Stepping into the world of proteins

1. Stepping into the world of proteins INTRODUCTION TO STRUCTURAL BIOLOGY AND X-RAY CRYSTALLOGRAPHY Montserrat Fàbrega Ferrer Miquel Coll’s lab – Structural biology of protein & nucleic acid complexes and molecular machines

2. Outline A. Structural biology 1. Definition 2. Proteins and its structure 3. Maintechniques B. X-ray crystallography 1. General scheme of work 2. From the gene to the protein 3. From the protein to the crystal 4. From the crystal to the structure C. Biomedical applications: structure-based drug design D. Practical session

3. A. Structural biology

4. Definition of structural biology • WHAT? • Structural biology is a branch of molecular biology, biochemistry and biophysics that studies: • The molecular structure of biological macromolecules (proteins and nucleic acids) • The acquisition of the structures • Structure and function relationships • WHY? • Macromolecules are only able to carry out most of the their functions when they are coiled into specific three-dimensional shapes.

5. Definition of structural biology • HOW? • Experimental techniques • Bioinformatic predictions

6. Proteins • Proteins are large biological molecules consisting of one or more chains of amino acids. • Proteins perform many different functions within living organisms: • Metabolic reactions • Replication of DNA • Response to stimuli • Transport of molecules • Maintenance of cell’s shape • Cell signaling • Immune responses • Cell adhesion • Cell cycle

7. Protein structure: primary structure

8. Protein structure: secondary structure RANDOM COIL Unfolded polypeptide chain lacking any fixed three-dimensional structure.

9. Protein structure: tertiary structure Myoglobin

10. Protein structure: quaternary structure Hemoglobin

11. Basic vocabulary about protein structure STRUCTURAL DOMAIN Self-stabilizing element that folds independently. STRUCTURAL MOTIF Short segment of protein three-dimensional structure found in a large number of different proteins.

12. Basic vocabulary about protein structure SUPERSECONDARY STRUCTURE Specific combination of secondary structure elements. PROTEIN FOLD General protein architecture.

13. Techniques Indirect methods No atomic data Proteolysis assays Spectroscopic methods Atomic data

14. Techniques Spectroscopic methods X-ray crystallography 3·1016 – 3·1019 Hz Electrons Scattering Nuclear magnetic resonance (NMR) 6·104 – 1·106 Hz Atomic nucleus Absorbance and re-emission ofelectromagnetic radiation

15. B. X-ray crystallography

16. General scheme of work Gene Protein Structure Crystals

17. From the gene to the protein Gene Protein HETEROLOGOUS EXPRESSION Express a protein codified by a certain gene in a host organism which does not naturally have this gene Laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms RECOMBINANT DNA TECHNOLOGY Escherichia coli

18. From the gene to the protein Escherichia coli • Gram-negative, rod-shaped bacterium • Most widely studied prokaryotic organism • Can grow easily and inexpensively in a laboratory setting • Host organism for the majority of work with recombinant DNA

19. From the gene to the protein Cloning the gene Polymerase chain reaction (PCR) CLONING = Making several copies!

20. From the gene to the protein • Polymerase chain reaction (PCR) • We need: • DNA from the interest organism • Thermo resistant DNA polymerase • Magnesium (cofactor) • Primers • Deoxyribonucleotides (dNTPs) • Thermociclator http://www.youtube.com/watch?v=HMC7c2T8fVk

21. From the gene to the protein What happens in one cycle of PCR reaction?

22. From the gene to the protein Progression of the PCR reaction

23. From the gene to the protein Cloning the gene Expression vector: Plasmid or virus designed for protein expression in cells. The vector is used to introduce a specific gene into a target cell and take advantage of the cell’s mechanism for protein synthesis to produce the protein encoded by the gene. Your gene: codifies for the interest protein Promoter: drives expression in the host organism Ori: allows replication of the vector Antibiotic resistance gene: avoids contaminations and the loss of the plasmid

24. From the gene to the protein Transforming Escherichia coli Transformation: Genetic alteration of a cell resulting from the direct uptake of exogenous DNA from its surroundings.

25. From the gene to the protein Producing the protein Big culture Small preculture Colony Protein production is induced with IPTG (isopropyl β-D-1-thiogalactopyranoside) Antibiotic present in all the steps to avoid contaminations!

26. From the gene to the protein Purifying the protein Centrifuge at high speed to separate proteins from the other cellular components (membranes and DNA) Soluble protein Chromatography purification steps to separate the interest protein from the others High concentration Homogeneous and stable Considerable amount (mg) Pure protein

27. From the protein to the crystal Gene Protein Structure Crystals

28. From the protein to the crystal Crystallization principles INCREASE IN THE PROTEIN CONCENTRATION Vapor diffusion REDUCING THE PROTEIN SOLUBILITY Salt type and concentration Temperature pH Organic additives or polymeric precipitants Starting protein concentration

29. From the protein to the crystal Main crystallization techniques: vapor diffusion technique Hanging drop Sitting drop

30. From the protein to the crystal How to find the correct condition? 96 well plates Sitting drops (0.1 μL + 0.1 μL) TRYING LOTS OF CONDITIONS!! High-throughput screening

31. From the protein to the crystal Reproduction of the conditions at larger scale and optimization of the crystals 24 well plates Hanging or sitting drops (1 μL + 1 μL)

32. From the crystal to the structure Gene Protein Structure Crystals

33. From the crystal to the structure Fishing and freezing the crystals

34. From the crystal to the structure X-ray source X-rays Crystal Diffraction pattern Structure Data processing

35. C. Biomedical application: structure-based drug design

36. Drug discovery DRUG DISCOVERY: Process by which new candidate medications are discovered. RATIONAL DRUG DESIGN: Process of finding new medications based on the knowledge of a biological target.

37. Rational drug design LIGAND-BASED DRUG DESIGN: The structural details of the target are not known. Based on information about a know subset of ligands. STRUCTURE-BASED DRUG DESIGN: The target structure is known. The main technique used in this method is molecular docking. PDB file with the structure of the target protein X-ray crystallography NMR The first success: a peptide-based HIV protease inhibitor (1990)

38. D. Practical session

39. Experiment 1. Production of protein Small preculture Big culture • 1. Follow the growth of the cells • 2. Induce expression

40. Experiment 1. Production of protein 3. Collect and break the cells 4. Analyze expression and solubility of the protein (SDS-PAGE)

41. Experiment 2. Crystallization techniques Starting with a sample of pure protein and a known condition of crystallization • Hanging drops • Sitting drops 24 well plates

42. Thank you! Miquel Coll’s lab Structural biology of protein & nucleic acid complexes and molecular machines