Protein Methods; Protein Functions

Protein Methods;Protein Functions Andy HowardBiochemistry Lectures, Fall 2010 8 September 2010 Proteins: methods and functions

Visualization of structures (concl’d) The Protein Data Bank Protein Purification Salting Out Chromatography Electrophoresis Structural Methods X-ray crystallography Macromolecular NMR Other techniques Protein function Structure-function relationships PTM Zymogens and Post-translational modification Allostery Specific protein functions Distributions Plans for Today Proteins: methods and functions

Mostly helical:RecG - DNA Mixed: lysozyme Ribbon diagrams Proteins: methods and functions

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

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 Proteins: methods and functions

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 Proteins: methods and functions

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. Proteins: methods and functions

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 Proteins: methods and functions

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. Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

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) Proteins: methods and functions

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 Proteins: methods and functions

SDS PAGE illustrated Proteins: methods and functions

Isoelectric focusing • 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 pH where charge =0 (isoelectric point) • Sensitive to single changes in charge (e.g. asp  asn) • Readily used preparatively with samples that are already semi-pure Proteins: methods and functions

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 Proteins: methods and functions

Structure Methods! . . .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. Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

How are protein crystals unusual? • Aqueous interactions required for crystal integrity: they disintegrate if dried • Bigger unit cells (~10nm, not 1nm) • Intermolecular forces are weak ionic forces • 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 Proteins: methods and functions

Crystal structures: Fourier transforms of diffraction results • Positions of spots tells you how big the unit cell is • Intensities tells you what the contents are • We’re using electromagnetic radiation, which behaves like a wave,exp(2ik•x) = cos2k•x + isin2k•x • Therefore intensity Ihkl = C*|Fhkl|2 • 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) Proteins: methods and functions

How do Fourier sums work? • This is a lesson not only for crystallography, but rather for any instance where Fourier math applies: • All the atoms in the unit cell contribute to the intensity of any one diffraction spot • All the diffraction spots contain information about any particular atom • So don’t fall into the trap of thinking that any one spot is associated with any one atom Proteins: methods and functions

The phase problem Fhkl bhkl  ahkl • Note that we saidIhkl = C*|Fhkl|2 • That means we can figure out|Fhkl| = (1/C)√Ihkl • But we can’t figure out the direction ofF: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! Proteins: methods and functions

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 • H positions can be inferred, especially if you are able to get high-resolution data (see next slide) • Atomic mobility can estimated for intermediate to high resolution data Proteins: methods and functions

Limitations of resolution • 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; even some hydrogens Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

Results • 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 Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

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 Proteins: methods and functions

Sperm whale myoglobinPDB 2jho1.4Å16.9 kDa Poll question: discuss! • Which protein would yield a more interpretable CD spectrum? • (a) myoglobin • (b) Fab fragment of immunoglobulin G • (c) both would be fully interpretable • (d) CD wouldn’t tell us anything about either protein Anti-fluorescein FabPDB 1flr1.85 Å52 KDa Proteins: methods and functions

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 Proteins: methods and functions

Solution scattering • Proteins in solution scatter X-rays in characteristic, spherically-averaged ways • Low-resolution structural information available • Does not require crystals • Until ~ 2000 you needed high [protein] • Thanks to BioCAT, SAXS on dilute proteins is becoming more feasible • Hypothesis-based analysis Proteins: methods and functions

Fiber Diffraction • Some proteins, like many DNA molecules, possess approximate fibrous order(2-D ordering) • Produce characteristic fiber diffraction patterns • Collagen, muscle proteins, filamentous viruses Proteins: methods and functions

X-ray spectroscopy • All atoms absorb UV or X-rays at characteristic wavelengths • Higher Z means higher energy, lower for a particular edge • Perturbation of absorption spectra at E = Epeak +  yields neighbor information • Changes just below the peak yield oxidation-state information • X-ray relevant for metals, Se, I Proteins: methods and functions

Mass spectrometry as a structural tool • MS tells you molecular weights • Can give high precision in m/m • Not, strictly speaking, a way of determining structure • Can distinguish oligomeric state • Coupled with proteolytic digestion, it can be used to find interesting fragmentation patterns Proteins: methods and functions

Protein Function: Generalities • Proteins do a lot of different things. Why? • Well, they’re coded for by the ribosomal factories • … But that just backs us up to the question of why the ribosomal mechanism codes for proteins and not something else! Proteins: methods and functions

Proteins are chemically nimble • The chemistry of proteins is flexible • Protein side chains can participate in many interesting reactions • Even main-chain atoms can play roles in certain circumstances. • Wide range of hydrophobicity available (from highly water-hating to highly water-loving) within and around proteins gives them versatility that a more unambiguously hydrophilic species (like RNA) or a distinctly hydrophobic species (like a triglyceride) would not be able to acquire. Proteins: methods and functions

What proteins can do: I • Proteins can act as catalysts, transporters, scaffolds, signals, or fuel in watery or greasy environments, and can move back and forth between hydrophilic and hydrophobic situations. Proteins: methods and functions

What proteins can do: II • Furthermore, proteins can operate either in solution, where their locations are undefined within a cell, or anchored to a membrane. • Membrane binding keeps them in place. • Function may occur within membrane or in an aqueous medium adjacent to the membrane Proteins: methods and functions

What proteins can do: III • Proteins can readily bind organic, metallic, or organometallic ligands called cofactors. These extend the functionality of proteins well beyond the chemical nimbleness that polypeptides by themselves can accomplish • We’ll study these cofactors in detail in chapter 7 Proteins: methods and functions

Structure-function relationships I Proteins with known function:structure can tell is how it does its job • Example: yeast alcohol dehydrogenase • Catalyzesethanol + NAD+ acetaldehyde + NADH + H+ • We can say something general about the protein and the reaction it catalyzes without knowing anything about its structure • But a structural understanding should help us elucidate its catalytic mechanism Proteins: methods and functions

Why this example? • Structures of ADH from several eukaryotic and prokaryotic organisms already known • Yeast ADH is clearly important and heavily studied, but until 2006 there was no high-resolution structure of it! • We got crystals 11 years ago, but so far I haven’t been able to determine the structure PDB 2HCYSaccharomyces alcohol dehydrogenase 2.44Å37.4kDa monomerdimer shown Proteins: methods and functions

Protein Methods; Protein Functions