions requires the development and enhancement of software capable of carrying out molecular modelling, structure visualization, structure prediction, and protein folding and energetic calculations. Bioinformatics is a new area, comprising the analysis of biological sequence information, recovery of evolutionary patterns, prediction of gene function and ‘silicon- based biology’. The amount of information in the bioinformatics databases requires sophisticated tools to deal with it. Predictive methods allow researchers to understand the possible structure and function of proteins whose existence is only hypothesized based on genomic sequence data, and which are not even known to exist otherwise. Molecular modelling is still an art form that cannot be applied well outside of specialized research groups. The successes, in particular molecular simulation and structure prediction, have dramatically increased. Nevertheless, the technology developed by the computational biology community should be known by experimental biomedical researchers. With the development of user-friendly interfaces and documentation, a growing number of biological scientists can capitalize on the wealth of structure analytical functions provided by molecular modelling tools. Homology modelling facilitates the creation of the hypothetical structure for an unknown protein using as a template another protein that shares many of the sequence features and whose structure is known. Docking involves tools to model both protein-protein and protein-ligand interactions. Finally, The new drug development is now based on a thorough understanding of the relationship between the drug and its target protein, whether it is an enzyme, receptor, structural, or transport protein. Detailed information regarding the physical, chemical, biochemical, genetic, and physiological characteristics of that relationship must be obtained. Molecular modeling serves several functions in research today: it provides a mechanism to visualize structure in three dimensions; allows researchers to analyze elements of structure and make protein- to - protein comparisons; predicts the structures of proteins; and facilitates computer-aided drug design (CADD). Combinatorial chemistry and high-throughput screening offer new opportunities in drug design. The use of computational tools increases the effectiveness of these methods. Combinatorial chemistry generates a multitude of chemically related compounds, derived from a common scaffold substituent variation, so-called “combinatorial libraries”. The high-throughput screening (HTS) means testing large number of compound (libraries) in the shortest possible time and is routinely used to identify novel drug molecules in most pharmaceutical companies. Computational tools are required because more compounds are potentially available than can be screened cost-effectively Virtual screening is the computational equivalent of biological testing and can be used to select compound for screening and to design combinatorial libraries. Virtual screening is fast and cheap compared to “wet chemistry. Docking generate 3D coordinates for compound in library; find best orientation within protein active site and score and rank compound according to predicted binding affinity
8:55- 9:00 - Introduction to 4th day: Computational methods (Cynthia Ebert)
9:00-10:00 - Drug target selection and drug development in the post-genome era (Laszlo Patthy)
10:00-10:30 - Coffee break
10:30-11:30- Molecular biology databases, database searching and alignment techniques (Federico Fogolari)
11:30-12:30- Methods in drug design (Gabriele Cruciani)
12:30-14:00 - Lunch
14:00-15:00- Molecular dynamics simulations of biological systems (Paolo Carloni)
15:00-16:00- Molecular modelling of proteins (Rebecca Wade)
16:00-16:30 - Coffee break
16:30-17:30- Artificial Metalloproteins: from Structure to Function (Angela Lombardi)
17:30-18:30- Computer Assisted Combinatorial Chemistry (Stanislav Miertus)
18:30-19:00 - Open discussion
19.00 - Closer course
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