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Protein Structure: Nature’s Robots Lectures 7 and 8 Chem 464 Spring 2017 Abrol Section 1 CHAPTER 4 Proteins: Structure, Function, Folding Learning goals: – Structure and properties of the peptide bond – Structural hierarchy in proteins • Primary, Secondary, Tertiary, Quaternary • Primary and Secondary structure building blocks. – Structure and function of fibrous proteins – Structure analysis of globular proteins – Protein folding and denaturation Structure of Proteins • Unlike most organic polymers, protein molecules adopt a specific three-dimensional conformation. • This structure is able to fulfill a specific biological function • This structure is called the native fold • The native fold has a large number of favorable interactions within the protein • There is a cost in conformational entropy of folding the protein into one specific native fold Favorable Interactions in Proteins • Hydrophobic effect – Release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy • Hydrogen bonds – Interaction of N-H and C=O of the peptide bond leads to local regular structures such as α-helices and β-sheets • London dispersion – Medium-range weak attraction between all atoms contributes significantly to the stability in the interior of the protein • Electrostatic interactions – Long-range strong interactions between permanently charged groups – Salt-bridges, esp. buried in the hydrophobic environment strongly stabilize the protein Levels of Protein Structure Sequence Structure Globular Proteins Membrane Proteins Primary Structure of Proteins Primary Structure of Proteins Lone Amino Acid Amino Acid In Protein Structure of the Peptide Bond • Structure of the protein is partially dictated by the properties of the peptide bond • The peptide bond is a resonance hybrid of two canonical structures • The resonance causes the peptide bonds – to be less reactive compared to esters, for example – to be quite rigid and nearly planar – to exhibit a large dipole moment in the favored trans configuration Resonance in the Peptide Bond Was amide C-N bond length a clue? Bond Bond Length C-C 1.54 Å C-C Benzene 1.40 Å C=C 1.34 Å C≡C 1.20 Å C-N 1.49 Å C-N Amide 1.32 Å C=N 1.27 Å C≡N 1.14 Å Bond Type Single Resonant Double Triple Single Resonant Double Triple The Rigid Peptide Plane and the Partially Free Rotations • Rotation around the peptide bond is not permitted • Rotation around bonds connected to the alpha carbon is permitted • φ (phi): angle around the α-carbon—amide nitrogen bond • ψ (psi): angle around the α-carbon—carbonyl carbon bond • In a fully extended polypeptide, both ψ and φ are 180° The polypeptide is made up of a series of planes linked at α carbons Secondary Structures • Secondary structure refers to a local spatial arrangement of the polypeptide backbone • Two regular arrangements are common: • The α helix – stabilized by hydrogen bonds between nearby residues • The β sheet – stabilized by hydrogen bonds between adjacent segments that may not be nearby • Irregular arrangement of the polypeptide chain is called the random coil The α Helix • Helical backbone is held together by hydrogen bonds between the backbone amides of an n and n+4 amino acids • Right-handed helix with 3.6 residues (5.4 Å) per turn • Peptide bonds are aligned roughly parallel with the helical axis • Side chains point out and are roughly perpendicular with the helical axis What is a right-handed helix? The α Helix: Top View • The inner diameter of the helix (no side chains) is about 4–5 Å • Too small for anything to fit “inside” • The outer diameter of the helix (with side chains) is 10–12 Å • Happens to fit well into the major groove of dsDNA • Residues 1 and 8 align nicely on top of each other • What kind of sequence gives an α helix with one hydrophobic face? Secondary Structure of Proteins: α-Helix C=O (nth) --- H—N (n+4th) hydrogen bond n+4 = α Helix n+3 = 310 Helix n+5 = π Helix Sequence affects helix stability • Not all polypeptide sequences adopt α-helical structures • Small hydrophobic residues such as Ala and Leu are strong helix formers • Pro acts as a helix breaker because the rotation around the N-Ca bond is impossible • Gly acts as a helix breaker because the tiny R-group supports other conformations • Attractive or repulsive interactions between side chains 3–4 amino acids apart will affect formation The Helix Dipole • Recall that the peptide bond has a strong dipole moment – Carbonyl O negative – Amide H positive • All peptide bonds in the α helix have a similar orientation • The α helix has a large macroscopic dipole moment • Negatively charged residues often occur near the positive end of the helix dipole α-Helix: Dipole Moment i i+4 α-Helix: Coiled-Coil Structure Residues a and d : Nonpolar Residues b, c, e, f, g : Polar β Sheets • The planarity of the peptide bond and tetrahedral geometry of the α-carbon create a pleated sheet-like structure • Sheet-like arrangement of backbone is held together by hydrogen bonds between the backbone amides in different strands • Side chains protrude from the sheet alternating in up and down direction Secondary Structure of Proteins: β-Sheet Parallel and Antiparallel β Sheets • Parallel or antiparallel orientation of two chains within a sheet are possible • In parallel β sheets the H-bonded strands run in the same direction – Resulting in bent H-bonds (weaker) • In antiparallel β sheets the H-bonded strands run in opposite directions – Resulting in linear H-bonds (stronger) Secondary Structure of Proteins: β-Sheet Antiparallel β sheet Parallel β sheet β Turns • β turns occur frequently whenever strands in β sheets change the direction • The 180° turn is accomplished over four amino acids • The turn is stabilized by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence • Proline in position 2 or glycine in position 3 are common in β turns Ramachandran Plot for Secondary Structures ψ φ G.N. Ramachandran, C. Ramakrishnan & V. Sasisekharan (1963): Stereochemistry of polypeptide chain configurations. In: J. Mol. Biol. vol. 7, p. 95-99. Distribution of φ and ψ Dihedral Angles • Some φ and ψ combinations are very unfavorable because of steric crowding of backbone atoms with other atoms in the backbone or side chains • Some φ and ψ combinations are more favorable because of chance to form favorable H-bonding interactions along the backbone • A Ramachandran plot shows the distribution of φ and ψ dihedral angles that are found in a protein • shows the common secondary structure elements • reveals regions with unusual backbone structure Ramachandran Plot Secondary Structure: Amino Acid Propensities Helix Breakers Proline Isomers • Most peptide bonds not involving proline are in the trans configuration (>99.95%) • For peptide bonds involving proline, about 6% are in the cis configuration. Most of this 6% involve β-turns • Proline isomerization is catalyzed by proline isomerases Circular Dichroism (CD) Analysis • CD measures the molar absorption difference ∆ε of leftand right-circularly polarized light: ∆ε = εL – εR • Chromophores in the chiral environment produce characteristic signals • CD signals from peptide bonds depend on the chain conformation Group Discussion • There is a transmembrane α-helix with 28 amino acids that spans the cell membrane exactly. What is the width of the cell membrane? • Why can the Proline residue be considered a helix breaker? Draw to explain your answer. • Why is large portion of Ramachandran space empty for proteins? Protein Tertiary Structure • Tertiary structure refers to the overall spatial arrangement of atoms in a protein • Stabilized by numerous weak interactions between amino acid side chains. − Largely hydrophobic and polar interactions − Can be stabilized by disulfide bonds • Interacting amino acids are not necessarily next to each other in the primary sequence. • Two major classes – Fibrous and globular (water or lipid soluble) Fibrous Proteins Fibrous Proteins Globular Proteins Water-soluble (Soluble Proteins) Lipid-soluble (Membrane Proteins) Fibrous Proteins: From Structure to Function Structure of α-Keratin in Hair Chemistry of Permanent Waving Structure of Collagen • Collagen is an important constituent of connective tissue: tendons, cartilage, bones, cornea of the eye • Each collagen chain is a long Gly- and Pro-rich lefthanded helix • Three collagen chains intertwine into a right-handed superhelical triple helix • The triple helix has higher tensile strength than a steel wire of equal cross section • Many triple-helices assemble into a collagen fibril Collagen Fibrils Silk Fibroin • Fibroin is the main protein in silk from moths and spiders • Antiparallel β sheet structure • Small side chains (Ala and Gly) allow the close packing of sheets • Structure is stabilized by – hydrogen bonding within sheets – London dispersion interactions between sheets Spider Silk • Used for webs, egg sacks, and wrapping the prey • Extremely strong material – stronger than steel – can stretch a lot before breaking • A composite material – crystalline parts (fibroin-rich) – rubber-like stretchy parts Supersecondary Structures (Motifs or Folds) • Specific arrangement of several secondary structure elements – All alpha-helix – All beta-sheet – Both • Motifs can be found as reoccurring structures in numerous proteins • Proteins are made of different motifs folded together Supersecondary Structures (Motifs) β-hairpin Greek Key Helix-turn-Helix Jelly Roll β−β−α (Zinc Finger) Conservation of Supersecondary Motifs (Helix-loop-Helix) Yeast (Green) Drosophila (Red) DNA Binding Domains (Homeodomains) of organisms separated by a BILLION years! Black dots (•) mark conserved residues. Secondary Structure Packing (Tertiary Structure Classification) 1. Alpha proteins (α) 2. Beta proteins (β) 3. Alpha and beta proteins (α/β) Mainly parallel beta sheets (betaalpha-beta units) (α+β) Mainly antiparallel beta sheets (segregated alpha and beta regions) 4. Multi-domain proteins (alpha and beta) Folds consisting of two or more domains belonging to different classes 5. Membrane and cell surface proteins and peptides Driving Force for Tertiary Structures (Globular Proteins) I. Compact structures that minimize the exposure of hydrophobic residues to solvent. (the hydrophobic effect) Driving Force for Tertiary Structure (Globular Proteins) II. Buried H-bonding and salt-bridge groups should be paired up. Tertiary Structure Highlights Serine proteases Myoglobin NAD Binding Domain and Immunoglobin Quaternary Structure • Quaternary structure is formed by the assembly of individual polypeptides into a larger functional cluster Quaternary Structures Same driving forces apply as in tertiary structure formation. Hydrophobic Effect Polar Residue Pairing More H-bonds found buried! Nature uses symmetry so optimal use of resources! Quaternary Structure Highlights • Two identical protein subunits binding together to form a symmetric protein dimer. • The Cro repressor protein from bacteriophage lambda binds to DNA to turn off viral genes. • Its two identical subunits bind head-to-head, held together by a combination of hydrophobic forces (blue) and a set of hydrogen bonds (yellow region). • Hemoglobin made of 2 α-globin (red) and 2 β-globin (blue) units. • Heme group that binds to Oxygen shown in Green. Quaternary Structure Highlights The Src protein with four different domains • Two of the domains form a protein kinase enzyme. • The other two (SH2 and SH3) domains perform regulatory functions. • ATP substrate in RED. • The site that binds ATP is positioned at the interface of the two domains that form the kinase. Quaternary Structure Highlights The enzyme neuraminidase exists as a ring of four identical polypeptide chains. The small diagram shows how the repeated use of the same binding interaction forms the structure. Four fibronectin type 3 modules from the extracellular matrix molecule fibronectin form a linear quaternary structure. Viruses (Quaternary and a lot more!) Capsids of some viruses: (A) Tomato bushy stunt virus; (B) poliovirus (C) simian virus 40 (SV40); (D) satellite tobacco necrosis virus. Structure of a Spherical Virus Identical protein subunits pack together to create a spherical shell (a capsid) that encloses the viral genome, composed of either RNA or DNA. For geometric reasons, no more than 60 identical subunits can pack together in a precisely symmetric way. The tomato bushy stunt virus (TBSV) shown here, for example, is a spherical virus about 33 nm in diameter formed from 180 identical copies of a 386 amino acid capsid protein plus an RNA genome of 4500 nucleotides. To construct such a large capsid, the protein must be able to fit into three somewhat different environments, each of which is differently colored in the virus particle shown here. Protein Structure Methods: X-Ray Crystallography Steps needed • Purify the protein • Crystallize the protein • Collect diffraction data • Calculate electron density • Fit residues into density Pros • No size limits • Well-established Cons • Difficult for membrane proteins • Cannot see hydrogens Structure Methods: Biomolecular NMR Steps needed • Purify the protein • Dissolve the protein • Collect NMR data • Assign NMR signals • Calculate the structure Pros • No need to crystallize the protein • Can see many hydrogens Cons • Difficult for insoluble proteins • Works best with small proteins Intrinsically Disordered Proteins • Contain protein segments that lack definable structure • Composed of amino acids whose higher concentration forces less-defined structure – Lys, Arg, Glu, and Pro • Disordered regions can conform to many different proteins, facilitating interaction with numerous different partner proteins Intrinsically Disordered Proteins Proteostasis Maintenance of cellular protein activity is accomplished by the coordination of many different pathways. Protein Stability and Folding • A protein’s function depends on its 3D-structure • Loss of structural integrity with accompanying loss of activity is called denaturation • Proteins can be denatured by: • heat or cold • pH extremes • organic solvents • chaotropic agents: urea and guanidinium hydrochloride Ribonuclease Refolding Experiment • Ribonuclease is a small protein that contains 8 cysteines linked via four disulfide bonds • Urea in the presence of 2-mercaptoethanol fully denatures ribonuclease • When urea and 2-mercaptoethanol are removed, the protein spontaneously refolds, and the correct disulfide bonds are reformed • The sequence alone determines the native conformation • Quite “simple” experiment, but so important it earned Chris Anfinsen the 1972 Chemistry Nobel Prize How can proteins fold so fast? • Proteins fold to the lowest-energy fold in the microsecond to second time scales. How can they find the right fold so fast? • It is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest-energy one is found (Levinthal’s paradox) • Search for the minimum is not random because the direction toward the native structure is thermodynamically most favorable Proteins folding follow distinct paths Proteins folding follow distinct paths Protein Folding Movie Simulation of millisecond protein folding: NTL9 (from Folding@home) Chaperones prevent misfolding Chaperonins facilitate folding Protein misfolding is the basis of numerous human diseases Group Discussion • Curling or straightening of hair is permanent. Why? • Second "For Thought" Video: Simulation of millisecond protein folding: NTL9 (from Folding@home) If one of the amino acids in this protein was mutated to a different amino acid (like in a disease), how may this mutation affect the protein structure as well as function or even cause disease? Ser Thr Ser Pro Leu Ile Ala Ser Asp Glu Asp Arg Gly Ala Trp Phe Group Discussion: Effect of mutation Ser Thr Ser Pro Leu Ile Ala Ser Asp Glu Asp Arg Gly Ala Trp Phe Chapter 4: Summary • the two most important secondary structures – α helices – β sheets • tertiary and quaternary structures • how properties and function of fibrous proteins are related • how to determine three-dimensional structures of proteins • how proteins fold?