Lecture 11Protein and Peptide ChemistryMargaret A. DaughertyFall 2003BIOC 205Purification: Step 1• Cells: Break them open!Big Problem: Crude extract is not the “natural” environmentIf you break open a cell, proteins are exposed to proteases, otherproteins that “specialize” in protein degradation.Crude ExtractTotal contents of cellBIOC 205Factors in Stabilizing ProteinsDuring Purification•pH– Requires the use of a buffer to maintain the pH in aphysiological range (~7.0) where the protein will not denature(affects titratable amino acids)• Temperature– Thermal stability of proteins varies. Many proteins irreversiblydenature (lose 3o structure) at higher temperatures. Affectsweak interactions. Therefore, most purifications are carriedout at between 0-4ûC.• Degradative enzymes– Proteases chew up proteins. Nucleases chew up nucleic acids.These can be “controlled” by pH, temperature and inhibitors.(Temperature is a factor - proteases less active at 4C)• Adsorption to surfaces– Fewer steps the better! The air-liquid interface is a prime spotfor denaturation.BIOC 205PROTEINS HAVE AN OPTIMAL pH RANGE FOR THE NATIVE STATEPart of TCA cycle - convertsmalate to fumarate; found inmitochondriaNative;folded;activeDenatured;unfolded;inactivePurification: Step 2getting rid of “cellular debris”Separate “cellular debris” (membranes, insolubleproteins, organelles….) from soluble molecules (whichhopefully includes your protein).Crude extract -----> contains your protein of interest+Other Proteins, Nucleic acids,Polysaccharides, Lipids , Cellular membranes,Organelles...,BIOC 205CENTRIFUGATIONSeparation method that involves applying a centrifugalforce to a particle. Separation based on mass of particle.F = mω2rm = mass of objectr = distance from center of rotationω = rotor speed (radians/sec)Two objects of differing mass willexperience different centrifugal forcesat the same rotor speedWe can usehigh speedsto get ridof cellulardebrisBIOC 205How do we know where ourprotein is? protein assay- Spectroscopic– Functional Assay• Enzyme activity• DNA binding• Substrate binding– Immunoassay• ElisaMost important piece of any purification protocol!BIOC 205Purification: Solubility• Protein solubility --> complex function of pH,temperature, salt concentration, composition, etc.;• Differences in solubility of proteins often used asan initial purification step.• Ammonium Sulfate precipitationBIOC 205An example:Increasing (NH4)2SO4In general, lower solubility at high salt concentrations.“Salting out” of proteins. Specific control of theconcentration can selectively precipitate some andleave others in solution.Ammonium Sulfate PrecipitationPrinciples of Column ChromatographyPorous resin(solid phase) withcertain chemicalpropertiesResevoirwith buffer(mobilephase) ofinterestEffluent: whatcomes out of thecolumnProtein appliedin mobile phaseProteinsmigratedifferentiallydependingupon (1).theirpropertiesand (2) thatof the resin.BIOC 205Size Exclusion ChromatographyBIGsmallBIOC 205Ion-exchange Chromatography++++Resins used: polyanionic: used to separate positively charged molecules(e.g., cation-exchange chromatography!) separates molecules based on charge of moleculespolycationic: used to separate negatively charged molecules (e.g., anion-exchange chromatography!)BIOC 205+12345678910Ion-exchange Chromatography+Resin: anionic resinpasses throughSaltgradientLoading bufferLow saltStep 1: low salt bufferStart salt gradientStep 2: NaCl gradientelutesExample: three proteins of different charge++++ elutesthen+Why this order?BIOC 205Protein Purification1). Lyse cells2). Separate out cellular debrisCentrifugation3). Purify based on Size: Size exclusion chromatographySolubility: Ammonium Sulfate PrecipitationCharge: Ion exchange chromatographyBinding ability: Affinity chromatography4). Follow purificationSpectroscopic Assays: UV/Vis spectroscopyFunctional Assays: Based on biological function Immunoassays: ELISABIOC 205PROTEIN CHARACTERIZATIONWhat is the amino acid content?What is the amino acid sequence?What is the molecular weight?What is its pI?Does the protein self-associate (have quaternary structure?)If so, what is association stoichiometry?(monomer-dimer, monomer-trimer….)What is the association equilibrium constant?What is the structure of the molecule? NMR & crystallographyBIOC 205Electrophoresis:analytical method to characterize proteins• Separates components of a mixturebased upon their charge and/or size– Paper electrophoresis– Polyacrylamide gel electrophoresis(PAGE)– SDS-PAGE– Isoelectric focusing (IEF)BIOC 205PAGE: polyacrylamide gel elecrophoresis• Cross-linked acrylamide gels act as amolecular sieve• An electric field is used to separatethe proteins;• Migration ∝ charge-to-mass ratio• Electrophoretic mobility:• Small molecules >> large molecules(with the same charge density)• pH of the buffer and protein mixtureis high (~9) so that the proteinscarry a net-negative charge;• Molecules of similar size and chargemove through the gel as a band.BIOC 205Visualization• Most frequently,Coomasie Brilliant Blueor Silver stain is used;• These stains bind to theproteins, not the gel.• Can use this to monitor proteinpurification - # of bands shoulddecrease with each purificationstep.BIOC 205Step 1LysateStep 2Step 3Step4SDS both denatures & binds the proteinDetermining Molecular WeightsSDS-PAGE• Disulfides are reducedwith β-mercaptoethanol;• All proteins assume arod like shape with auniform charge/massratio;Identificationof MW ofan unknownNet result - protein is negatively chargedBIOC 2052. Separation by cation-exchange chromatography;--detection with fluorophores (10-18 M; attamolar!)Amino Acid Analysisacidic basicFirst step on any newly discovered protein1. Hydrolyze peptide bonds; HCl/110C/24hrs harsh: trp destroyed/ser,thr,tyr often degraded asn & gln hydrolyzed to asp & glu --> glx/asx content3. Quantitation of amino acids; areas under peaks proportional to concentrationBIOC 205Protein Sequencing• Once you have obtained a PUREprotein, it can be sequenced;• Intact proteins may have blockedN-termini, disulfide bonds, etcmaking characterization difficult;• “Divide and conquer” strategy isnormally employed-- (1) fragmentthe protein into peptides and do(2) amino acid analysis.BIOC 205Protein Fragmentation(Hint: know these!)• Chemical– Cyanogen bromide (CNBr): cleaves after Met•