Biocrystallography course: from gene to drug
8 - 11 September 2003
Trieste, Italy

Introduction:

The four-day course "Biocrystallography course: from gene to drug", organized by the Centre of Excellence in Biocrystallography (CEB) of the University of Trieste and sponsored by the European Science Foundation (Programme on Integrated Approaches for Functional Genomics) and by the University of Trieste (Consorzio per lo Sviluppo Internazionale dell'Università di Trieste and Centre of Excellence in Biocrystallography), was held on 8-11 September 2003 in the Lecture Hall B3 of the H3 building of the University of Trieste.

The course was organized to discuss on issues related to protein expression (1 day), X-ray structure determination (2 days) and in silico analysis (1 day). The course has covered the main methodologies essential to follow the whole process that transform the gene information into knowledge of functional properties of the protein through its three-dimensional atomic structure. In particular, the course has dealt with methods for over-expression of proteins in prokaryotic and eukaryotic systems, purification and crystallization techniques, X-ray diffraction and strategies for solving the crystal structure, new biological knowledge derived by structural biology, computational methods in structural genomic, molecular modelling and computer-aided design.

This "Biocrystallography course: from gene to drug" has fitted very well with the ESF programme on integrated approaches for functional genomics, programme area "Structural genomics-protein structure determination, classification, modelling and docking".

Protein crystallography is a highly interdisciplinary field and this course has permitted to people from many different backgrounds (students of biochemistry, chemistry, biology, biophysics and crystallographers) to become more familiar with molecular biology and protein biochemistry as well as crystallography, structural biology and computational methods. The course was intended for students in the early stages of their research career. In order to offer as many applicants as possible the opportunity to attend the Course, the Organizing Committee (Prof. Silvano Geremia, Prof. Cynthia Ebert, Dr. Gianluca Tell) has increased from 40 to 60 the maximum number of students. The course has involved 61 students and 23 speakers coming from several European countries. The possibility to join together teachers of different fields and students with a variety of backgrounds in the same place for four days has advance collaboration and research in the interdisciplinary field of Structural Biology. As supporting material for the course, a book of about 200 pages, with the printed copy of the slides (six frames per page) projected during the lessons, was given to attendants. The printed copy of slides has considerably helped the students to follow the lessons. Furthermore, the students have easily integrated this material during the lecture course. The final programme, a summary of the discussed topics, the statistical information on participants and the list of participants are reported below.

Report

1st day: large-scale protein production and purification for structural biology

This part of the course was aimed at giving a general overview of several aspects of large-scale protein production and purification, starting from the different host cell systems currently used to overexpress recombinant proteins to modern techniques used for biochemical purification and quality control.

High-throughput protein expression and purification plays a pivotal role in structural genomics. In fact, the production of protein, suitable for crystallographic studies, on the scale required to generate tens to hundreds of different proteins per day, is one of the greatest obstacles for the conversion of protein structure determination to a high-throughput format. These requirements are today addressed by employing different heterologous 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) mass spectrometry can be used for purity assessment. Dynamic light scattering measurements can be used to verify sample dispersity and degree of aggregation.

The first talk entitled "Bacterial expression and E. coli as an expression host", by Dr. Gunter Stier from EMBL Heidelberg, was dedicated to give some detailed aspects in the problematic of recombinant protein expression in the most used E. coli system. One hour was dedicated to describe advantages and disadvantages of using Yeast and Baculovirus as expressing hosts by Dr. Sergey Fedorov from Aarhus University, Denmark in the lecture entitled 'Foreign gene expression in yeast and Baculovirus". Dr. Stefano Marchetti, from the University of Udine, gave some interesting suggestions in a developing expression system such as Plants in his lecture entitled "The plant expression system". In the afternoon, Dr. Antonio Leonardi, from the University of Naples, in his lecture entitled "Stable Gene expression in mammalian cell lines" spoke about the problems that make the mammalian cell system unfruitful for large-scale protein expression but useful only for functional studies on proteins of interest. The last part of the day was dedicated to face the problem of biochemical purification by gel chromatography, two hours by Prof. Jan-Christer Janson from the University of Uppsala in his talk entitled "Protein purification I and II", and quality control by means of Mass Spectrometry analysis by Dr. Andrea Scaloni from the Italian National Research Council, Naples.

During all their lessons, speakers gave the opportunity to the students to expose questions, problems and doubts.

2nd day: crystallization of the protein, crystallographic data collection and structure determination

This part of the course has been organized with the aim of giving a theoretical and applied overview of several aspects of protein X-ray crystallography, starting from the crystallization techniques and diffraction data collection to structure solution techniques.
One of the most important factors limiting the rate at which protein structures are determined by X-ray crystallography is the difficulty to obtain high-quality crystals. Two hours have been dedicated to this bottleneck for protein structure determination. In the first lesson "Crystallization of biological macromolecules", Prof. Adriana Zagari (University of Naples "Federico II") has presented the main properties of protein crystals, some theoretical aspect of protein crystallization and the principal methods for protein crystallization. In the second lesson "Crystal growth and crystal improvement strategies" held by Dr. Naomi Chayen (Imperial College, London) several new very interesting approaches to aid protein crystallization have been presented.

The main aspects of a diffraction experiment have been illustrated by Dr. Alberto Cassetta (Istituto di Cristallografia, CNR, Trieste) on his talk "X-ray sources and data diffraction from protein crystals". The diffraction theory, methods for solving the phase problem, and structure refinement were presented by Prof. Louise Johnson (University of Oxford) in a two hours lesson "Fundamentals of macromolecule structure determination, part I and II". The discussion on the other bottleneck for protein structure determination, the phase problem, has clearly evidenced that biocrystallography is a highly interdisciplinary field. Heavy atoms derivatives are essential to solve a structure by MAD or SAD methods and for example, to make selenomethionine containing protein, special techniques of molecular biology are necessary. On the other hand, a good starting model of the structure is necessary for phasing a structure by the Molecular Replacement method. Database searching thought alignment techniques and Molecular Modelling are essential for this purpose.

The day dedicated to X-ray protein crystallography was concluded with a visit to the X-ray diffraction beam line at the Elettra Synchrotron. The speakers got a chance to speak to many people and answer questions during the tour to the synchrotron and in the restaurant in the evening dinner.

3rd day: new biological knowledge derived by structural biology

The goal of structural biology is the understanding of the structure-function relationship of biological molecules. The X-ray diffraction from protein crystals is the principal technique for determination of very large structures to atomic resolution. The capability of this technique for the elucidation of the structure of biopolymers and their complexes is now very high (ribosome and viruses) and it has an immediate fall out on knowledge of biological systems. This part of the course has been organized with the aim of giving an overview of several aspects of the structural biology, through a collection of selected arguments.

Prof. Louise Johnson (University of Oxford) in her introductive lesson, "Overview on structural biology" has presented an historical excursus and the state of art of structural biology. Dr. Andrea Musacchio (European Institute of Oncology, Milan) in "Structural biology of cell cycle regulatory protein" has told about the spindle checkpoint proteins, MAD and their role in the mitosis process. Dr. Kristina Djinovic (Elettra Synchrotron, Trieste) in "Structural biology of cytoskeleton" has presented the structural results obtained for some domains of the -actinin , the actin binding domain (ABD) and the -actinin central repeats. Dr. Doriano Lamba (Istituto di Cristallografia, CNR, Trieste) in "Structural studies of proteins of medical and biotechnological interest" has presented three examples of proteins: i) A third-generation thrombolytic drug, a hybrid plasminogen activator (K2tu-PA), Amediplase; ii) An adhesion molecule involved in muscular dystrophies, dystroglycan domains; iii) A microbial hemicellulase, Acetyl-Xylan Esterase. Prof. Stefano Mangani (University of Siena) in "Advantages of 3rd and 4th generation synchrotron light in the structure determination of metallo-proteins" has presented the characteristics of the 3rd generation synchrotron light and several examples of metalloprotein structures determined using these sources: E. Coli and rat CutA1, aspecific bacterial phosphatase from E. coli, AphA enzyme, the metallo-membrane proteasi MMP10 and MMP1. Dr. Massimo Degano (S. Raffaele Scientific Institute, Milan) in "Structural Basis of T cell-mediated immune response" has presented the structural basis for T cell recognition of antigens. In particular, he has illustrated the structures of Class I or Class II MHC bound to peptide antigens and the structure of CD1, a molecule that bind lipid and glycolipid antigens, and present them to T cells for recognition via the TCR. Prof. Hugo L. Monaco (University of Verona) in "The liver basic Fatty Acid-Binding Protein" has told the long and interesting story of the "basic" Fatty Acid-Binding Protein.

The day was concluded with an open discussion on presented subjects and discussion continued also at the social dinner.

4th day: computational methods in structural biology

This part of the course presented the opportunities offered by "in silico" methods, with particular attention to the construction of 3D models of proteins (or protein aggregate) of interest for pharmaceutical field and to the screening of ligands for specific targets.

The computer-aided drug design is based on a detailed understanding of the relationship between the drug and its target, generally a protein. Prof. Laszlo Patthy (Hungarian Academy of Sciences, Budapest) illustrated the rationale for the choice of the proteic target and how the architecture of the protein is conserved in the different organisms. The molecular simulation and structure prediction requires an effort in the organization of the knowledge, and dr. Federico Fogolari (University of Verona) described the feature of the principal molecular biology databases, and how to extract the maximum of the information from these sources. Molecular modelling techniques allows: a) to compare protein- to - protein; b) to predict the structures of proteins; c) to suggest a mechanism for the interaction with ligand and other protein in the cells. The computational chemistry offer reliable models for the study of the function of the protein, and prof. Rebecca Wade (European Molecular Biology Laboratory, Heidelberg) in her lesson highlighted the problems that a researcher face to obtain the best output. Prof. Gabriele Cruciani (University of Perugia) showed how the computational methods allow the drug development according the requirement of the protein target. With this approach it is possible the screening of very large data set, according with different requirement of synthesis, bioavailability, and pharmacokinetic. Ab initio molecular dynamics (MD) allows realistic simulations of biological systems without adjustable parameters. Prof. Paolo Carloni (Sissa - International School for Advanced Studies, Trieste) described the principles on which ab initio MD is based. By a survey of recent applications, he provided a perspective for the advancement of methodological approaches in biomolecular modelling. In the study of the function of protein the metalloproteins represent an open question because the properties of a single cofactor are tuned by the structure. Prof. Angela Lombardi (University "Federico II" of Napoli) described how to design and realize minimal proteins that emulate the native biomolecule. With this approach it is possible the have a deeper insight in the principles of protein folding and stabilization. The opportunities offered by combinatorial chemistry in drug development were illustrated by prof. Stanislav Miertus (International Centre for Science and High Technology - ICS- Unido, Trieste). This approach generate a multitude of chemically related molecules and test "in silico" large number of compound (libraries) in the shortest possible time. This "Virtual Screening" is fast and cheap compared to "wet chemistry".