1. CH339K Proteins: Higher Order Structure

2. Higher Levels of Protein Structure

3. Repetitive background: -N-C-C-N-C-C- Side chains hang off the backbone

4. The shape of the peptide chain can be defined by the three consecutive bond torsional angles BondRotationTorsion angle defined NH to C  freephi C  to C=Ofreepsi C=O to NHrigid planaromega

5. Since  is constrained, only  and  can vary There are steric restrictions on what values they can assume

6. Permissable  Angles (Ramachandran Plot)

7. Secondary Structures Represent interactions among backbone atoms Examples   -helices  Other helices   -sheets   - and  -turns  These structures have characteristic  and  angles

8. H bonds between carbonyl O of residue n amide H of residue n+4  - helix

9. R/V Alpha Helix Woods Hole Oceanographic Institute 1966-2011

10. Helical parameters – Pitch and Rise

11. Backbone forms helix Side chains extend outwards  ≈ -57 o  ≈ -47 o 3.6 residues/turn

12. Helix Types  -helix: C=O H-bonded to NH of residue n+4 (aka 3.6 13 helix) 3 10 helix: C=O H-bonded to NH of residue n+3 –(  ≈ -49 o  ≈ -26 o )  -helix: C=O H-bonded to NH of residue n+5 (aka 4.1 16 helix) (  ≈ -57 o  ≈ -80 o )

13. Helix terminology H-bond makes a closed loop from amide H through backbone through carbonyl O Define helix by (a) Nbr of residues per turn (e.g. 3.6 for  -helix) (b) Nbr of atoms in the loop (e.g. 13 for  -helix)

15.  -Sheets Can be thought of as helix with two residues per helix Backbone atoms run in a plane Side chains extend up and down from plane   ≈ -110 o to -140 o   ≈ +110 o to +135 o

20. C=O of residue n with N-H of residue n+3

21. Gamma Turns: C=O of residue n with N-H of residue n+2

22.  Angles for Secondary Structures NOTE: Left-handed  -helix has  = +57,  = +47

23. Ramachandran Plot: Blue areas are permitted  and  angles

24. Ramachandran plot for pyruvate kinase

25. Tertiary Structures Determined by side chain interactions –Salt links –H-Bonds –Disulfides –Hydrophobic interactions Fibrous Proteins Globular Proteins

26. Fibrous Proteins Keratin  -keratin: hair, horns, and hoofs of mammals  -keratin: scales, claws and shells of reptiles, beaks and claws of birds, porcupine quills

27.  -keratin Lots of Ala, Gly, Cys All a-helix Right handed Left handed

29. Disulfides in the Barber Shop Sodium thioglycolateVarious peroxides

30. Fibrous Proteins - Fibroin 75-80% Ala/Gly 15% Ser

33. Within a fiber: crystalline regions are separated by amorphous regions.

34. Fibrous Proteins - Collagen Left handed helix of tropocollagen forms right handed triple helix of collagen.

36. Hydroxyproline participates in H-bonding between tropocollagen chains

37. In the absence of vitamin C, reaction 2 oxidizes Fe 2+ to Fe 3+. (1) (2)

38. Lack of hydroxyls causes serious destabilization of the triple helix

39. Scurvy Weakness Paleness Sunken eyes Tender gums and/or tooth loss Muscular pain Reopening of old wounds or sores Internal bleeding Loss of appetite Bruising easily Weight loss; inability to gain weight Diarrhea Increased heart rate Fever Irritability Aching and swelling in joints Shortness of breath Fatigue Arrrrr…

40. British Empire at its Peak A healthy navy is a victorious navy (of course, my ancestors were less than thrilled…)

41. Protein structure cartoons  -helixAntiparallel  -sheet

42. Globular Proteins (examples)

44. Motifs – common stable folding patterns Found in proteins w/ different functions result from the physics and chemistry of the structure

45. More motifs

46. Ricin B chain Two domains Each domain is a trefoil 3 repeats of a sheet-loop structure i.e. 6 repeats of a primitive fold Domains – Common patterns found in different proteins Typically have similar function Caused by evolution (gene recombination / duplication)

47. C-rich Domain of Earthworm Mannose Receptor Fibroblast Growth Factor

48. Domains can be shared among proteins

50. Quaternary Structure (Hemoglobin)

51. Folding Energetics Favoring FoldingFavoring Unfolding -  H from formation of interchain H- bonds and salt links High –  S from going from unfolded  folded state +  S from disulfide formationHigh +  from breaking H-bonds with solvent Enormous +  S from burial of hydrophobic side chains in the interior

52. Denaturation

53. Denaturants Heat (increases negative T  S contribution) Cold (H 2 O becomes less disordered) Pressure High and low pH (electrostatic effects) Low-polarity and non-polar solvents (e.g. EtOH) Chaotropes (urea, guanidinium chloride)

54. Milliseconds to seconds Rapid nucleation and hydrophobic collapse to “molten globule” Slower compaction into the native state Disulfides lessen negative  S Larger proteins often have multiple structural domains Each domain folds by mechanisms similar to those above. Once folded, domains reshuffle to form the final native structure. Protein Folding

55. Effects of disulfides on folding Denaturation of gelsolin with (open circles) and without (solid circles) 1 mM dithiothreitol From: Isaacson, Weeds, and Fersht (1999) Proc. Nat. Acad. Sci. 96: 11247-11252.

56. Rapid 2 o structure formation Collapse to molten globule Reshuffle to final state

58. Heat Shock Proteins Nucleotide binding domain – binds ATP and hydrolyzes it to ADP. Protein binding domain – contains a groove with an affinity for neutral, hydrophobic amino acid residues. The groove can interact with peptides up to seven residues in length. C-terminal domain –acts as a 'lid' for the substrate binding domain. When an Hsp70 protein is ATP bound, the lid is open and peptides bind and release relatively rapidly. When Hsp70 proteins are ADP bound, the lid is closed, and peptides are tightly bound to the protein binding domain.

59. Chaperonins - GroEL

61. Simpler Picture of GroEL Action

62. A Problem in Folding Creutzfeldt-Jakob Disease, Mad Cows, and the Laughing Disease of the New Guinea Cannibals Initially, persons may have difficulty sleeping, experience depression, problems with muscular coordination, impaired vision, and personality and behavioral changes such as impaired memory, judgment, and thinking. As the disease progresses, mental impairment becomes severe and involuntary muscle jerks (myoclonus) often occur along with blindness. Eventually, the ability to move or speak is lost and the person enters a coma until death occurs. (100% fatal)

63. Kuru Scrapie BSE

64. Spongioform Encephalopathy – your brain on CJD NormalModerateSevere

65. Brain atrophy in CJD – you’re usually dead before it reaches this stage

66. Prion Proteins PrPc Normal cellular prion protein (PrPc) – mostly  - helical C-terminal domain

67. Prion Proteins – C terminal region PrPcPrPsc

68. Vrious Mutations in CJD Prion Proteins CodonAmino acid changeReference 178aspartate to asparagineGoldfarb 1991b 180valine to isoleucineKitamoto 1993a 188threonine to alanineCollins 2000 196glutamate to lysinePeoc’h 2000 200**glutamate to lysineGoldgaber 1989 203valine to isoleucinePeoc’h 2000 208arginine to histidineMastrianni 1996 210valine to isoleucinePocchiari 1993 211glutamate to glutaminePeoc’h 2000 232methionine to arginineKitamoto 1993a