MDC Connects: Proteins, structures and how to get them
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This document discusses protein production and structure determination. It covers expressing recombinant proteins in common systems like E. coli, insect, and mammalian cells. Purification methods like affinity and size-exclusion chromatography are described. Quality control steps involving SDS-PAGE, mass spectrometry, and functional assays are outlined. Protein crystallography and X-ray crystal structure determination are summarized, including crystallization screening, diffraction data collection, phasing methods, and model building and refinement. The importance of protein structures for drug design is also briefly mentioned.
MDC Connects: Proteins, structures and how to get them
- 1. Proteins, structures and how to get them.
- 2. Proteins, structures and how to get them. • Protein production • Protein structure determination by X-ray crystallography A very brief introduction to:
- 3. Proteins as drug targets • Proteins are the targets of the vast majority of marketed drugs. • Target-based drug development therefore requires an good supply of high quality, purified proteins for: Assay development High throughput screening Biophysical analysis eg SPR, ITC Structure determination eg X-ray, NMR, Cryo-EM • Proteins themselves are also used as important biological therapeutics eg, mAbs, vaccines, TNF
- 4. Protein production: Expression systems • Historically proteins were obtained from natural sources eg. animal organs, blood, plants. • Proteins today are generated recombinantly. • Synthetic & codon optimised genes for the target protein cloned into a vector or plasmid & transfected into cultured cells for overexpression. • Common expression cell systems: E.coli eg. BL21(DE3) Baculovirus/insect cells eg. Sf9 or Sf21 Mammalian cells eg. HEK 293 • Depending on needs and yield grow volumes can vary from 10’s ml to 1000’s litres.
- 5. Protein production: Construct design • Protein construct design is often the critical step in generating proteins as required. • Optimisation of protein constructs involves careful choice of: What form of the protein is required? eg. full-length, catalytic/functional domain, protein in complex, PTM’s (phosphorylation, glycosoylation) required for function? Which expression system vectors to use? eg. E.coli, insect, mammalian, yeast. Affinity tags/fusion proteins +/- protease cleavage sites, to i) enable ease of purification eg. 6His, GST, stepavidin. ii) improve solubility eg. MBP, SUMO iii) enable assay eg. Avi-tag + biotioylation Reduction of structural heterogeneity & disorder – important for X-ray crystallography eg. N & C-term truncations, deletion of mobile domains or loops.
- 6. Protein production: Purification • Purification is carried out mostly by liquid chromatography methods based on the physical properties of the proteins. • Affinity chromatography – separation based on affinity of protein itself or to fused tags eg. His6, GST, MBP, FLAG (tags often removed subsequently by protease treatment) • Ion-exchange chromatography – separation based on protein charge. • Size-exclusion chromatography – separation based on molecular size. • Purification of membrane proteins (eg GPCR’s) can be very difficult due to their hydrophobicity and their instability out of membrane environment. Requires careful use of lipids & detergents and often stabilising mutations.
- 7. Protein production: Quality Control • SDS gels – gives indication of purity & approx. molecular size of denatured protein. • A280 – gives good estimate of protein concentration. • Analytical size-exclusion chromatography - molecular size of protein or complex in solution. • Mass Spectrometry (MS): i) Intact MS – accurate molecular mass + PTM’s ii) Peptide-mass fingerprinting – confirmation of protein ID. • Functional assay – Is the protein catalytically active?
- 8. Protein structure: Reasons for protein structure • Once a purified protein is available we may want to determine it’s structure – X-ray crystallography, Cryo-EM, NMR. • Protein structures themselves can inform on function. • High-resolution structures (≤ 2.5 Å) of protein- ligand complexes enables structure-based ligand: explain SAR, aid chemistry design. • Protein structures are essential for successfully progressing fragment-based ligand-design (FBLG) projects.
- 9. Protein crystallography: X-rays 1nm 10nm 100nm 1mm 10mm 0.1 mm atomic protein bonds proteins cell crystals limit of light microscope (0.4mm) wavelength of X-rays (0.15nm = 1.5Å) N O H • X-ray crystallography is an experimental method that exploits the fact that X-rays can be diffracted by the periodic assembly of atoms or molecules in a crystal. • X-rays have the appropriate wavelength (1-2 Ång) to be scattered by the electron clouds of atoms of comparable size. • By recording the X-ray diffraction patterns the electron density within the crystal can be reconstructed mathematically.
- 10. Protein crystallography : Synchrotron X-ray sources Diamond Light Source i04 beamline
- 11. Protein crystallography : Crystallisation • There is no way to predict the conditions that will crystallise a protein from it’s amino acid sequence. • Crystallisation screening : Empirical process of incubating large numbers of cocktails of precipitants, buffer and salts with the protein of interest to try and find conditions that produce crystals that diffract well. • Usually carried out robotically in 96-well plates using volumes of few 100nl per well. • Common methods: vapour diffusion, microbatch and microdialysis. “Think of it as finding a recipe where we have no idea what ingredients we need are”
- 12. Protein crystallography : Crystals
- 13. Protein crystallography : Diffraction expt. • Generally the only information obtained from a diffraction image is i) the position & ii) the intensity of the diffraction maxima ie. spots or reflections. • This is not sufficient information to reconstruct the electron density within the crystal. • Also requires phase angle of each reflection – but this is not directly observed in diffraction images. • This is the so called “Phase problem”.
- 14. Protein crystallography: Phases The necessary phase info can be obtained in a number of ways: • Molecular Replacement (MR): Use structure of similar/homologous proteins (>30% sequence ID): • Multiple Isomorphous Replacement (MIR). Soak atoms of heavy metals into crystals– eg. U, Hg, Au salts etc: • Multiple Anomolous Dispersion (MAD). Replace Met residues with selenoMet when expressing the protein & tune X-ray wavelength to selenium absoption edge. • Sulphur SAD: Rely on native sulphur atom anomolous signal.
- 15. Protein crystallography : Electron density to model Calculate 3D electron density map Build atomic model into electron density Refine model data against experimental data w Fo -k Fcå / FoåR-factor = R-free: cross validation value
- 16. Protein crystallography : Validation/QC Quality of Model : • Stereochemistry ie. bond lengths, angles – rms deviations • Main chain dihedral angles f & y: Ramachandran plots – number of violations • Side chain rotamers – no. of deviations from preferred conformations. • Planarity of aromatic rings and peptide groups – rms • MolProbity clash scores – number of close non-bonding contacts/1000 Quality of Experimental X-ray Data: • Resolution (<3.0Ang) • Completeness (>90%) • Redundancy (>5x) • Signal-to-noise (I/sI > 2) • Merging statistics eg. Rmeas, Rpim (≤10%), CC1/2
- 17. End
- 18. Protein production: Construct design strategy • Often best strategy is to work in parallel with as many constructs as practicable or affordable. • Constructs can initially be assessed quickly at small scale (~150ml) by expression level– good surrogate of protein behavior. • Once purified can score by catalytic/functional activity or by protein stability (thermal shift assay). • If required can use results to refine the constructs in subsequent rounds of design. • Repeat until successful!