Slide 1Slide 2The Alpha HelixThe b Strand and b Pleated SheetSlide 5Slide 6Secondary StructuresProtein SizeSlide 9Slide 10Slide 11Methods for Determining Protein Structure - NMRSlide 13Slide 14Slide 15Slide 16Structural MotifsGCN4 – A “Leucine Zipper” or “Coiled Coil”Slide 19Tropomyosin – A Coiled-Coil Protein in MuscleSlide 21Slide 22i434 Repressor – A Helix-Turn-Helix DNA Binding ProteinMembrane ProteinsK+ Channel – A Helical Transmembrane ProteinPorin - A b Barrel Transmembrane ProteinSlide 27The Folding ProblemThe Folding ProblemProtein FoldingSlide 32General Concepts in Protein FoldingGeneral Concepts in Protein FoldingSlide 35More General Concepts for Protein FoldingSometimes Proteins Don’t Fold ProperlyChaperonesSlide 39Slide 40GroE Helps Proteins FoldPeptide Bonds in Proteins are Almost Always TransCis-Trans Peptide bondsSlide 44Proteins may be posttranslationally modifiedSlide 46Reminder: EXAM 1 is one week from today.•The exam will cover material presented up to and including protein purification. (Should be in Lecture 8 on Friday, Sept. 13.)•The material starting at DNA supercoiling (should be presented on Monday, Sept. 16) will be covered on Exam II.•A practice exam will be posted on the website. There will NOT be a practice exam for Exam II.MCB 250 - Lecture 7Protein StructureProtein FoldingThe 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 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 or strand can be packed into the protein core or placed in a phospholipid bilayer.Protein Size•Biologically active polypeptides come in a wide variety of sizes–Small – Thyrotropin Releasing Hormone•3 amino acids – Gln-His-Pro, a peptide, not a protein–Huge – Titin, a molecular spring in cardiac muscle•~30,000 amino acids – 3450 kilodaltons (kd)–Median Size•~260 amino acids – 30 kd•How many different 260 amino acid proteins are possible–20260 = 10338 (Atoms in the universe ~ 1080), so only a tiny fraction of the possible amino acid sequences are actually usedMethods for Determining Protein Structure - X-ray Crystallography90% of the structures we have were determined by this method.Rate limiting step is usually getting crystals. Some proteins don’t crystallize.Provides the structure of a crystallized protein which may differ from the structure in solution. Improved instruments (e.g., Advanced Photon Source at Argonne National Lab) and computational methods are leading to a rapid increase in the number of structures determined. (Now more than 40,000 structures in the Protein Data Base.)Lots of mathX-Ray CrystallographyMethods for Determining Protein Structure - NMR•NMR - Nuclear Magnetic Resonance•Provides structure in solution•Only feasible for relatively small proteinsWays of Representing Protein StructuresSome proteins are mostly helical. Note- 4 subunits (a2 b2).a is red, bis blue. The green molecules are hemes. Hemoglobin is a heterotetramer.HemoglobinExamples of proteins composed of b-sheetsStructural MotifsA few structural motifs are present in many different proteins.a. Coiled coilb. Helix-turn-helixc. There are others that will come up later.GCN4 – A “Leucine Zipper” or “Coiled Coil”The Leucine Zipper MotifNote “heptad repeat”The leucine zipper forms a dimerization interface.Tropomyosin – A Coiled-Coil Protein in MuscleHelix-turn-helix motif in a DNA binding protein. Many different sequence-specific DNA binding proteins have HTH motifs.Helix-turn- helix: another viewi434 Repressor – A Helix-Turn-Helix DNA Binding ProteinMembrane ProteinsProteins with hydrophobic surfaces can enter phospholipid membranes.a. Amphipathic helicesb. b-SheetsK+ Channel – A Helical Transmembrane ProteinMembraneTop ViewPorin - A b Barrel Transmembrane Protein Top ViewMembraneClicker QuestionSmall ions such as Na+ or Cl- cannot pass through a lipid bilayer, but rather manage to cross cell membranes by flowing through the pore of an 'ion channel protein'. Ion channel proteins are typically composed of a-helices. What would you predict about the structure of the pore?A) The pore runs down the center of a single a-helix, which has nonpolar R groups facing out into the lipids.B) Multiple amphipathic a-helices surround the pore, with nonpolar R groups facing the lipids and polar/charged R groups facing into the pore.C) Multiple a-helices are oriented such that all of their R groups (nonpolar) point out into the lipids. The pore is lined by their polar peptide backbones.The Folding ProblemHow do you get from this:Hemoglobin b-subunit:1 MVHLTPEEKS AVTALWGKVN VDEVGGEALG RLLVVYPWTQ RFFESFGDLS TPDAVMGNPK VKAHGKKVLG AFSDGLAHLD NLKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG KEFTPPVQAA YQKVVAGVAN ALAHKYH 147a-subunit:1 MVLSPADKTN VKAAWGKVGA HAGEYGAEAL ERMFLSFPTT KTYFPHFDLS HGSAQVKGHG KKVADALTNA VAHVDDMPNA LSALSDLHAH KLRVDPVNFK LLSHCLLVTL AAHLPAEFTP AVHASLDKFL ASVSTVLTSK YR142To this:The Folding ProblemHow do you get from this:Hemoglobin b-subunit:1 MVHLTPEEKS AVTALWGKVN VDEVGGEALG RLLVVYPWTQ RFFESFGDLS TPDAVMGNPK VKAHGKKVLG AFSDGLAHLD NLKGTFATLS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG KEFTPPVQAA YQKVVAGVAN ALAHKYH 147a-subunit:1 MVLSPADKTN VKAAWGKVGA HAGEYGAEAL ERMFLSFPTT KTYFPHFDLS HGSAQVKGHG KKVADALTNA VAHVDDMPNA LSALSDLHAH KLRVDPVNFK LLSHCLLVTL AAHLPAEFTP AVHASLDKFL ASVSTVLTSK YR142To this:Protein Folding•All of the information for proper folding is present in the amino acid sequence.•Given the free rotation around the a-carbon, proteins can assume an essentially infinite number of conformations.•Some conformations are much more energetically favorable than others.•Usually only one conformation, the one with the lowest free