Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 6 Proteins: Secondary, Tertiary, and Quaternary Structure to.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 6 Proteins: Secondary, Tertiary, and Quaternary Structure to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 6.1 The Weak Forces What 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/mol

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 6.2 The Role of the Sequence in Protein Structure All of the information necessary for folding the peptide chain into its "native” structure is contained in the primary amino acid structure of the peptide.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 help

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 6.3 Secondary Structure The 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 backbone

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Consequences of the Amide Plane Two 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.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Steric Constraints on phi & psi Unfavorable 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Steric Constraints on phi & psi G. 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 structures

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Classes of Secondary Structure All 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Alpha Helix Read 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-bonds

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Alpha Helix Know 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 crystallographers

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Beta-Pleated Sheet Composed 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 strands –3.25 Angstroms for parallel strands –Each strand of a beta sheet may be pictured as a helix with two residues per turn

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The 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 turns

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Tertiary 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 contact

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Fibrous Proteins Much 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 nature

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Alpha Keratin Read 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!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Beta Keratin Proteins 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)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Collagen - A Triple Helix Principal 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Collagen The 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.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Collagen Triple Helix A 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/HyP

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Collagen Fibers Staggered 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Structural basis of the collagen triple helix Every 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Globular Proteins Some 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 cavities

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Globular Proteins More 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Globular Proteins The 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"

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company A New Way to Look at Globular Proteins Look 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 solvent

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Classes of Globular Proteins Jane Richardson's classification Antiparallel alpha helix proteins Parallel or mixed beta sheet proteins Antiparallel beta sheet proteins Metal- and disulfide-rich proteins

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Antiparallel Alpha Helical Proteins See 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 proteins

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Parallel or Mixed Beta Sheet Proteins See 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Antiparallel Beta Sheets See 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Metal-Rich and Disulfide-rich Proteins See Figure 6.35 Usually 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 reduced

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Thermodynamics of Folding Read 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 solvent

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Molecular Chaperones Why 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)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Protein Modules An 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 nature

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Predictive Algorithms If 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)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 6.5 Quaternary Structure What are the forces driving quaternary association? Typical K d for two subunits: 10 -8 to 10 -16 M! 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!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 6 Proteins: Secondary, Tertiary, and Quaternary Structure to.

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 6 Proteins: Secondary, Tertiary, and Quaternary Structure to.

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