Proteins the primary biological macromolecules of living organisms

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1 Proteins the primary biological macromolecules of living organisms

2 Protein structure and folding

3 Primary Secondary Tertiary Quaternary structure of proteins

4 Structure of Proteins Protein molecules adopt a specific 3-dimensional conformation in the aqueous solution 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 interactions within the protein There is a cost in conformational entropy during folding of the protein into one specific native fold

5 A simulated folding pathway

6 Interactions in Proteins Hydrophobic effect Hydrogen bonds London dispersion Electrostatic interactions

7 Structure of the Peptide Bond Structure of the protein is partially dictated by the properties of the peptide bond

8 The planar peptide bond Each peptide bond has some (~ 40%) double-bond character due to resonance and cannot rotate. The peptide bond is a resonance hybrid of two canonical structures

9 Structure of the Peptide Bond The resonance causes the peptide bonds be less reactive compared to e.g. esters be quite rigid and nearly planar exhibit large dipole moment

10 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

11 The Rigid Peptide Plane and the Partially Free Rotations Φ (phi): angle around the - carbon amide nitrogen bond (C-C α -N-C) y (psi): angle around the - carbon carbonyl carbon bond (N-C-C α -N) In fully extended polypeptide, both y and f are 180

12 Distribution of f and y Dihedral Angles Some f and y combinations are very unfavorable because of steric crowding of backbone atoms with other atoms in the backbone or side-chains Some f and y combinations are more favorable because of chance to form favorable H-bonding interactions along the backbone Ramachandran plot shows the distribution of f and y dihedral angles that are found in a protein shows the common secondary structure elements reveals regions with unusual backbone structure

13 Ramachandran Plot

14 Ramachandran Plot

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16 Secondary Structures Secondary structure refers to a local spatial arrangement of the polypeptide chain Two regular arrangements are most common: The helix stabilized by hydrogen bonds between nearby residues The sheet stabilized by hydrogen bonds between adjacent segments that may not be nearby Irregular arrangement of the polypeptide chain is called the random coil

17 The helix

18 The helix Right-handed helix with 3.6 residues (5.4 Å) per turn Helical backbone is held together by hydrogen bonds between the nearby backbone amides Side chains point out and are roughly perpendicular with the helical axis

19 The 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 Å Happens to fit well into the major groove of dsdna

20 Sequence Affects Helix Stability Not all polypeptide sequences adopt -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-C a bond is impossible Gly acts as a helix breaker because the tiny R- group (hydrogen) allows other conformations

21 Sheets

22 Sheets The backbone is more extended with dihedral angles in the range of ( 90 < y < 180 )

23 Sheets The planarity of the peptide bond and tetrahedral geometry of the -carbon create a pleated sheet-like structure Sheet-like arrangement of backbone is held together by hydrogen bonds between the more distal backbone amides Side chains protrude from the sheet alternating in up and down direction

24 Parallel and Antiparallel Sheets In parallel sheets the H-bonded strands run in the same direction

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26 Parallel and Antiparallel Sheets In antiparallel sheets the H-bonded strands run in opposite directions

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29 Turns -turns occur frequently whenever strands in 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 -turns

30 Structures of β turns

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32 Protein Tertiary Structure Tertiary structure refers to the overall spatial arrangement of atoms in a polypeptide chain or in a protein One can distinguish two major classes fibrous proteins typically insoluble; made from a single secondary structure globular proteins water-soluble globular proteins lipid-soluble membraneous proteins

33 Structure of collagen fibrous protein

34 Structure of whale myoglobin globular protein

35 An ABC transporter of E. coli globular membrane protein

36 Motifs (folds) - Protein Folding Patterns Common arrangements of several secondary structure elements

37 How many folds? The number of unique folds in nature is fairly small (possibly a few thousands) 90% of new structures submitted to PDB in the past decade have similar structural folds in PDB, practically no new folds in the last three years

38 Quaternary Structure Quaternary structure is formed by spontaneous assembly of individual polypeptide subunits into a larger functional cluster

39 Dimer Cro protein of bacteriophage lambda two identical subunits Tetramer Human hemoglobin two alpha(red) two beta(yellow) subunits 4 heme groups

40 Primary Secondary Tertiary Quaternary structure Summary (a) Linear Sequence of amino acids. (b) Local folding into specific peptide backbone conformations. Stabilized by h- bonding and other non-covalent interactions between atoms in peptide backbone. (c) Final folded 3-dimensional structure of a single polypeptide chain. Stabilized by noncovalent interactions between amino acid side chain residues. (d) Specific aggregation of two or more polypeptide chains. Often characterized by its symmetry.

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42 Protein Stability and Folding A protein s function depends on its threedimensional 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 reduction of disulfide bonds by Mercaptoethanol

43 Renaturation of unfolded, denatured protein ribonuclease

44 Ribonuclease Refolding Experiment Ribonuclease is a small protein that contains 8 cysteins 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

45 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)

46 Levinthal s paradox: There are approximately possible conformations for a typical protein (~125 amino acids). Even if it took only sec to try out each conformation, it would take years to try a significant fraction of them. Obviously folding does not happen randomly direction toward the native structure is thermodynamically most favorable

47 The thermodynamics of protein folding depicted as a free-energy funnel

48 Chaperonins Special class of molecular chaperones that facilitate protein folding About 10 to 15% of proteins in E. coli require chaperonins to right folding Require ATP

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50 Other examples of assisted molecular processes in folding and posttranslational modification of proteins Disulfide crosslinking bonding disulfide isomerase cis trans isomerization of proline prolyl cistrans isomerase Hydroxylation of Proline in collagen prolyl 4- hydroxylase (ascorbate demand)

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52 Protein function

53 Functions of Globular Proteins Storage of ions and molecules myoglobin, ferritin Transport of ions and molecules hemoglobin, serotonin transporter Defense against pathogens antibodies, cytokines Muscle contraction actin, myosin Biological catalysis - enzymes chymotrypsin, lysozyme

54 Example of Protein Function and Ligand Binding Myoglobin single polypeptide oxygen storage in tissues, muscle Hemoglobin tetramer of two alpha and two beta subunits transports oxygen from lungs to tissues

55 Ligand Binding Binding - reversible, transient process of chemical equilibrium: A + B AB A molecule that binds is called a ligand (typically a small molecule) A region in the protein where the ligand binds is called the binding site Ligand binds via non-covalent forces, which enables the interactions to be transient

56 Function of Myoglobin Organisms need to store oxygen for metabolism Generaly - protein side-chains lack affinity for O 2 Heme - Fe 2+ in free heme could be oxidized to Fe 3+ Heme is bound to protein, Fe 3+ is protected by a His residue In mammals, myoglobin is the main oxygen storage protein

57 Structures of Porphyrin and Heme

58 Structure of Myoglobin

59 Binding of Carbon Monoxide CO has similar size and shape to O 2 ; it can fit to the same binding site CO binds to heme over 20,000 times better than O 2 because the carbon in CO can be donated a lone electron pair to vacant d-orbitals on the Fe 2+ Protein pocket decreases affinity for CO, but is still binds about 250 times better than oxygen CO is highly toxic as it competes with oxygen. It blocks the function of myoglobin, hemoglobin, and mitochondrial cytochromes that are involved in oxidative phosphorylation

60 Example of a Binding Pocket O 2 and CO in hemoglobin and myoglobin

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62 Could Myoglobin Work as Good O 2 Transporter? po 2 in lungs is about 13 kpa: it sure binds oxygen well po 2 in tissues is about 4 kpa: it will not release it!

63 Simple change in the affinity would help but is not the ideal solution Cooperation of two binding (affinity) states is indeed better solution

64 For Effective Transport Affinity Must Vary with po 2 po 2 in lungs is about 13 kpa: it sure binds oxygen well po 2 in tissues is about 4 kpa: it releases about half of it at ph 7.6

65 How Can Affinity to Oxygen Change Like This? Must be a protein with multiple binding sites Binding sites must be able to interact with each other This phenomenon is called cooperativity Positive cooperativity can be recognized by sigmoidal binding curves

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67 Hemoglobin Hemoglobin is a tetramer of two different subunits ( 2 2) Each subunit is similar to myoglobin

68 Hemoglobin Binding sites must be able to interact with each other

69 Subunit Interactions: Details Binding sites must be able to interact with each other Example of some interactions Interactions can be between subunits and also within one subunit

70 Conformational Change is Triggered by Oxygen Binding

71 Hemoglobin is an allosteric protein allosteric proteins change their activity due to induced conformational changes Hemoglobin - two affinity states induced by ligand binding

72 Molecular disease of Hemoglobin

73 Sickle-Cell anemia Mutation of single amino acid Val instead of Glu at position 6 in beta chain Replacement of charged Glu for Val makes hydrophobic contact on the surface Hemoglobin aggregates

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76 Another examples of proteins

77 Fibrous Proteins: From Structure to Function Function Structure Example Tough, rigid, Cross-linked -helixes -keratin hard (nails, horns) Rigid linker (S S) Tensile strength, Cross-linked triple-helixes Collagen non-stretching Flexible linker (Lys-HyLys) (tendons, cartilage) Soft, flexible Non-covalently held -sheets non-stretchy van der Waals interaction Silk fibroin (egg sac, nest, web)

78 Structure of -Keratin in Hair

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80 Chemistry of Permanent Waving

81 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 left-handed helix Three collagen chains intertwine into a righthanded superhelical triple helix The triple helix has higher tensile strength than a steel wire of equal cross section Many triple-helixes assemble into a collagen fibril

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83 4-Hydroxyproline in Collagen Forces the proline ring into a favorable folding Offer more hydrogen bonds between the three strands of collagen The post-translational processing is catalyzed by prolyl hydroxylase and requires -ketoglutarate, molecular oxygen, and ascorbate (vitamin C)

84 Silk Fibroin Fibroin is the main protein in silk from silk moths and spiders Antiparallel 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

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