Biochemistry: Concepts and Connections Dean R. Appling Spencer J. Anthony-Cahill Christopher K. Mathews Chapter 6 The Three Dimensional Structure of Proteins
Cartoon representation of myoglobin, showing α-helical secondary structure Linus Pauling: Rules for secondary structure Bond angles and lengths should be like those for respective free amino acids (as determined from X-ray structures of the amino acids) No atoms should approach one another more closely than allowed by their van der Waals radii Amide group must remain planar Noncovalent bonds (particularly hydrogen bonds) stabilize 3-D structure
Rotation around bonds in a polypeptide backbone The positions of the six atoms in each amide plane are essentially fixed; thus, free rotation is allowed about the C α carbons. However, this rotation is restricted by steric interactions. Such rotation is described by two angles termed ϕ (between N and C α ) and ψ (between C α and the carbonyl C).
The four levels of protein structure Primary (1 ) structure refers to the amino acid sequence that makes up the protein Secondary (2 ) structure refers to local areas of repeating main chain structure
The four levels of protein structure Tertiary (3 ) structure refers to the spatial arrangement of the scondary structural elements in the polypeptide chain Quaternary (4 ) structure refers to the spatial arrangement of multiple polypeptide chains to form multisubunit complexes
Secondary structure elements α helix β sheet 3 10 helix
Describing secondary structures in proteins α helix: side chains radiate out from helix axis; H bonds are nearly parallel to the helix axis; often amphiphilic, with distinct hydrophilic and hydrophobic faces β sheet: stabilized by interchain H bonds; side chains alternate sides of the sheet Strands in β sheet can be either parallel (both strands N C) or antiparallel (one strand N C, the other strand C N) The 3 10 helix meets the Pauling criteria but is not frequently found in proteins Parameters that define secondary structures:
β strands may have parallel or antiparallel orientations in a β sheet
The polypeptide II helix The polypeptide II helix (often called a polyproline II helix because ~1/3 of the residues in this secondary structure element are prolines) forms a left handed helix and is not stabilized by hydrogen bonds. This structure is prevalent in collagen.
Side chain positions in an α helix In an α helix, the side chains radiate away from the helical axis. The center of the α helix consists backbone atoms, closely packed. The hydrogen bonds that stabilize the helix are shown as yellow dashes.
Side chain positions in a β sheet In a β sheet, neighboring side chains are located on opposite faces of the sheet, which is stabilized by main-chain hydrogen bonds between adjacent β strands.
Steric interactions determine peptide conformation Certain ϕ and ψ angles result in steric clashes, where atoms are closer than their van der Waals radii. These conformations are not allowed. The backbone of an α helix results in closely packed atoms that do not sterically clash.
Ramachandran plot The Ramachandran plot shows sterically allowed ϕ and ψ angles. Shown here are allowed angles for poly-l-alanine in each of various conformations. White areas correspond to sterically allowed conformations based on theoretical predictions. The allowed regions would be smaller for amino acids with larger R groups.
Observed values for ϕ and ψ Actual ϕ and ψ angles for 30,692 residues in 209 proteins for which highresolution structures were available. S. A. Hollingsworth, D. S. Berkholz, and P. A. Karplus, Protein Science 18:1321 (2009)
Ramachandran plots for glycine and proline glycine proline Proline (right) has a far more limited range of permissible angles than glycine (left).
Fibrous protein structure Fibrous proteins are elongated molecules with well-defined secondary structures Examples include: keratin - hair, fingernails, feathers, scales, or intermediate filaments (intracellular) fibroin silk cocoons collagen - abundant connective tissue protein; matrix material in bone on which mineral components precipitate
α-keratin In keratin, large hydrophobic residues repeat every four positions; The α helix has 3.6 residues/turn, giving each helix a hydrophobic side, which defines the interface between two long helices in the coiled-coil structure typical of keratin.
Silk fibroin The extensive close-packed β sheet of fibroin is interrupted by compact folded regions which provide some elasticity.
Collagen Collagen, abundant connective tissue protein; matrix material in bone, on which mineral components precipitate; triple-strand left-handed helix Contains hydroxyproline (Hyp) and hydroxylysine G-X-Y tripeptide motif, where X is Pro and Y is Pro or Hyp, lends itself to triple-strand structure Polypeptide chains crosslinked and glycosylated Vitamin C (ascorbic acid) is a cofactor required for proline hydroxylation; vitamin C deficiency (scurvy) leads to collagen degeneration
Collagen
Three ways to represent the 3D structure of the small, single-chain protein ubiquitin Solvent-accessible surface model Cartoon Stick model and close-up
Globular proteins fold into defined structures Proteins have diverse structures, with varying amounts of helix, sheet and loop regions Larger proteins often contain two or more distinct domains of compact folded structure A typical protein domain is ~200 amino acids and will fold independently A domain frequently possesses some defined function (e.g., DNA recognition, oligomerization, cofactor binding, etc.)
Classification of protein structure Protein tertiary structure is characterized by the content of helix and sheet secondary structures as well as defined turns that link these secondary structures Some proteins are predominantly helix or sheet, others possess a mixture of helix and sheet, or very little defined secondary structure Not all parts of a globular protein structure can be categorized as helix, sheet, or turn. Such regions are often called random coil or, more properly, irregularly structured regions.
Common features of folded globular proteins Globular proteins have a nonpolar (hydrophobic) interior and a more hydrophilic exterior β sheets are usually twisted or wrapped into barrel structures. The polypeptide chain can turn corners, e.g., β turns:
Distribution of hydrophobic and hydrophilic residues in myoglobin
Most of the information specifying the tertiary structure is carried in the amino acid sequence, as shown by Anfinsen s experiment with bovine ribonuclease A
Heat-induced denaturation of RNase A Denaturation was measured by viscosity ( ), optical rotation (O), or UV absorbance (Δ). The thermodynamic hypothesis of protein folding is supported by the reversibility of folding shown by renaturation followed by repeat denaturation (closed triangles).
Three thermodynamic factors influence folding and stability of proteins Favorable intramolecular enthalpic interactions charge-charge interactions (ionic bonds; salt bridges) intermolecular hydrogen bonds van der Waals interactions; proteins are densely packed Unfavorable loss of conformational entropy Unfolded state has many conformations (high entropy) Folded state has only a few closely related conformations (low entropy) Favorable gain of solvent entropy from burying hydrophobic groups (the hydrophobic effect ) As hydrophobic side chains cluster in the interior they release ordered solvent molecules from clathrate structures
Proteins vary in the extent to which the folded state is favored by enthalpic or entropic factors Thermodynamic parameters for folding of selected proteins
Detail of H-bonding in a typical protein
Disulfide bonds greatly increase stability Bovine trypsin inhibitor: 3 S-S bonds Effect is partly entropic; S S reduce the number of conformations possible in the unfolded state Proteins with disulfide bonds primarily exist and function extracellularly
Dynamics of globular protein structure: protein folding Levinthal s paradox: If protein samples all possible conformations, rejecting each until it reaches the most stable conformation, folding a protein could take 10 30 years! But a small protein like RNase A (124 residues) folds in about a minute Not all states are sampled. Intermediate states encountered, including the molten globule: a compact partially folded state that has nativelike secondary structure and folding topology but lacks the defined tertiary structure interactions.
Protein folding energy landscapes Idealized funnel Rugged landscape 2-state folding landscape Landscape with metastable intermediates
Intermediate and off-pathway states in protein folding Isomerization of prolyl bonds is catalyzed by peptide prolyl isomerase; the cis isomer is disfavored by 1000-fold vs. the trans isomer The formation of non-native disulfide bonds is corrected by protein disulfide isomerase, which catalyzes the reduction and reoxidation of disulfide bonds
Chaperones promote proper folding of proteins and thereby prevent the formation of aggregated states associated with disease
Chaperonins: Protein complexes that facilitate protein folding The GroEL/ES chaperone of E. coli Why needed? 1. Intracellular protein concentrations are very high; GroEL/ES helps prevent intermolecular interactions that lead to protein aggregation 2. Protection needed when cell is heatstressed and proteins begin to unfold
Schematic of chaperonin function Interior is lined with hydrophobic groups early in the process After ATP and GroES bind, a conformational change occurs and the interior presents hydrophilic groups promoting the proper folding of the protein
Several diseases involve misfolding of normal proteins to give amyloid fibrils or amyloid plaques Highly ordered amyloids form from non-native folding intermediates or disordered aggregate states Prions: Infectious agents that cause disease by inducing amyloid formation on contact Amyloid fibrils of insulin
Prediction of secondary structure from amino acid sequence Empirical methods are ~ 80% accurate Based on observed distributions of amino acids in helix vs. sheet conformations (e.g., Ala prefers helix, Val prefers sheet, etc.) Amphiphilic α helix shows repeating patterns of side chain polarity every 3-4 residues Amphiphilic β strand shows repeating patterns of side chain polarity every other residue
Prediction of tertiary structure from amino acid sequence Critical need for accurate prediction from sequence, since structures are known for only ~ 1% of all known sequences Prediction of tertiary structure is difficult due to the need to correctly predict interactions between residues that are far apart in the primary structure Current computational methods are ~ 60% accurate
Quaternary structure Example: hemoglobin, an α 2 β 2 tetramer, is heterotypic Shown here: homotypic interactions among identical asymmetric subunits (b) to (h): examples of point group symmetry C 2 : one axis of symmetry (twofold) C 3 : one threefold axis D 2 : three twofold axes D 4 : one fourfold axes and two twofold axes
Structures with helical symmetry Two structures of helical symmetry, actin and the Tobacco mosaic virus These structures are capable of indefinite growth in length
C 2 symmetry example In the transthyretin dimer, the two monomers combine to form a β-sandwich. The dimer has twofold symmetry about the C 2 axis perpendicular to the image.
Interaction between trypsin and bovine pancreatic trypsin inhibitor Heterotypic proteinprotein interactions Complementary surfaces determine specific interactions
Ultraviolet-visible (UV-Vis) light absorption Proteins and nucleic acids absorb in the UV A (absorbance) = log I 0 /I Beer-Lambert Law: A = εlc; A is directly proportional to concentration
Fluorescence emission of fluorescent proteins Green fluorescent protein (GFP) was first isolated from a jellyfish Gene for GFP can be fused to the gene for a protein of interest, often with minimal effect on protein function or localization within a cell; thus, the fusion protein can often be used to monitor protein localization in cells
SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis)
α-helices May be Polar, Nonpolar or Amphiphilic Figure 6.22 The so-called helical wheel presentation can reveal the polar or nonpolar character of α-helices.
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