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lec6 - Lecture 6: Protein/ Structure/ Composi4on ...

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Unformatted text preview: Lecture 6: Protein / Structure / Composi4on Ter4ary / Quaternary Structure Key Concepts •  •  •  Tertiary and quaternary structures result from folding of primary structure (and secondary structural elements) in 3 dimensions. Tertiary and quaternary structures are stabilized (“held together”) by noncovalent interactions (all types) and in extracellular proteins, sometimes also by disulfide bonds. Tertiary structure –  Most proteins' tertiary structures are combinations of α helices, β sheets, and loops and turns. –  Larger proteins often have multiple folding domains. –  Folding of H2O-soluble, globular proteins into their native structures follows some basic rules/ principles: •  •  •  minimization of solvent-accessible surface area (burying hydrophobic groups) maximization of intraprotein hydrogen bonds chirality (right-handed twist and connectivity) of the polypeptide backbone –  Fibrous proteins have repeating secondary structure •  Quaternary structure –  Some proteins have multiple polypeptide chains (quaternary structure). –  Arrangement of polypeptides in multimeric proteins is generally symmetrical. –  Quaternary structure can play important functional roles for multi-subunit proteins, especially in regulation. •  •  Structure are obtained from X-ray Crystallography and NMR Proteins are dynamics Learning Objec4ves •  Outline 3 principles guiding folding of water-soluble globular proteins and the generalizations about protein structure resulting from those principles. Relate the principles to real protein structures. •  Explain the term amphipathic, with an amphipathic protein α helix as an example. •  How hydrophobic side chains are buried? •  Recognize examples (ribbon diagrams) of such common folding motifs (frequently encountered combinations of secondary structures) as coiled coils of α-helices, stacked β-sheets, βαβ elements, β-barrels •  Explain the term tertiary structure. •  Define the terms domain and subunit as they relate to protein structure. Be able to recognize different domains in a ribbon diagram of a single polypeptide chain with 2 or more domains. •  Describe the structure of fibrous proteins. How are such structures stabilized ? Learning Objec4ves, con4nued •  Describe in general terms the structure of the polypeptide chain of myoglobin. •  Describe the general structure of a protein, including where in the structure you would expect to find hydrophobic groups and where you would expect to find polar/charged groups. •  Explain the term quaternary structure (of a protein), and be able to describe a protein in terms like "homotetramer", "heterodimer", etc. •  Explain simple rotational symmetry for an oligomeric protein such as a homodimer like the Cro protein or a heterotetramer like hemoglobin. –  Be able to use (correctly) the terms "2-fold", "3-fold", etc. to refer to simple rotational axes of symmetry and recognize that simple level of symmetry in a protein structure. •  Methods used for protein structure determination / Protein Data Bank Levels of protein structure 3 ­dimensional conforma4on of a whole polypep4de chain in its folded state (includes not only posi4ons of backbone atoms, but of all the sidechain atoms as well) Globular protein structures generally 4ghtly packed, compact units Principles guiding folding of water ­soluble, globular proteins 1.  Minimiza4on of solvent ­accessible surface area –  The polypep4de chain takes the conforma4on that buries the maximum amount of hydrophobic surface area in the protein interior. This tends to collapse the polypep4de chain into a small volume. –  Burial of hydrophobic R groups away from water requires at least 2 interac4ng secondary structural elements, e.g., 2 α helices, or a β ­α ­β loop (uses α helix to connect 2 parallel β strands), or 2 β sheets, etc. 2.  Maximiza4on of hydrogen bonding within the protein –  Hydrogen bond donors and acceptors must be bonded. On the protein exterior water is available but in the interior they must sa4sfy each other. The need to form hydrogen bonds can be sa4sfied by secondary structure forma4on. 3.  Chiral effect –  Backbones of L ­amino acids tend to twist in a right ­handed direc4on. Both α ­ helices and β ­sheets twist to the right, affec4ng the connec4ons between secondary structure elements. Hydrophobic effect •  burying as many hydrophobic groups as possible the most important driving force in folding of water ­soluble protein – why ? •  Secondary structural elements (α ­helices and β sheets) o\en amphipathic –  R groups on one side hydrophobic (and face interior of protein) –  R groups on other side hydrophilic (and face aqueous environment, outside) Hydrophobic core Burying hydrophobic side chains 1) Amphipathic helices used to bury hydrophobic R groups toward interior of protein on 1 side of helix while other side of helix interacts with water 2) Associa1ons of β ­strands chains Face ­to ­face: Mediated by side ­chains (hydrophobic interac4ons) Interac4ons could be between parts of same chain or between different 3) Associa1ons of helices and β ­strands Loca4ons of hydrophobic and hydrophilic side chains h_p://www.biochem.arizona.edu/classes/bioc462/462a/jmol/sidechain/sidechain.html Structural mo4fs •  recognizable pa_erns of combina4ons of secondary structural elements •  bury hydrophobic R groups in between “layers”/elements •  Others mo4fs: –  coiled coils of 2 or more α helices (αα) / stacks of β ­ sheets / β barrels (folds/ twists into a cylinder) / β saddles (twisted β sheet) βαβ β hairpain αα Greek key mo4f: ββββ Maximizing hydrogen bonds •  Polar side chains can be buried, if their polar groups are hydrogen ­bonded •  Polar backbone groups and side chains tend to be either –  in contact with water (hydra4on) –  hydrogen ­bonded with OTHER PROTEIN GROUPS (e.g., in secondary structures like α ­helices and β sheets) Others interac4ons Metal Salt Bridges CYS ­CYS interac4ons Effect of muta4ons on protein structure LEU ASP: ARG LYS : LEU PHE: ARG GLU: GLU ARG SER GLU SER ALA: SER THR: Proteins •  Every protein has a unique three dimensional structure made up of a variety of helices, β ­sheets and non ­regular regions, which are folded in a specific manner •  Classified as either fibrous or globular depending on their morphology  ­  provide structural support for cells and 4ssues.  ­  contain high propor4ons of regular secondary structure (o\en repe44ve sequences), such as α ­ helix or β pleated sheet.  ­  have a structural rather than dynamic role because of their rodlike or sheetlike shapes  ­  Func4ons involve the precise binding of small ligand or large macromolecules.  ­  Roughly spherical shapes, may contain several types of regular secondary structure elements. A por4on of the protein’s may also be irregular or unique  ­ Generally not repe44ve sequence  ­ Include enzymes, regulatory proteins water ­soluble (unless they are located in biological membranes) Fibrous proteins •  The helices can wind around each other, forming a supercoil or coiled ­coil. •  “Heptad” repeat: •  Great tensile strength •  Le\ ­handed supercoils (of right ­handed α ­helices) are found in nature e.g. : –  Kera4n, the main component of hair, quills, and horns –  Myosin, an important muscle protein By which type of interactions are the 2 helices held together ? Replace Leu on the left by Glu, which AA would you expect on the right ? A coiled coil •  Most abundant proteins of mammals •  Major protein of car4lage, skin, bone, tendon, and teeth •  Repea4ng tripep4de seq. Gly–X–Pro or Gly–X–4 ­Hyp adopts a le\ ­handed helical structure with 3 residues per turn •  Pro are converted to 4 ­Hyp by prolyl hydroxylase (requires vitamin C) •  Skin lesions, fragile blood vessels, poor wound healing and ul4mately death Collagen α chain of collagen Three α helices wrap around one another with a right-handed twist Why is GLY (in red) found where the three chains are in contact ? Fibroin •  produced by insects (e.g. silk moths) and spiders •  primary structure: rich in Gly and Ala (some4mes Ser) alterna4ng –Gly ­Ala ­Gly ­Ala ­Gly ­Ala ­ •  secondary structure: an4parallel β ­sheet Proteins with a lot of α ­helices Myoglobin •  First high ­resolu4on crystal structure •  Oxygen storage – binds O2 in muscle cells •  Single domain protein •  Heme is buried in hydrophobic core •  Fe2+ ion in heme binds oxygen •  ~70% α ­helical (8) / ~30% turns & loops at surface Ribbon rendi4on: the polypep4de backbone tracing in space is shown Space filling Myoglobin structure cross ­sec4onal view showing interior of protein no charged residues surface view few hydrophobic residues mostly charged residues only 2 HIS (polar) for binding the heme and O2. many hydrophobic residues h_p://www.biochem.arizona.edu/classes/bioc462/462a/jmol/myoglob/myoglob.html Proteins with β conforma4on Fa_y acid binding protein (mostly β conforma4on; β sheet in a “clam” mo4f) Green fluorescent protein (β barrel; used as a “reporter” in molecular gene4cs experiments) NOBEL PRICE 2008 h_p://www.biochem.arizona.edu/classes/bioc462/462a/jmol/beta_domain/beta_domain.html Globular proteins have a variety of Ter4ary structures Which protein does this Ramachandran diagram correspond to ? +180 ψ 0  ­180  ­180 0 +180 φ Amino Acid sequence and Protein Structure Ile – Ala – His –Thr –Tyr – Gly – Pro – Phe – Glu - Ala Ala – Met – Cys –Lys –Trp – Glu – Ala – Gln – Pro - Asp Gly – Met – Glu –Cys –Ala – Phe – His – Arg Where might bends or β turns occur ? Disulfide ? Location of Asp, Ile, Thr, Ala, Gln, Lys ? Protein Domains •  Domains: structurally independent folding units looking like separate globular proteins but all part of same polypep4de chain •  Larger proteins: consist of 2 or more domains linked together in a single chain Coiled-coil domain (2 helices) Have a hydrophobic core, where most of the hydrophobic residues are sequestered away from water β-sandwich domain (2 sheets) Protein Domains •  the “immunoglobulin fold”, a “β sandwich” domain •  a cell surface protein (CD4), with 4 similar domains (each in a different color). –  The folding mo4f of each of the 4 domains is the same. •  Each domain consists of 2 an4parallel β sheets, with loops between β strands: mo4f = the "immunoglobulin fold". Berg et al., Fig. 2-52 Interac4ons Between Domains and Proteins •  The same kinds of bonds and interac4ons that hold secondary structure elements together to form domains also serve to hold complete domains together as larger structures. •  Shape complementarity becomes an increasingly important factor in higher order structure, allowing many Van der Waals interac4ons. H-bonds Surfaces fit together Electrostatic interactions ++ -Hydrophobic patches Quaternary Structure •  3 ­dimensional rela4onship of the different polypep4de chains (subunits) in a mul4meric protein •  Terminology: each polypep4de chain in a mul4chain protein = a subunit •  2 ­subunit protein = a dimer, 3 subunits = trimeric protein, 4 = tetrameric, etc. •  Quaternary structure is stabilized by the same types of forces as ter4ary structure: noncovalent interac4ons, or for extracellular proteins some4mes disulfide bonds Hemoglobin: Heterotetramer Quaternary structure α2β2 Phage λ Cro protein: Homodimer Quaternary structure α2  ­ homo (dimer or trimer etc.): iden4cal subunits  ­ hetero(dimer or trimer etc.): noniden4cal subunits Berg, Tymoczko & Stryer Fig. 3.48 Berg, Tymoczko & Stryer Fig. 3.49 Symmetry in quaternary structures •  Individual subunits can be superimposed on other iden4cal subunits (brought into coincidence) by rota4on about one or more rota4onal axes. •  If the required rota4on = 180° (360°/2), protein has a 2 ­fold axis of symmetry (e.g., Cro repressor protein above). •  If the rota4on = 120° (360°/3), e.g., for a homotrimer, the protein has a 3 ­fold symmetry axis. Rota4onal symmetry in proteins: all subunits are related by rota4on about a single n ­fold rota4on axis (C2 symmetry has a 2 ­fold axis, 2 iden4cal subunits; C3 symmetry has a 3 ­fold axis, 3 iden4cal subunits, etc.) Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 4-24a Higher order symmetry Protein Structure Determina4on: X ­ray crystallography Diffraction pattern •  Crystallography is a technique that directly images molecules •  Suitable for any size molecule. Requires crystals, which are o\en hard to grow •  Late 50s: first X ­ray of Myoglobin •  Nobel Price Chemistry 1962 “for their studies of the structures of globular proteins” Azurin Protein Crystals Flavodoxin Rubredoxin Azidomet Hemoglobin Bacteriochlorophyll a Protein Structure Determina4on: NMR structure determina4on Nuclear Magne4c Resonance: Measures proper4es of atoms inside proteins using giant magnet. Determines which residues are in close proximity cross peak: close •  No crystals needed, but limited to ~30kD •  Nobel Price Chemistry 2002: Kurt Wuthrich “for his development of nuclear magne6c resonance spectroscopy for determining the three ­dimensional structure of biological macromolecules in solu6on” Protein Data Bank (PDB) •  www.pdb.org ...
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