1 / 37

Protein Methods

Protein Methods. Andy Howard Introductory Biochemistry Fall 2010, IIT. Proteins are worth studying. We’ll perform a quick overview of methods of studying proteins Purification methods Analytical methods Structural methods. The Protein Data Bank. http://www.rcsb.org/

alijah
Télécharger la présentation

Protein Methods

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Protein Methods Andy Howard Introductory BiochemistryFall 2010, IIT

  2. Proteins are worth studying • We’ll perform a quick overview of methods of studying proteins • Purification methods • Analytical methods • Structural methods Protein Methods and Function

  3. The Protein Data Bank • http://www.rcsb.org/ • This is an electronic repository for three-dimensional structural information of polypeptides and polynucleotides Protein Methods and Function

  4. What it contains • 68000 structures as of September 2010 • Most are determined by X-ray crystallography • Smaller number are high-field NMR structures • A few calculated structures, most of which are either close relatives of experimental structures or else they’re small, all-alpha-helical proteins Protein Methods and Function

  5. What you can do with the PDB • Display structures • Look up specific coordinates • Run clever software that compares and synthesizes the knowledge contained there • Use it as a source for determining additional structures Protein Methods and Function

  6. Protein Purification • Why do we purify proteins? • To get a basic idea of function we need to see a protein in isolation from its environment • That necessitates purification • An instance of reductionist science • Full characterization requires a knowledge of the protein’s action in context Protein Methods and Function

  7. Salting Out • Most proteins are less soluble in high salt than in low salt • In high salt, water molecules are too busy interacting with the primary solute (salt) to pay much attention to the secondary solute (protein) • Various proteins differ in the degree to which their solubility disappears as [salt] goes up • We can separate proteins by their differential solubility in high salt. Protein Methods and Function

  8. How to do it • Dissolve protein mixture in highly soluble salt like Li2SO4, (NH4)2SO4, NaCl • Increase [salt] until some proteins precipitate and others don’t • You may be able to recover both: • The supernatant (get rid of salt; move on) • The pellet (redissolve, desalt, move on) • Typical salt concentrations > 1M Protein Methods and Function

  9. Dialysis • Some plastics allow molecules to pass through if and only ifMW < Cutoff • Protein will stayinside bag, smaller proteins will leave • Non-protein impurities may leave too. Protein Methods and Function

  10. Gel-filtration chromatography • Pass a protein solution through a bead-containing medium at low pressure • Beads retard small molecules • Beads don’t retard bigger molecules • Can be used to separate proteins of significantly different sizes • Suitable for preparative work Protein Methods and Function

  11. Ion-exchange chromatography • Charged species affixed to column • Phosphonates (-) retard (+)charged proteins:Cation exchange • Quaternary ammonium salts (+) retard (-)charged proteins:Anion exchange • Separations facilitated by adjusting pH Protein Methods and Function

  12. Affinity chromatography • Stationary phase contains a species that has specific favorable interaction with the protein we want • DNA-binding protein specific to AGCATGCT: bind AGCATGCT to a column, and the protein we want will stick; every other protein falls through • Often used to purify antibodies by binding the antigen to the column Protein Methods and Function

  13. Metal-ion affinity chromatography • Immobilize a metal ion, e.g. Ni, to the column material • Proteins with affinity to that metal will stick • Wash them off afterward with a ligand with an even higher affinity • We can engineer proteins to contain the affinity tag:poly-histidine at N- or C-terminus Protein Methods and Function

  14. High-performance liquid chromatography • Many LC separations can happen faster and more effectively under high pressure • Works for small molecules • Protein application is routine too, both for analysis and purification • FPLC is a trademark, but it’s used generically Protein Methods and Function

  15. Electrophoresis • Separating analytes by charge by subjecting a mixture to a strong electric field • Gel electrophoresis: field applied to a semisolid matrix • Can be used for charge (directly) or size (indirectly) Protein Methods and Function

  16. SDS-PAGE • Sodium dodecyl sulfate: strong detergent, applied to protein • Charged species binds quantitatively • Denatures protein • Good because initial shape irrelevant • Bad because it’s no longer folded • Larger proteins move slower because they get tangled in the matrix • 1/Velocity  √MW Protein Methods and Function

  17. SDS PAGE illustrated Protein Methods and Function

  18. Isoelectric focusing I • Protein applied to gel without charged denaturant • Electric field set up over a pH gradient (typically pH 2 to 12) • Protein will travel until it reaches the pH where charge =0 (isoelectric point) Protein Methods and Function

  19. Isoelectric focusing II • Sensitive to single changes in charge (e.g. asp -> asn) • Can be readily used preparatively with samples that are already semi-pure Protein Methods and Function

  20. Ultraviolet spectroscopy • Tyr, trp absorb and fluoresce:abs ~ 280-274 nm; f = 348 (trp), 303nm (tyr) • Reliable enough to use for estimating protein concentration via Beer’s law • UV absorption peaks for cofactors in various states are well-understood • More relevant for identification of moieties than for structure determination • Quenching of fluorescence sometimes provides structural information Protein Methods and Function

  21. Warning: Specialty Content! • I determine protein structures (and develop methods for determining protein structures) as my own research focus • So it’s hard for me to avoid putting a lot of emphasis on this material • But today I’m allowed to do that, because it’s one of the stated topics of the day. Protein Methods and Function

  22. How do we determine structure? • We can distinguish between methods that require little prior knowledge (crystallography, NMR, ?CryoEM?)and methods that answer specific questions (XAFS, fiber, …) • This distinction isn’t entirely clear-cut Protein Methods and Function

  23. Crystallography: overview • Crystals are translationally ordered 3-D arrays of molecules • Conventional solids are usually crystals • Proteins have to be coerced into crystallizing • … but once they’re crystals, they behave like other crystals, mostly Protein Methods and Function

  24. How are protein crystals unusual? • Aqueous interactions required for crystal integrity: they disintegrate if dried • Bigger unit cells (~10nm, not 1nm) • Small # of unit cells and static disorder means they don’t scatter terribly well • So using them to determine 3D structures is feasible but difficult Protein Methods and Function

  25. Crystal structures: Fourier transforms of diffraction results • Experiment: • Grow crystal, expose it to X-ray • Record diffraction spots • Rotate through small angle and repeat ~180 times • Position of spots tells you size, shape of unit cell • Intensity tells you what the contents are • We’re using electromagnetic radiation, which behaves like a wave, exp(2ik•x) • Therefore intensity Ihkl = C*|Fhkl|2 Protein Methods and Function

  26. What are these Fhkl values? • Fhkl is a complex coefficient in the Fourier transform of the electron density in the unit cell:(r) = (1/V) hklFhkl exp(-2ih•r) • Critical point: any single diffraction spot contains information derived from all the atoms in the structure; and any atom contributes to all the diffraction spots Protein Methods and Function

  27. The phase problem Fhkl • Note that we saidIhkl = C*|Fhkl|2 • That means we can figure out|Fhkl| = (1/C)√Ihkl • We can’t figure out the direction of F:Fhkl = ahkl + ibhkl = |Fhkl|exp(ihkl) • This direction angle is called a phase angle • Because we can’t get it from Ihkl, we have a problem: it’s the phase problem! bhkl  ahkl Protein Methods and Function

  28. What can we learn? • Electron density map + sequence  we can determine the positions of all the non-H atoms in the protein—maybe! • Best resolution possible: Dmin =  / 2 • Often the crystal doesn’t diffract that well, so Dmin is larger—1.5Å, 2.5Å, worse • Dmin ~ 2.5Å tells us where backbone and most side-chain atoms are • Dmin ~ 1.2Å: all protein non-H atoms, most solvent, some disordered atoms; some H’s Protein Methods and Function

  29. What does this look like? • Takes some experience to interpret • Automated fitting programs work pretty well with Dmin < 2.1Å ATP binding to a protein of unknown function: S.H.Kim Protein Methods and Function

  30. How’s the field changing? • 1990: all structures done by professionals • Now: many biochemists and molecular biologists are launching their own structure projects as part of broader functional studies • Fearless prediction: by 2020: • crystallographers will be either technicians or methods developers • Most structures will be determined by cell biologists & molecular biologists Protein Methods and Function

  31. Macromolecular NMR • NMR is a mature field • Depends on resonant interaction between EM fields and unpaired nucleons (1H, 15N, 31S) • Raw data yield interatomic distances • Conventional spectra of proteins are too muddy to interpret • Multi-dimensional (2-4D) techniques:initial resonances coupled with additional ones Protein Methods and Function

  32. Typical protein 2-D spectrum • Challenge: identify whichH-H distance is responsible for a particular peak • Enormous amount of hypothesis testing required Prof. Mark Searle,University of Nottingham Protein Methods and Function

  33. Results of NMR studies • Often there’s a family of structures that satisfy the NMR data equally well • Can be portrayed as a series of threads tied down at unambiguous assignments • They portray the protein’s structure in solution • The ambiguities partly represent real molecular diversity; but they also represent atoms that area in truth well-defined, but the NMR data don’t provide the unambiguous assignment Protein Methods and Function

  34. Comparing NMR to X-ray • NMR family of structures often reflects real conformational heterogeneity • Nonetheless, it’s hard to visualize what’s happening at the active site at any instant • Hydrogens sometimes well-located in NMR;they’re often the least defined atoms in an X-ray structure • The NMR structure is obtained in solution! • Hard to make NMR work if MW > 35 kDa Protein Methods and Function

  35. What does it mean when NMR and X-ray structures differ? • Lattice forces may have tied down or moved surface amino acids in X-ray structure • NMR may have errors in it • X-ray may have errors in it (measurable) • X-ray structure often closer to true atomic resolution • X-ray structure has built-in reliability checks Protein Methods and Function

  36. Cryoelectron microscopy • Like X-ray crystallography,EM damages the samples • Samples analyzed < 100Ksurvive better • 2-D arrays of molecules • Spatial averaging to improve resolution • Discerning details ~ 4Å resolution • Can be used with crystallography Protein Methods and Function

  37. Circular dichroism • Proteins in solution can rotate polarized light • Amount of rotation varies with  • Effect depends on interaction with secondary structure elements, esp.  • Presence of characteristic  patterns in presence of other stuff enables estimate of helical content Protein Methods and Function

More Related