Lecture 5Outline of Last Lecture1. Review of Peptides2. Properties of the Peptide Bond3. Secondary Structure of Proteins4. Tertiary Structure of Proteins5. Quaternary Structure of Proteins6. The Biological Protein Folding ProblemOutline of Current Lecture1. Protein Structure Review2. Protein PurificationCurrent Lecture1. Protein Structure ReviewPrimary Structure (amino cid sequence  Secondary structure (α-Helix)  Tertiary Structure (polypeptide chain)  Quaternary Structure (assembled subunits)Important advances that established the modern era of biochemistry:1953 – Watson & Crick deduced DNA structure implicit in their proposal was the idea that the sequence of nucleotide units genetically encoded information for proteins in DNA duplex1953 – Sanger determind first complete amino acid sequence for any protein (Insulin)1957 – Anfinsen proposed that primary protein sequence dictated its 3D native structure1959 – Perutz & Kendrew determined first high resolution structures of proteins – myoglobin and hemoglobin1970 – Smith discovered restriction enzymes – ushering in modern molecular biology1980 – Roche invented and marketed practical H-s-affinity tagsPrimary structural analysis – what amino acids could be importantExamples of Modified Residues:Phosphorylation – a common reversible protein modificationSerine (S), threonine (T), tyrosine (Y)(Large negative charge, large electrostatic potential)Lysine (K) – methylation, acetylation, ubiquitylation (finished protein—time for degradation), sumoylation (used to signal degradation)Mono, di, triSerine (S), Threonine(T), Asparagine (N) – Glycosylation (sugar)Myristoylation – addition of fatty acid by amid linkage on N-terminus. Irreversible (FA are hydrophobic) important in targeting proteins to membranesPalmitoylation – C-terminus and cysteines – addition of fatty acid. Reversible modification, aids in trafficking proteins to appropriate vesicles in organelles. Also targets proteins to membranesAA sequence motifs can be signatures for functionExamples:Signal peptide- hydrophobic resides near N-termins may target protein for export from cell or to intracellular membrane locationNuclear localization signal (NLS)- sequence that targets transport of euk proteins from the cytosol into nucleus (PKKKRKV)Binding motifs – TEV protease – binds to peptide (ENLYFQG)Sequence homologsCompare with other protein sequencesProteins with similar sequences are likely to have similar 3D structures and may have similar functions (at least in part)Is the protein a member of some est. family?Compare with other protein sequencesProteins with similar sequences are likely to have similar 3D structures and may have similar functionsDetermine molecular mechanisms of some disease – sickle cell condition results from change in a single hemoglobin AAE6VRead as Glu  ValChange at position 6With normal Hb, erythrocytes discs shaped and flexible at all pressures of oxygenE6V—stiff and irregular at low pressures of oxygen, and are fragile, blood Hb content low  anemiaPredict disease/sequence of a protein/function of a proteinLook for homolog protein in multiple states, compare the states to look at motions within the protein, compare to your structure and begin to understand your protein and how it worksMap the location of residues that are mutated in the diseased state on the surface of your protein structure. Does it explain the disease state?2. Protein PurificationProteins can be separated from each other and purified individually but most protein molecules are marginally stable, so proteins must be handled very gentlyExploit differences in proteins based on:Solubility in salts or solventsNet molecular chargeSurface regions of +/- chargeOverall size and shapeHydrophobic interactionsSpecific interactions with ligandsProtein Purification: Prepare a starting extract – usually purification requires soluble crude extractChoose a buffersaltspHadditives (substrate, product, ATP, protease, inhibitors, etc.)thiol compounds (maintain reduced Cys or break disulfides)Inhibits oxidation of proteinsHomogenization – breaks the cells openDifferential centrifugationFractionation based on physical or biochemical propertiesRelatively fast and effectivea. Precipitation methodsAmmonium sulfatePolyethyleneimineb. Chromatographic methodsion exchange chromatographyPack a column with beadsSoluble crude extract goes inMany protein fractions come outI.E.C. behavior depends on the net charge on a protein (surface exposed)The pI is the pH at which the net charge on the protein is 0.Whether to use a cation exchange or anion exchange depends on what the pI isSee slides for exampleSize exclusionAffinity purification – including tagsHydrophobic interactionsc. Centrifugationequilibrium densityvelocity sedimentationd. ElectrophoresisNative and denaturing PAGIsoelectric focusingFPLC Chromatography – fast protein liquid chromatographyGel Filtration (permeation) chromatographyDepends on size and shape – NOT chargeLarger proteins can only fit around the beadsSmaller proteins can fit inside all of the holes inside the beadsConsider rod-shaped vs globularProtein mixture is added to column containing cross-linked polymerAffinity Chromatography – most selective of common purification strategiesTaking advantages of specific ligand binding characteristicsSpecific interactions with functional groups immobilized on beads.Protein of interest is eluted by ligand solutionAntibody based affinity chromatography – attach antibody to solid support, 1 protein is reagent, change pH, strip off antibody complexes, his-6 tag (imidazole) coordinate metal (Ni2+)Adds an affinity – predicts the behaviorSDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)SDS –artificial fatty acid – sulfuric acid derivationHydrophobic characterBinds avidly and consistently to most proteins at fairly consistent ratioEstimate the size of a protein relative to proteins of known sizeEach polypeptide runs as a discrete band, detect by non-specific stain to see nearly any protein; complex proteins fall apart into their separate polypeptidesNative gel electrophoresisEstimate native Mr but remember it may not be accurate charge, size, and shape influence migration (as for gel filtration)Useful mostly for looking at protein:protein interactionsSedimentation analysisLarge particles sediment more rapidly than small particles, assuming same shape and same fractional coefficientSpherical