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. Pauling, Corey, and Branson (1951) H bonds between carbonyl O of residue n amide H of residue n+4 Each amino acid is rotated 100 o from the previous one. 3.6 amino acids per turn  - 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. Idealized Helices

16.  - 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

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

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

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

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

25. Ramachandran plot for pyruvate kinase

26. Tertiary Structures Three dimensional folding Determined by side chain interactions –Salt links –H-Bonds –Disulfides –Hydrophobic interactions Fibrous Proteins Globular Proteins

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

28.  -keratin Lots of Ala, Gly, Cys All  -helix (well, almost) Right handed Left handed

30. Disulfides in the Barber Shop Sodium thioglycolateVarious peroxides

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

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

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

37. Hydroxyproline participates in H-bonding between tropocollagen chains

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

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

40. 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…

41. The Brits Found the Link Between Fruits and Veggies and Healthy Sailors Walk wide o' the Widow at Windsor, For 'alf o' Creation she owns: We 'ave bought 'er the same with the sword an' the flame, An' we've salted it down with our bones. (Poor beggars! -- it's blue with our bones!) The Widow at Windsor – Kipling We broke a King and we built a road -- A court-house stands where the reg'ment goed. And the river's clean where the raw blood flowed When the Widow give the party. (Bugle: Ta--rara--ra-ra-rara!) The Widow’s Party - Kipling

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

43. Protein structure cartoons  -helixAntiparallel  -sheet

44. Globular Proteins (examples)

46. Structural Motifs – “supersecondary structures” common stable folding patterns Formed from consecutive sequences Found in proteins w/ different functions result from the physics and chemistry of the structure

47. Greek Key Motif (antiparallel  -sheets) a)Schematic of motif b)Staphylococcus nuclease protein

48. More motifs

49. 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 – Stable, independently folded, globular units Common patterns found in different proteins Typically have similar function Caused by evolution (gene recombination / duplication) Frequently (not always!) correspond to exons in genes

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

51. Domains can be shared among proteins

53. Quaternary Structure (Hemoglobin)

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

55. Denaturation

56. 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)

57. 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

58. 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.

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

61. 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.

62. Chaperonins - GroEL

64. Simpler Picture of GroEL Action

65. 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)

66. Prion Diseases Human Prion Diseases Creutzfeldt-Jakob Disease (CJD) Variant Creutzfeldt-Jakob Disease (vCJD) Gerstmann-Straussler-Scheinker Syndrome Fatal Familial Insomnia Kuru Animal Prion Diseases Bovine Spongiform Encephalopathy (BSE) Chronic Wasting Disease (CWD) Scrapie Transmissible mink encephalopathy Feline spongiform encephalopathy Ungulate spongiform encephalopathy

67. Kuru Scrapie BSE

68. Spongioform Encephalopathy – your brain on CJD NormalModerateSevere

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

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

71. Prion Proteins – C terminal region PrPcPrPsc

72. Infectious Proteins The presence of one misfolded PrPsc causes adjacent PrPc to toggle into the misfolded state.

73. Various 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