BIOC 460 Summer 2011
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 Some structural motifs found in proteins: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/motif/motif.htm Locations of hydrophobic and hydrophilic side chains: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/sidechain/sidechain.html 5 different domains in one subunit of pyruvate kinase: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/proteindomains/domain1.htm Myoglobin: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/myoglob/myoglob.html Structures of ab proteins: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/alpha_beta/alpha_beta.html Hemoglobin: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/hemoglobin/newhb.html 1
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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 a helices, b 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 multi- 2 subunit proteins, especially in regulation.
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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, b-sheets and non-regular regions, which are folded in a specific manner.
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Generalizations about H2O-soluble, globular protein structure – *minimization of solvent-accessible surface area – maximization of hydrogen bonding and van der Walls interactions 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 are tightly packed, compact units • Secondary structural elements ( -helices and 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
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1. Minimizing surface (burying hydrophobic side chains) • •
Amphipathic secondary structural elements Burial of hydrophobic R groups away from H 2O requires at least 2 interacting secondary structural elements, e.g., 2 a helices, or a b-abloop (uses ahelix to connect 2 parallel b strands), or 2 b sheets, etc. • How can 2 a 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
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Berg et al., Fig. 2.44
a-helical coiled coil (2 ahelices coiled around each other) of a leucine zipper motif (heptad repeat) • “Helical wheels" projections down helix axes What is chemical nature of Leu?
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 -helix will be amphipathic if one side of the helix is in a polar environment and the other side is in a hydrophobic environment. 6
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2. Maximizing hydrogen bonds within the protein • especially important in "driving"/stabilizing formation of secondary structures like a-helices and b sheets – “burying” polar N-H and C=O groups of backbone in nonpolar protein interior is thermodynamically more favorable • 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 a-helices and b sheets) How can H-bonding within an a-helix decrease polarity of peptide backbone? 7
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: 1. a helices of L-amino acids tend to be right-handed. 2. b-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 bab structure, are usually right-handed.
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Structural motifs • • •
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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 (aa) - stacks of b-sheets -bab elements (often found in parallel b-sheets) -b-barrels (b sheet folds/twists into a cylinder) -b saddles (twisted b sheet) Jmol routine: some structural motifs found in proteins http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/motif/motif.htm
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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. 9
Water-soluble globular protein tertiary structures Examples: 1. Myoglobin (Mb): the globin fold • Jmol structure of Mb: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/myoglob/myoglob.html
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water soluble protein that binds O2 in muscle cells for storage and for intracellular transport, using a heme group very compact structure (almost no empty space inside) mostly (70%) a-helical, little to no bstructure; rest is turns & loops (at surface) All amphipathic helices 8 a-helices, designated by letters A - H, from N to C terminus 5 Pro residues, 4 in turns (Pro is a helix “breaker”) 10
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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)11
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.
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Berg et al., Fig. 2-49
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2. Triose phosphate isomerase, an ab barrel protein an enzyme in the glycolytic pathway) Jmol structures of ab proteins: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/alpha_beta/alpha_beta.html
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an (ab)8 or TIM barrel (parallel 8-stranded barrel on interior, surrounded by helices, a structural motif found in many different enzymes
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Examples:
What are orientations of b strands: parallel or anti-parallel? 13
•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 14
Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 4-19
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3. Protein domains, continued • •
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the “immunoglobulin fold”, a “b 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 sheets, with loops between strands: motif = the "immunoglobulin fold".
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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 barrel (big antiparallel 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 16
Berg et al., Fig. 2-50
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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 b conformation, how are adjacent side chains oriented? 17
Amino acid sequence of a porin b strands are indicated, with diagonal lines indicating direction of hydrogen bonding along the 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 strands (adjacent R groups project out from sheet on opposite sides). 18
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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)
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different subunits designated with Greek letters –e.g., subunits of a heterodimeric protein = the “a subunit" and the “b 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 ahelix and bconformation. Quaternary structure is stabilized by the same types of forces as tertiary structure: noncovalent interactions, or for extracellular proteins sometimes disulfide bonds. 19
Examples of quaternary structure in proteins •
Cro protein from bacteriophage lambda (), a homodimer • Hemoglobin, a heterotetramer (a2b 2) • 2 identical subunits (red) structurally similar to 2 identical subunits (yellow) •a and b 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 http://www.biochem.arizona.edu/classes/bioc462/4 62a/jmol/hemoglobin/newhb.html
Protein Tertiary and Quaternary Structure
Berg et al., Fig. 2-53
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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?
21 Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 4-24a
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.
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Explain the term amphipathic, with an amphipathic protein a helix as an example.
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Recognize examples (ribbon diagrams) of such common folding motifs (frequently encountered combinations of secondary structures) as coiled coils of a-helices, stacked b-sheets, bab elements, b-barrels, and b saddles.
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Explain the term tertiary structure.
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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.
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Describe in general terms the structure of the polypeptide chain of myoglobin. 22
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Learning Objectives, continued •
Describe the structure of the “immunoglobulin fold” (single domain).
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Describe the general structure of an ab barrel, including where in the structure you would expect to find hydrophobic groups and where you would expect to find polar/charged groups.
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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. 23
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