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BIOC 460, spring 2008 Lecture 6 Protein Tertiary and Quaternary Structure Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 2, pp. 44-53, 61-62; Chapter 12, pp. 337-338 Directory of Jmol structures of proteins: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/routines/routines.html Jmol routine: some structural motifs found in proteins: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/motif/motif.htm Jmol routine showing locations of hydrophobic and hydrophilic side chains: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/sidechain/sidechain.html Jmol routine -- 5 different domains in one subunit of pyruvate kinase: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/proteindomains/domain1.htm Jmol structure of myoglobin: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/myoglob/myoglob.html Jmol structures of αβ proteins: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/alpha_beta/alpha_beta.html Jmol structure of hemoglobin http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/hemoglobin/newhb.html Key Concepts • Tertiary and quaternary structures result from folding of primary structure (and secondary structural elements) in 3 dimensions. • 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 • 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 multisubunit proteins, especially in regulation. LEC 6, Protein Tertiary and Quaternary Structure 1 BIOC 460, spring 2008 Learning Objectives • 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. • 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, and β saddles. • 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 in general terms the structure of the polypeptide chain of myoglobin. Learning Objectives, continued • Describe the structure of the “immunoglobulin fold” (single domain). • Describe the general structure of an αβ barrel, including where in the structure you would expect to find hydrophobic groups and where you would expect to find polar/charged groups. • Describe the general structure (arrangement of hydrophobic vs. polar R groups) of a globular protein that is embedded in a lipid bilayer (membrane). – Specifically, describe how the primary and secondary structures of a bacterial porin relate to the tertiary structure (and function) of a single porin subunit. • 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. LEC 6, Protein Tertiary and Quaternary Structure 2 BIOC 460, spring 2008 Tertiary Structure • 3-dimensional conformation of a whole polypeptide chain in its folded state (includes not only positions of backbone atoms, but of all the sidechain atoms as well) • Most water-soluble and membrane proteins are globular (compact and roughly spherical). • 3-D structures determined by – x-ray diffraction of protein crystals, or – NMR spectroscopy of protein in solution (for proteins that aren’t too large). • 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. 3 principles guiding folding of H2O-soluble, globular proteins & consequences of those principles • Generalizations about H2O-soluble, globular protein structure – *minimization of solvent-accessible surface area – maximization of hydrogen bondin within the protein – chiral effect * 1. minimization of solvent-accessible surface area • burying as many hydrophobic groups as possible • the most important driving force in folding of water-soluble proteins • Globular protein structures generally tightly packed, compact units • Secondary structural elements (α-helices and β sheets) often amphipathic – R groups on one side hydrophobic (and face interior of protein) – R groups on other side hydrophilic (and face aqueous environment, outside) • Jmol routine showing locations of hydrophobic and hydrophilic side chains http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/sidechain/sidechain.html LEC 6, Protein Tertiary and Quaternary Structure 3 BIOC 460, spring 2008 1. Minimizing surface (burying hydrophobic side chains) • Amphipathic secondary structural elements • Burial of hydrophobic R groups away from H2O requires at least 2 interacting 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. • How can 2 α helices get together to bury hydrophobic R groups, if there's water around them? amphipathic helices -- used to bury hydrophobic R groups toward interior of protein on 1 side of helix while other side of helix interacts with H2O Berg et al., Fig. 2.44 α-helical coiled coil (2 α helices coiled around each other) of a leucine zipper motif (heptad repeat) • schematic diagram ("helical wheels" projections down helix axes) Fig. 16-30 from Stryer, Biochemistry, 4th ed.(1995) • Residues a and d of each strand pack tightly together to form a hydrophobic core. • If residues b, c, and f on periphery are polar or charged, the helices are amphipathic helices. • Note: Any protein α-helix will be amphipathic if one side of the helix is in a polar environment and the other side is in a hydrophobic environment. LEC 6, Protein Tertiary and Quaternary Structure 4 BIOC 460, spring 2008 2. Maximizing hydrogen bonds within the protein • especially important in "driving"/stabilizing formation of secondary structures like α-helices and β sheets – makes “burying” polar N-H and C=O groups of backbone in interior of protein more favorable thermodynamically • polar side chains sometimes also buried, if their polar groups are hydrogen-bonded • Polar backbone groups and side chains tend to be either – in contact with water (hydration) OR – hydrogen-bonded with OTHER PROTEIN GROUPS (e.g., in secondary structures like α-helices and β sheets) 3. the chiral effect • tendency of extended backbone structural arrangements to be righthanded as a result of having all L-amino acids • Consequences: twist and connectivity – twist: • α helices of L-amino acids tend to be right-handed. • β-conformation strands (and sheets) of L-amino acids tend to twist in a right-handed direction, forming saddles or barrels. – connectivity: • crossovers between adjacent secondary structural elements, e.g., in βαβ structure, are usually right-handed. LEC 6, Protein Tertiary and Quaternary Structure 5 BIOC 460, spring 2008 Structural motifs • recognizable patterns of combinations/groupings of secondary structural elements • bury hydrophobic R groups in between “layers”/elements • Examples of motifs: – coiled coils of 2 or more α helices (αα) – stacks of β-sheets – βαβ elements – β barrels (β sheet folds/twists into a cylinder) – β saddles (twisted β sheet) • Jmol routine: some structural motifs found in proteins http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/motif/motif.htm • Some motifs have functional significance, such as the helix-loop-helix (helix-turn-helix) DNA-binding motif or the EF hand calcium-binding motif. Others serve only a structural role. water-soluble globular protein tertiary structures Examples: 1. Myoglobin (Mb): • Jmol structure of Mb: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/myoglob/myoglob.html • • • • • • • • binds O2 in muscle cells for storage and for intracellular transport, using a heme group mostly (70%) α-helical; rest mostly turns & loops (at surface) first high-resolution crystal structure of a protein ever determined very compact structure (almost no empty space inside) very water-soluble 5 Pro residues, 4 in turns 8 α helices, designated by letters A - H, from N to C terminus Helices amphipathic (surface sides hydrophilic R groups, buried sides more hydrophobic R groups) LEC 6, Protein Tertiary and Quaternary Structure 6 BIOC 460, spring 2008 Myoglobin structure Berg et al., Fig. 2.48B; heme black with purple Fe2+ Nelson & Cox, Lehninger Principles of Biochemistry, Fig. 4-16 (heme in red; blue residues: Leu, Ile, Val, Phe) Myoglobin structure, continued • Distribution of Amino Acids in Mb structure • (hydrophobic residues in yellow, charged residues in blue, others in white) A. surface view ; B. cross-sectional view showing interior of protein • NOTE: many charged residues on surface, none in interior • many hydrophobic residues in interior, but also a few on surface • The only polar residues inside are 2 His residues involved in binding the heme and O2. Berg et al., Fig. 2-49 LEC 6, Protein Tertiary and Quaternary Structure 7 BIOC 460, spring 2008 2. Triose phosphate isomerase, an αβ barrel protein • an enzyme in the glycolytic pathway) • Jmol structures of αβ proteins: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/alpha_beta/alpha_beta.html • an (αβ)8 barrel (parallel 8-stranded β barrel on interior, surrounded by α helices, a structural motif found in many different enzymes • Some βαβ enzymes (triose phosphate isomerase, and one domain of pyruvate kinase): •Garrett & Grisham, Biochemistry, 3rd ed., Fig. 6-30 3. Protein Domains • Domains: structurally independent folding units looking like separate globular proteins but all part of same polypeptide chain • connected in same primary structure • Larger proteins often have 2 or more domains. • Jmol routine -- 4 different domains in one subunit of pyruvate kinase: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/proteindomains/domain1.htm •Troponin C, a protein found in muscle •2 domains, all one polypeptide chain Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 4-19 LEC 6, Protein Tertiary and Quaternary Structure 8 BIOC 460, spring 2008 3. Protein domains, continued • the “immunoglobulin fold”, a “β sandwich” domain • a cell surface protein (CD4), with 4 similar domains (each in a different color). – The folding motif of each of the 4 domains is the same. • Each domain consists of 2 antiparallel β sheets, with loops between β strands: motif = the "immunoglobulin fold". Berg et al., Fig. 2-52 4. Porins (example of a membrane protein) • found in outer membranes of bacteria and in outer mitochondrial membranes • channel-forming proteins - permit passage of ions and small molecules across membrane • globular, but their "solvent" is NOT water it’s a membrane • lipid core of membrane like a very nonpolar solvent • structure of each chain of porin mainly a large β barrel (big antiparallel β sheet, 16 strands, folded into a cylinder) • structure sort of like an "inside out" watersoluble protein • hydrophobic residues on outer surface, interacting with hydrophobic lipid core of membrane • inner side of the barrel forms water-filled channel across membrane; has more hydrophilic (charged and polar) R groups Berg et al., Fig. 2-50 LEC 6, Protein Tertiary and Quaternary Structure 9 BIOC 460, spring 2008 Structure of one subunit of a bacterial porin • left: side view, in plane of membrane; right: view from periplasmic space (from inside, looking out through pore in outer membrane) Berg et al., Fig. 12-20 On a single strand in β conformation, where are the side chains of the amino acid residues? (all on one side? Alternating sides? 3 residues’ R groups on one side, 1 on the other side?) Amino acid sequence of a porin • β strands are indicated, with diagonal lines indicating direction of hydrogen bonding along the β sheet • hydrophobic residues (F, I, L, M, V, W and Y) shown in yellow Berg et al., Fig. 12-21 •Note the more or less alternating hydrophobic and hydrophilic residues in the β strands (adjacent R groups project out from sheet on opposite sides). LEC 6, Protein Tertiary and Quaternary Structure 10 BIOC 460, spring 2008 Quaternary structure (4° structure) • 3-dimensional relationship of the different polypeptide chains (subunits) in a multimeric protein, the way the subunits fit together and their symmetry relationships • only in proteins with more than one polypeptide chain; proteins with only one chain have no quaternary structure.) Terminology • Each polypeptide chain in a multichain protein = a subunit • 2-subunit protein = a dimer, 3 subunits = trimeric protein, 4 = tetrameric • homo(dimer or trimer etc.): identical subunits • hetero(dimer or trimer etc.): more than one kind of subunit (chains with different amino acid sequences) • different subunits designated with Greek letters – e.g., subunits of a heterodimeric protein = the "α subunit" and the "β subunit". – NOTE: This use of the Greek letters to differentiate different polypeptide chains in a multimeric protein has nothing to do with the names for the secondary structures α helix and β conformation. • Some protein structures have very complex quaternary arrangements; e.g., mitochondrial ATP synthase, viral capsids…. Examples of quaternary structure in proteins • Cro protein from bacteriophage lambda (λ), a homodimer Berg et al., Fig. 2-53 • Hemoglobin, a heterotetramer (α2β2) • 2 identical α subunits (red) structurally similar to 2 identical β subunits (yellow) • α and β also very similar to structure of myoglobin (both primary and tertiary structure) • gene duplication of single ancestral gene and subsequent divergent evolution of sequences --> different globin genes • tertiary "fold" conserved through evolution Berg et al., Fig. 2-54 • Jmol structure of hemoglobin LEC 6, Protein Tertiary and Quaternary Structure 11 BIOC 460, spring 2008 Symmetry in quaternary structures • simplest kind of symmetry = rotational symmetry • Individual subunits can be superimposed on other identical subunits (brought into coincidence) by rotation about one or more rotational axes. • If the required rotation = 180° (360°/2), protein has a 2-fold axis of symmetry (e.g., Cro repressor protein above). • If the rotation = 120° (360°/3), e.g., for a homotrimer, the protein has a 3-fold symmetry axis. Rotational symmetry in proteins: Cyclic symmetry: all subunits are related by rotation about a single n-fold rotation axis (C2 symmetry has a 2-fold axis, 2 identical subunits; C3 symmetry has a 3-fold axis, 3 identical subunits, etc.) What type of rotational axis of symmetry is apparent in the hemoglobin structure above? LEC 6, Protein Tertiary and Quaternary Structure Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 4-24a 12