Biochemistry - I SPRING Mondays and Wednesdays 9:30-10:45 AM (MR-1307) Lectures 3-4. Based on Profs. Kevin Gardner & Reza Khayat

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1 Biochemistry - I Mondays and Wednesdays 9:30-10:45 AM (MR-1307) SPRING 2017 Lectures 3-4 Based on Profs. Kevin Gardner & Reza Khayat 1

2 Outline Overview of protein structure Peptide bonds Secondary structure The Ramachandran plot Protein tertiary and quaternary structures Multi-domain proteins Protein complexes and symmetry Protein Folding Circular dichroism (CD) Determination of 3D protein structure Molecular Chaperones Defects in protein folding and human diseases 2

3 Four Levels of Protein Structure Also β-sheet, random coil Established by covalent interactions: peptide bonds Chapter 3: Amino acids, peptides and proteins 3

4 Overview of Protein Structure 3D structure of protein is determined by AA sequence Structure determines function Most isolated proteins adopt a stable fold Common structural patterns/folds define the 3D protein space Folding: Non-covalent interactions maximized 4

5 Peptide Bond: Structural Implications The peptide bond has some double bond character due to resonance; hence, the bond is not free to rotate and is therefore almost planar Proline: 6% in cis configuration 5

6 Steric Restrictions on the Peptide Bond The planar nature of the peptide bond restricts the possible conformations that a polypeptide chain can assume. A dihedral angle is defined between three successive chemical bonds Two dihedral angles are sufficient to define the conformation of the polypeptide chain - phi (φ) and psi (ψ) C N ±180 N C Omega (ω) is between C-N = peptide bond Å (Angstrom) = 0.1 nm (nanometer); 10-9 meter = nm N (c) (d) 0 N 6

7 Dihedral Angle - Definition Dihedral angle: The angle between two planes, where each plane passes through three atoms. Determined from four atoms. β plane: Passes through C 3, C 2, and H 4 Describes the angle between planes α and β, or the angle between the C3-H4 and C2-H1 bonds α plane: Passes through C 3,C 2, and H 1 Due to steric hindrance between the AA backbone and sidechains, many of the dihedral angles found in AAs are limited to specific ranges 7

8 Steric Clashes Restrict the Dihedral Angles Conformation in which both ϕ and φ are zero degrees is prohibited due to steric clash 8

9 Four Levels of Protein Structure Also β-sheet, random coil established by non-covalent interactions: H-bonds and steric clashes Chapter 3: Amino acids, peptides and proteins 9

10 Secondary Structure Polypeptide chains adopt limited conformations due to the planar arrangement of the polypeptide chain and steric interactions between the main- and side-chains In 1951 Linus Pauling, Robert Corey, and Herman Branson proposed two general conformations as the structural conformations of a protein - secondary structure A helical conformation referred to as α-helix An extended conformation referred to as β-strand Both types of structure satisfy hydrogen bonding requirements of protein backbone atoms 10

11 Secondary structure: α-helix The α-helix (right-handed) is shown wrapped around an imaginary axis R-groups protrude outward The repeating unit is a single turn of the helix extending ~ 5.4 A 3.6 AA residues per turn, 1.5 A rise per AA H-bonds between amide protons of residue n and carbonyl of residue n+3 or n+4 Every peptide bond has potential to participate in H-bonding different representations: A. ball-and-stick with ribbon outline B. ball-and-stick looking down helix C. space-filling model D. helical wheel 11

12 Secondary structure: α-helix Helix-breaking residues (1) adjacent turns of Glu/Asp (negative charges repel) (2) adjacent turns of Lys/Arg (positive charges repel) (3) bulky R-groups (4) Pro - unable to H-bond and restricted dihedral angles 12

13 Secondary Structure: α-helix The electric dipole of a peptide bond (below, left) is transmitted along the α-helix (below, right) resulting in an overall helix dipole H-bond Protein structures have evolved to take advantage of this dipole moment for protein function by properly placing α-helices 13

14 Secondary Structure: α-helix left right C-term α-helices are handed - only right handed helices are seen in proteins with L-amino acids N-term 14

15 Amphipathic Helix Front. Cell. Infect. Microbiol., 2014, 4:167 All hydrophobic residues face in one direction and all polar residues face in the opposite direction 15

16 Secondary Structure: β-strand and β-sheet The backbone is extended in a zigzag shape Hydrogen-bonds bonds are formed between adjacent segments of polypeptide chain The chains can be parallel or antiparallel (same or different amino to carboxyl orientation), and may form with one or more peptide chains Peptide plane 1 amino acid has 2 hydrogen-bonds to a single amino acid 1 amino acid has 2 hydrogen-bonds to two amino acids 16

17 Secondary Structure: β- and γ-turns Globular proteins have numerous turns where the polypeptide reverses direction β-turns type I and II have 4 AA that make a 180 o turn γ-turn has 3 AA (not shown) Gly (flexible) and Pro (assumes cis configuration) are well suited for a tight turn Predominantly found on protein surface as residues 2 and 3 need to H-bond with water ω=180 ω=0 17

18 Ramachandran Plots Simple way to represent the backbone dihedral angles found in a protein Initially calculated for small model system: N-acetyl-L-alanine-methylamide 18

19 Ramachandran Plots Derived in 1963 from hard-sphere calculations (left) of small polypeptides to predict allowed dihedral angles for predicted secondary structures Hundreds of high resolution protein crystal structures (right) support the calculations from 1963 The Ramachandran plot is required to test the modeled geometry of structure Describes the dihedral angles phi (φ) and psi (ψ) of a polypeptide 19

20 Four Levels of Protein Structure Also β-sheet, random coil established by non-covalent interactions (exception: disulfide bonds) Chapter 3: Amino acids, peptides and proteins 20

21 Partitioning of Hydrophobic, Hydrophilic AA The hydrophobic effect is the strongest driving force for protein folding 21

22 Forces Leading to Protein Folding Electrostatic/ionic Hydrogen bonds Disulfide bonds Dispersion forces 22

23 Factors Contributing to Protein Folding Rigidity of backbone determines the secondary structure Torsion or rotation around dihedrals C-N bond: phi (φ) C-C bond: psi (ψ) Steric hindrance Most Pro Least Gly Interactions among amino acids Electrostatic interactions Hydrogen bonds Disulfide bonds Dispersive forces Steric hindrance (Van Der Waals Forces) Amino acid properties Hydropathy index for side chains. A number representing the hydrophobic or hydrophilic properties of an amino acids side chain Hydrophobic residues prefer to be buried inside of proteins, and hydrophilic residues are located on the surface of proteins interacting with the solvent (water) Helix propensity: A number representing the propensity of an AA to be in a helix 23

24 Globular proteins Cytoplasmic (soluble) proteins 75% is packed 25% is cavities (filled with water or empty) Unique tertiary structures (folds/motifs) Different combinations of β- sheets and α-helices can be present in a protein β-sheets and α-helices are found in different layers because they can not H-bond to one another using backbone H-bond donors or acceptors A domain is a compact, independent, and stable ensemble of packaged secondary structure elements Multiple domains can exist in a protein structure 24

25 Multi-domain Proteins CO2 NH3 NH3 Cat muscle pyruvate kinase CO2 Maltodextrin binding protein NH3 CO2 NH3 CO2 The different colored regions correspond to different domains Domains can have different structures and different stability Domains can be inserted within other domains, or be completely independent. 25

26 Fibrous Proteins: Keratins Mainly serve structural roles A single secondary structure element makes a tertiary quaternary structure Insoluble in water High hydrophobic AA content. Packed intra- and inter-molecularly α-keratins: Dry weight of hair, wool, nails, claws Right handed helix with a tighter turn due to hydrophobic AA packing intra- and inter-molecularly Helices pack to form left-handed super helix Stabilized by disulfide bonds 26

27 Fibrous Proteins: Collagen Collagen: Connective tissue such as tendons, cartilage, etc. Three left handed helices with 3 amino acids per turn pack together Requires repeating motif: Gly-X-Pro or Gly-X-(Pro-OH) - prolyl hydroxyls Final structure is right-handed super helix (2.9 Å rise per AA) 27

28 Protein Structure Representation Ribbon Representation Surface Representation Surface colored by electrostatic potential 28

29 Protein Denaturation Proteins can be denatured by heat, extremes of ph, organic solvents, urea, or guanidinium HCl In 1955, ribonuclease could be denatured and renatured to a catalytically active form in vitro Many proteins do not refold correctly in vitro Urea: Disrupts hydrophobic and H-bond interactions β-mercaptoethanol: cleaves four disulfide bonds to yield eight Cys residues Unfolding: Put enzyme in dialysis bag with urea and β-mercaptoethanol, enzyme denatures Refolding: Put bag in buffer without denaturing agents, enzyme refolds (concentration dependent). 8 Cys residues could form four disulfide bonds in 105 different ways (7*5*3*1=105), so weak bonding interactions must correctly position peptide to regenerate correct conformation. 29

30 Theories of Protein Folding Theory 1 (right): Local structures such as α-helices and β-sheets form spontaneously into super-secondary structures. This promotes long range interactions to form structure. Theory 2 (left): Spontaneous collapse driven by release of water molecules forming cage around hydrophobic AA to molten globule state, then other AA find correct conformation by sampling the energy landscape depicted on the left. Native structure has the lowest energy. 30

31 Molecular Chaperones: Type I The Hsp70 family, heat shock proteins of ~70 kda, refold heat denatured proteins Proteins that bind hydrophobic AA of unfolded globular proteins in solution, or on the ribosome then use ATP hydrolysis to refold the protein. These are simple systems composed of few polypeptide chains. 31

32 Molecular Chaperones: Type II These are sophisticated multicomponent systems. GroEL/GroES = chaperonins are required by the 10-15% of proteins that do not fold spontaneously; this number increases to approximately 30% after heat shock. Unfolded proteins go inside large cavity in GroEL and GroES complexes (heptamers), mechanism poorly understood. ATP hydrolysis is used to promote conformational changes of GroEL Surface and cut-away image 32

33 Most Proteins are Marginally Stable As proteins fold, strong forces favor, and oppose, the folding process For proteins to fold, the net sum of these forces must be energetically favorable (ΔG < 0). However, they are usually only slightly favorable: Typical protein stability: ~5-10 kcal/mol Thermal room temperature: ~0.5 kcal/mol Most proteins can be unfolded by: Slight changes in solution conditions (e.g. temperature, ph, salt), often leading to an irreversible loss of structure and function Amino acid mutations that remove favorable interactions (or generate unfavorable interactions) In either case, this often leads to an irreversible loss of structure and function 33

34 Protein Folding & Disease Inability to fold Toxic alternative folds cancer(s) p53 scrapie prion protein cystic fibrosis Marfan syndrome ALS scurvy MSUD CFTR Alzheimer s disease β-amyloid fibrillin cataracts crystallins superoxide dismutase collagen α-ketoacid DH Tay-Sachs disease retinitis pigmentosa Mislocation β- hexoamidase rhodopsin 34

35 Computer Simulations of Protein Folding 35

36 Four Levels of Protein Structure Also β-sheet, random coil established by non-covalent interactions (exception: disulfide bonds) Chapter 3: Amino acids, peptides and proteins 36

37 Quaternary Structure: Protein Complexes Multiple polypeptide chains (same or different) that oligomerize into a quaternary structure. Protomers in multimers may be arranged with ROTATIONAL or HELICAL symmetry, meaning that the protomers can be superimposed by rotation on rotational axis or by helical rotation. single rotational axis Cyclical symmetry (Cn) n is the highest symmetry group 37

38 Circular Dichroism (CD) Spectroscopy Structural asymmetry in a molecule gives rise to difference in absorption (via peptide bond) of left-handed versus right-handed plane-polarized light. Measurement of this difference is called circular dichroism (CD) spectroscopy. If the protein is folded into an orderly manner it will have peaks and regions of positive and negative values as shown below corresponding to the type of structure present. 38

39 X-ray Crystallography Electrons scatter X-rays and the amplitude of the wave scattered by an atom is proportional to its number of electrons. The scattered waves recombine -constructive interference. The ways in which the scattered waves recombine depends on the atomic arrangement. Diffraction pattern observed Strength: High resolution Weakness: Static structure 39

40 Nuclear Magnetic Resonance (NMR) Spectroscopy NMR-active nuclei - most commonly, 1 H, 13 C and 15 N will have a distinct chemical shift depending on its environment (covalent and non-covalent interactions) Specific magnetization is transferred into sample to probe particular environments Nuclei-nuclei distances can be measured and used to calculate the structure Strengths: Records dynamics in solution at individual atoms Weakness: Low sensitivity & signal overlap for large proteins 40

41 Cryo-Electron Microscopy (CryoEM) Images of protein/complex suspended in vitrified ice are taken using an electron microscope The images are averaged and used to calculate a 3D model of the protein or protein complex Strength: Records dynamics and high resolution Weakness: Large macromolecular complexes 41