Bài giảng Biochemistry 2/e - Chapter 6: Proteins: Secondary, Tertiary, and Quaternary Structure

Outline 6.1 Forces Influencing Protein Structure 6.2 Role of the Amino Acid Sequence in Protein Structure 6.3 Secondary Structure of Proteins 6.4 Protein Folding and Tertiary Structure 6.5 Subunit Interactions and Quaternary Structure

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Chapter 6Proteins: Secondary, Tertiary, and Quaternary Structureto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Outline6.1 Forces Influencing Protein Structure 6.2 Role of the Amino Acid Sequence in Protein Structure 6.3 Secondary Structure of Proteins 6.4 Protein Folding and Tertiary Structure 6.5 Subunit Interactions and Quaternary Structure 6.1 The Weak ForcesWhat are they? What are the relevant numbers? van der Waals: 0.4 - 4 kJ/mol hydrogen bonds: 12-30 kJ/mol ionic bonds: 20 kJ/mol hydrophobic interactions: <40 kJ/mol6.2 The Role of the Sequence in Protein StructureAll of the information necessary for folding the peptide chain into its "native” structure is contained in the primary amino acid structure of the peptide.How do proteins recognize and interpret the folding information?Certain loci along the chain may act as nucleation points Protein chain must avoid local energy minima Chaperones may help6.3 Secondary StructureThe atoms of the peptide bond lie in a plane The resonance stabilization energy of the planar structure is 88 kJ/mol A twist about the C-N bond involves a twist energy of 88 kJ/mol times the square of the twist angle. Twists can occur about either of the bonds linking the alpha carbon to the other atoms of the peptide backboneConsequences of the Amide PlaneTwo degrees of freedom per residue for the peptide chain Angle about the C(alpha)-N bond is denoted phi Angle about the C(alpha)-C bond is denoted psi The entire path of the peptide backbone is known if all phi and psi angles are specified Some values of phi and psi are more likely than others.The angles phi and psi are shown hereSteric Constraints on phi & psiUnfavorable orbital overlap precludes some combinations of phi and psi phi = 0, psi = 180 is unfavorable phi = 180, psi = 0 is unfavorable phi = 0, psi = 0 is unfavorable Steric Constraints on phi & psiG. N. Ramachandran was the first to demonstrate the convenience of plotting phi,psi combinations from known protein structures The sterically favorable combinations are the basis for preferred secondary structuresClasses of Secondary StructureAll these are local structures that are stabilized by hydrogen bonds Alpha helix Other helices Beta sheet (composed of "beta strands") Tight turns (aka beta turns or beta bends) Beta bulge The Alpha HelixRead the box on page 167 First proposed by Linus Pauling and Robert Corey in 1951 Identified in keratin by Max Perutz A ubiquitous component of proteins Stabilized by H-bondsThe Alpha HelixKnow these numbers Residues per turn: 3.6 Rise per residue: 1.5 Angstroms Rise per turn (pitch): 3.6 x 1.5A = 5.4 Angstroms The backbone loop that is closed by any H-bond in an alpha helix contains 13 atoms phi = -60 degrees, psi = -45 degrees The non-integral number of residues per turn was a surprise to crystallographersThe Beta-Pleated SheetComposed of beta strands Also first postulated by Pauling and Corey, 1951 Strands may be parallel or antiparallel Rise per residue: 3.47 Angstroms for antiparallel strands3.25 Angstroms for parallel strandsEach strand of a beta sheet may be pictured as a helix with two residues per turnThe Beta Turn(aka beta bend, tight turn) allows the peptide chain to reverse direction carbonyl C of one residue is H-bonded to the amide proton of a residue three residues away proline and glycine are prevalent in beta turnsTertiary Structure Several important principles: Secondary structures form wherever possible (due to formation of large numbers of H-bonds) Helices and sheets often pack close together Tertiary Structure Several important principles: The backbone links between elements of secondary structure are usually short and direct Proteins fold to make the most stable structures (make H-bonds and minimize solvent contactFibrous ProteinsMuch or most of the polypeptide chain is organized approximately parallel to a single axis Fibrous proteins are often mechanically strong Fibrous proteins are usually insoluble Usually play a structural role in natureAlpha KeratinRead the box on page 175 Found in hair, fingernails, claws, horns and beaks Sequence consists of 311-314 residue alpha helical rod segments capped with non-helical N- and C-termini Primary structure of helical rods consists of 7-residue repeats: (a-b-c-d-e-f-g)n, where a and d are nonpolar. Promotes association of helices! Beta KeratinProteins that form extensive beta sheets Found in silk fibers Alternating sequence: Gly-Ala/Ser-Gly-Ala/Ser.... Since residues of a beta sheet extend alternately above and below the plane of the sheet, this places all glycines on one side and all alanines and serines on other side! This allows Glys on one sheet to mesh with Glys on an adjacent sheet (same for Ala/Sers) Collagen - A Triple HelixPrincipal component of connective tissue (tendons, cartilage, bones, teeth) basic unit is tropocollagen: three intertwined polypeptide chains (1000 residues each MW = 285,000 300 nm long, 1.4 nm diameter unique amino acid composition CollagenThe secrets of its a.a. composition... Nearly one residue out of three is Gly Proline content is unusually high Unusual amino acids found: 4-hydroxyproline 3-hydroxyproline 5-hydroxylysine Pro and HyPro together make 30% of res. The Collagen Triple HelixA case of structure following composition The unusual amino acid composition of collagen is unsuited for alpha helices OR beta sheets But it is ideally suited for the collagen triple helix: three intertwined helical strands Much more extended than alpha helix, with a rise per residue of 2.9 Angstroms 3.3 residues per turn Long stretches of Gly-Pro-Pro/HyPCollagen FibersStaggered arrays of tropocollagens Banding pattern in EMs with 68 nm repeat Since tropocollagens are 300 nm long, there must be 40 nm gaps between adjacent tropocollagens (5x68 = 340 Angstroms) 40 nm gaps are called "hole regions" - they contain carbohydrate and are thought to be nucleation sites for bone formation Structural basis of the collagen triple helixEvery third residue faces the crowded center of the helix - only Gly fits here Pro and HyP suit the constraints of phi and psi Interchain H-bonds involving HyP stabilize helix Fibrils are further strengthened by intrachain lysine-lysine and interchain hydroxypyridinium crosslinks Globular ProteinsSome design principles Most polar residues face the outside of the protein and interact with solvent Most hydrophobic residues face the interior of the protein and interact with each other Packing of residues is close However, ratio of vdw volume to total volume is only 0.72 to 0.77, so empty space exists The empty space is in the form of small cavitiesAn amphiphilic helix in flavodoxin:A nonpolar helix in citrate synthase:A polar helix in calmodulin:Globular ProteinsMore design principles "Random coil" is not random Structures of globular proteins are not static Various elements and domains of protein move to different degrees Some segments of proteins are very flexible and disordered Know the kinds and rates of protein motion Globular ProteinsThe Forces That Drive Folding Peptide chain must satisfy the constraints inherent in its own structure Peptide chain must fold so as to "bury" the hydrophobic side chains, minimizing their contact with water Peptide chains, composed of L-amino acids, have a tendency to undergo a "right-handed twist" A New Way to Look at Globular ProteinsLook for "layer structures"Helices and sheets often pack in layers Hydrophobic residues are sandwiched between the layers Outside layers are covered with mostly polar residues that interact favorably with solventClasses of Globular ProteinsJane Richardson's classification Antiparallel alpha helix proteins Parallel or mixed beta sheet proteins Antiparallel beta sheet proteins Metal- and disulfide-rich proteinsAntiparallel Alpha Helical ProteinsSee Figure 6.29 for some examples Simplest way to pack helices - short connecting loops and antiparallel packing The helix bundle often involves a slight (15 degree) left-handed twist The globin proteins - myoglobin and hemoglobin - are antiparallel alpha proteinsParallel or Mixed Beta Sheet ProteinsSee Figure 6.30, 6.31 Parallel beta sheets distribute nonpolar residues on both sides of the beta sheet This means that both faces of the sheet must be protected from solvent Thus parallel beta sheets are core structures Parallel beta barrels are in this class Doubly wound parallel beta sheets also Antiparallel Beta SheetsSee Figures 6.32, 6.33, 6.34 Antiparallel beta sheets place nonpolar residues on only one face of the sheet Only one face must be protected from solvent Thus antiparallel beta sheet proteins may contain as few as two layers Possibilities: barrels, beta sandwiches and sheets covered by helices on one face only Metal-Rich and Disulfide-rich ProteinsSee Figure 6.35Usually less than 100 residues Conformations usually heavily influenced by metals and/or disulfide bridges These proteins are usually unstable if the metals are removed or the disulfides are reducedThermodynamics of FoldingRead the box on page 192 Separate the enthalpy and entropy terms for the peptide chain and the solvent Further distinguish polar and nonpolar groups The largest favorable contribution to folding is the entropy term for the interaction of nonpolar residues with the solventMolecular ChaperonesWhy are chaperones needed if the information for folding is inherent in the sequence? to protect nascent proteins from the concentrated protein matrix in the cell and perhaps to accelerate slow steps Chaperone proteins were first identified as "heat-shock proteins" (hsp60 and hsp70)Protein ModulesAn important insight into protein structure Many proteins are constructed as a composite of two or more "modules" or domains Each of these is a recognizable domain that can also be found in other proteins Sometimes modules are used repeatedly in the same protein There is a genetic basis for the use of modules in naturePredictive AlgorithmsIf the sequence holds the secrets of folding, can we figure it out? Many protein chemists have tried to predict structure based on sequence Chou-Fasman: each amino acid is assigned a "propensity" for forming helices or sheets Chou-Fasman is only modestly successful and doesn't predict how sheets and helices arrange George Rose may be much closer to solving the problem. See Proteins 22, 81-99 (1995) Modeling protein folding with Linus (George Rose)Ken Dill’s folding funnel. Unfolded structures lie around the top. As the protein folds, it falls down the wall of the energy funnel to more stable conformations.The native, folded structure is at the bottom.Nature Structural Biol.4, 10-19 (1997).6.5 Quaternary StructureWhat are the forces driving quaternary association? Typical Kd for two subunits: 10-8 to 10-16M! These values correspond to energies of 50-100 kJ/mol at 37 C Entropy loss due to association - unfavorable Entropy gain due to burying of hydrophobic groups - very favorable! What are the structural and functional advantages driving quaternary association?Know these! Stability: reduction of surface to volume ratio Genetic economy and efficiency Bringing catalytic sites together Cooperativity
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