PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Main Types of Molecular Interactions Hydrogen Bonds N H - - - - N N-H + N low temperature high temperature N H - - - - O C strength is very dependent on geometry donor acceptor and distance (2.6-3.1 A) Hydrophobic Interactions (waxy residues: Ileu, Leu, Val, Phe, Trp) high salt high temperature low salt Ionic Interactions (charged residues:Asp- Glu- S- Lys+ Arg+ His+) low ionic strength high ionic strengthSlide 8Protein Properties - Handles for FractionationSlide 10Protein Properties-Handles for FractionationSeparation Processes that can be Used to Fractionate ProteinsSlide 13Protein Inactivation/Stabilization Buffers Solution ComponentsSlide 15Slide 16Slide 17What You Can Learn from Amino Acid Sequence 1. Molecular weight of the polypeptide chain 2. Charge versus pH; Isoelectric point 3. Extinction coefficient 4. Hydrophobicity & membrane spanning regions 5. Potential modification sites 6. Conserved motifs that suggest cofactor affinity What You Can’t Learn from Amino Acid Sequence 1. Function 2. 3-Dimensional structure; Shape 3. Multi-subunit features 4. Ammonium sulfate precipitation properties 5. Surface features (hydrophobic patches, charge distribution, antigenic sites) Conclusion: Protein Purification is still very empirical!Slide 19Slide 20Protein Purification(from a lecture by Dr. Richard Burgess, University of Wisconsin, Madison, at the CSH protein purification course).Object: to separate a particular protein from all other proteins and cell componentsThere are many proteins (over 4300 genes in E. coli)A given protein can be 0.001-20% of total proteinOther components: nucleic acids, carbohydrates, lipids, small moleculesEnzymes are found in different states and locations: soluble, insoluble, membrane bound, DNA bound, in organelles, cytoplasmic, periplasmic, nuclearStudy Question You are given a shoe box full of an assortment of small objects including:Ping Pong ballsSugar cubesPaper clips1/2” brass screwsIron filings1. List the properties of each of these components that might help you fractionate them.2. Devise the most efficient method you can for getting pure paper clips.20 Naturally-occurring Amino AcidsAcidic: D, E, (C, Y)Basic: K, R, HHydrophobic: I, L, V, W, FPolar: S, T, N, QOther: G, A, M, POverview of Protein PurificationTypes of SeparationsProtein PropertiesProtein Inactivation/StabilizationProtein Analysis and PurificationAnalytical SeparationsGel-electrophoresisIEF2D-gelsPreparative SeparationsVarious chromatographic methodsTotal E. coli Proteins - 2-Dimensional GelMain Types of Molecular Interactions Hydrogen BondsN H - - - - N N-H + N low temperature high temperature N H - - - - O C strength is very dependent on geometry donor acceptor and distance (2.6-3.1 A) Hydrophobic Interactions (waxy residues: Ileu, Leu, Val, Phe, Trp) high salt high temperature low salt Ionic Interactions (charged residues:Asp- Glu- S- Lys+ Arg+ His+) low ionic strength high ionic strength-+ - +Cl- Na+...H2OHHHHH HHHVariables that Affect Molecular ForcesTemperatureIonic strengthIon typePolarity of solvent (dielectric constant)pHProtein Properties - Handles for FractionationSize (110 Da/amino acid residue) smallest most proteins largestAmino acids: 30 100 1,000 15,000MW (kDa): 3.3 11 110 1,600 Multi-subunit complexes can contain 5-30 subunitsShape globular (sphere) asymmetric (cigar)Effects frictional properties, effective radius, movement through poresCentrifuge Gel filtrationSediments slowerAppears smallerElutes earlierAppears largerProtein Properties - Handles for FractionationNet chargeIonizable group pKa pH2 pH7 pH12C-terminal (COOH) 4.0 oooooooo----------------------------------------Aspartate (COOH) 4.5 oooooooooo-------------------------------------Glutamate (COOH) 4.6 ooooooooooo------------------------------------Histidine (imidazole) 6.2 +++++++++++++ooooooooooooooooooooN-terminal (amino) 7.3 +++++++++++++++ooooooooooooooooooCysteine (SH) 9.3 ooooooooooooooooooooooo-----------------Tyrosine (phenol) 10.1 oooooooooooooooooooooooooo-------------Lysine (amino) 10.4 ++++++++++++++++++++++++ooooooooArginine (guanido) 12.0 ++++++++++++++++++++++++++++++oIsoelectric pointpI = pH where protein has zero net chargeTypical range of pI = 4-9Charge distribution ++++----uniform++++----clusteredversusProtein Properties-Handles for FractionationHydrophobicity Hydrophobic residues usually are buried internally The number and distribution on the surface vary Can use Hydrophobic Interaction ChromatographySolubility Varies from barely soluble (<g/ml) to very soluble (>300 mg/ml) Varies with pH, ionic strength/type, polarity of solvent, temperature Least soluble at isoelectric point where there is least charge repulsionLigand and metal binding Affinity for cofactors, substrates, effector molecules, metals, DNA When ligand is immobilized on a bead, you have an affinity bead HHHhydrophobic patchSeparation Processes that can be Used to Fractionate Proteins Separation Process Basis of SeparationPrecipitation ammonium sulfate solubilitypolyethyleneimine (PEI) charge, sizeisoelectric solubility, pIChromatography gel filtration (SEC) size, shapeion exchange (IEX) charge, charge distribution hydrophobic interaction(HIC) hydrophobicityDNA affinity DNA binding siteimmunoaffinity (IAC) specific epitopechromatofocusing pIElectrophoresis gel electrophoresis (PAGE) charge, size, shapeisoelectric focusing (IEF) pICentrifugation sucrose gradient size shape, densityUltrafiltration ultrafiltration (UF) size, shapeTypical Protein Purification SchemeProtein Inactivation/StabilizationBuffers Solution ComponentsProtein Sources for PurificationTraditional natural sources Bacteria, animal and plant tissueCloning recombinant proteins into overexpression vector/host systems for intracellular production (E. coli the most used)In vitro protein synthesis Transcription/translation systemsTotal E. coli Proteins - 2-Dimensional GelDetermining the protein sequence from gel(proteomics)What You Can Learn from Amino Acid Sequence 1. Molecular weight of the polypeptide chain 2. Charge versus pH; Isoelectric point 3. Extinction coefficient 4. Hydrophobicity & membrane spanning regions 5. Potential modification