Slide 1OutlineSlide 3Restriction AnalysisInformation from Gel ElectrophoresisSlide 6Clicker QuestionSlide 8Southern BlotsSlide 10Slide 11Southern BlotAn L-Amino AcidThe Peptide BondSlide 15The Peptide Bond is RigidHydrophobic Side ChainsHydrophilic Side ChainsAcidic Side ChainsBasic Side GroupsAromatic Side ChainsSlide 22Disulfide BondsForces Affecting Protein StructureSlide 25Slide 26Peptide Bonds Rotate Freely Around the a-CarbonSecondary structureThe Alpha-HelixThe Alpha HelixSlide 31Clicker QuestionThe b Strand and b Pleated SheetSlide 34Slide 35Secondary StructuresProtein SizeSlide 38Slide 39Slide 40Methods for Determining Protein Structure - NMRA Little More DNAProtein StructureMCB 250 - Lecture 6Outline•Gel Electrophoresis•The peptide bond•Amino acids – Primary structure•Secondary structurea–helixb-sheet•Methods for determining protein structure–X-ray crystallography–NMR•Examples of protein structuresFigure 7-3Restriction AnalysisApaIXbaIKpnIXhoIMarker4833251710The “Marker” lane contains DNA fragments of known size allowing the sizes of the unknown fragments to be determined.Information from Gel Electrophoresis•Size–Distance migrated is inversely proportional to the logMW (or log(# basepairs).•Quantity–Binding of ethidium is directly proportional to MASS of DNA1011 Molecules x 10 Kb= 1012 Kb= ~100 ng2x1011 Molecules x 5 Kb= 1012 Kb= ~100 ng1012 Molecules x 1 Kb= 1012 Kb= ~100 ngQuantitation of DNA by Ethidium Bromide Staining10 Kb5 Kb1 KbMarker DNA -Known sizeand quantityThe “size” of the box is meant to reflect the “brightness” of the band.Clicker Question•You have purified your favorite plasmid from a strain of E. coli. You run an agarose gel with a standard of known concentration to quantitate the DNA. But what about protein contamination? •Does the presence of contaminating protein interfere with this procedure?A.YesB.NoThe Density of the Matrix Defines the Resolution of the Gel Percent AgaroseEthidium bromide stains all DNA fragments. How can we identify a particular DNA fragment in a complex mixture of fragments?Imagine that the E. coli chromosome (4,600 kb) has been digested with Eco RI. How many fragments do you expect? 4,600kb/4kb = 1150. Is it possible to identify a fragment that carries one particular gene?Southern blotting provides a method for doing exactly this.Southern BlotsTransfer DNAto membrane under denaturing conditionsAdd labeled piece of DNA and hybridizeDigest sample DNAwith restriction enzymesElectrophoreseSouthern BlotInvented by E. SouthernOxford UniversityEcoRI EcoRIProbexAGGCATTAG5’-3’TTTAACGCG5’-3’CCGGCTAAC5’-3’Labeled “probe” hybridizesto complementarysequenceTCCGTAATC***3’--5’TCCGTAATC***3’--5’After hybridization to the radioactive probe, the membrane is placed on a piece of photographic film. Radiation from the labeled probe will expose the film (“radioautography”). (Asterisks indicate probe is radioactively labeled.)Ethidium bromide stain - lots of fragments visibleAfter hybridization to a specific probe and radioautography - only the fragment containing the sequence complementary to the probe is visibleFig 7-6Southern BlotAn L-Amino AcidFig 6-1The Peptide BondFig 6-3Note that charges remain on N and C terminiConvention: proteins are designated N terminal  C terminal, i.e., the N-terminus is on the left.DipeptideA 'polypeptide' is the product of a single gene, a linear chain of amino acids linked together by peptide bonds.But in order for a polypeptide to become a mature functional protein, it must fold in 3-dimensions and adopt a specific conformation. Peptide bondsThe Peptide Bond is RigidHydrophobic Side ChainsHydrophilic Side ChainspKa 9 (-SH  -S-)Acidic Side ChainspKa 3.9 4.3Basic Side GroupspKa 10.4 12 6.2H+Aromatic Side ChainspKa 10 (-OH  -O-)Amino Acid Side Groups have Multiple Properties, All of Which Affect Protein Folding and PropertiesSmallTinyPositivePolarChargedNon-polarAromaticAliphaticDon’t memorize this – just think about it and appreciate it.Disulfide BondsHSCH2HSCH22H+SCH2SCH2+2 CysteineSidechainsTwo Cysteines joined by disulfide is calledCystineCan be spontaneous but interconversion is more likelyto be enzyme mediated.Can be intramolecular or intermolecularForces Affecting Protein StructureVan der WaalsBonds Hydrophobic ClusterStacked RingsNegatively ChargedAmino AcidsIonic BondsH BondsDisulfideBondSSLevels of Protein Structure and NomenclatureLarge Proteins Often Fold into DomainsDomainPeptide Bonds Rotate Freely Around the a-CarbonHowever, some conformations are energetically unfavorable.Secondary structureInvolves hydrogen bonding between -C=O and -NH groups that are part of the peptide backbone.Amino acid #1Amino acid #2Hydrogen bond acceptorHydrogen bond donorThe Alpha-Helix•Held together by H bonds•3.6 amino acid residues per turn•H bonds between the backbone C=O of residue n and the NH of residue n + 4–All backbone H bond donors and acceptors are joined in H bonds except the first 4 NH and the last 4 C=OAn amino acid in a protein is called a residue since it has lost water and is no longer an amino acid.3.6 Residues5.4 ALike Fig 6-8The Alpha HelixR GroupsFace Out123456789~3.5 AAs H H O H H O H H O H H O H H O H H O H H O H H O -N-C-C-N-C-C-N-C-C-N-C-C-N-C-C-N-C-C-N-C-C-N-C-C- R R R R R R R R 1 2 3 4 5 6 7 8~12 AThe properties of the helix are defined by the amino acid side chains. Example– an amphipathic helix.A “Coiled-Coil”Clicker Question•The alpha helix is the most stable secondary structure known. Are all amino acid sequences equally likely to form an alpha helix?A.YesB. NoClueProline"MALEK“ are favoredP is a breakerG is disfavored NOT a rule, just a tendencyThe b Strand and b Pleated Sheetb-Pleated SheetAntiparallelParallelRRR~7 A (2 residues)By convention the arrows point N-term to C-term- Note that Fig 6-8 in Watson is wrongNote that secondary structures are connected by loops. The loops may be as small as 2 amino acids or larger as in the structures on the previous slides.Secondary Structures•Compact and stable structures•Easy to pack together•All backbone H bonding donors and acceptors are satisfied so that if the side chains of that protein segment are hydrophobic then the protein segment is totally hydrophobic. • Hence, a helix