CH339K

1
CH339K

• Proteins Higher Order Structure

2
Higher Levels of Protein Structure
3
Side chains hang off the backbone
Repetitive background -N-C-C-N-C-C-
4
The shape of the peptide chain can be defined by
the three consecutive bond torsional angles
Bond Rotation Torsion angle definedNH to Ca
free phi Ca to CO free psiCO to NH rigid
planar omega
5
Since w is constrained, only f and y can
vary There are steric restrictions on what values
they can assume
6
Permissable F-Y Angles(Ramachandran Plot)
7
Secondary Structures

• Represent interactions among backbone atoms
• Examples
• a-helices
• Other helices
• b-sheets
• b- and g-turns
• These structures have characteristic f and y
  angles

8
a-helix

• Pauling, Corey, and Branson (1951)
• H bonds between
• carbonyl O of residue n
• amide H of residue n4
• Each amino acid is rotated 100o from the previous
  one.
• 3.6 amino acids per turn

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
• f -57o
• -47o
• 3.6 residues/turn

12
Helix Types

• a-helix CO H-bonded to NH of residue n4 (aka
  3.613 helix)
• 310 helix CO H-bonded to NH of residue n3
• (f -49o y -26o)
• p-helix CO H-bonded to NH of residue n5 (aka
  4.116 helix)
• (f -57o y -80o)

13
Helix terminologyH-bond makes a closed loop from
amide H through backbone through carbonyl
ODefine helix by (a) Nbr of residues per turn
(e.g. 3.6 for a -helix)(b) Nbr of atoms in the
loop (e.g. 13 for a -helix)
14
(No Transcript)
15
Idealized Helices
16
b-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
• f -110o to -140o
• y 110o to 135o

17
(No Transcript)
18
(No Transcript)
19
(No Transcript)
20
(No Transcript)
21
CO of residue n with N-H of residue n3
22
Gamma Turns
CO of residue n with N-H of residue n2
23
F-y Angles for Secondary Structures
NOTE Left-handed a-helix has f 57, y 47
24
Ramachandran Plot Blue areas are permitted F and
Y 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

a-keratin hair, horns, and hoofs of
mammals b-keratin scales, claws and shells of
reptiles, beaks and claws of birds, porcupine
quills
28

• a-keratin
• Lots of Ala, Gly, Cys
• All a-helix (well, almost)

Right handed
Left handed
29
(No Transcript)
30
Disulfides in the Barber Shop
Sodium thioglycolate
Various peroxides
31
Fibrous Proteins - Fibroin
75-80 Ala/Gly 15 Ser
32
(No Transcript)
33
(No Transcript)
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.
36
(No Transcript)
37
Hydroxyproline participates in H-bonding between
tropocollagen chains
38
(1)
(2)
In the absence of vitamin C, reaction 2 oxidizes
Fe2 to Fe3.
39
Lack of hydroxyls causes serious destabilization
of the triple helix
40
Scurvy
Arrrrr

• 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

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 flowedWhen
  the Widow give the party.(Bugle
  Ta--rara--ra-ra-rara!)
• The Widows 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
a-helix
Antiparallel b-sheet
44
Globular Proteins (examples)
45
(No Transcript)
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 b-sheets)

1. Schematic of motif
2. Staphylococcus nuclease protein

48
More motifs
49

• 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

• 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

50
C-rich Domain of Earthworm Mannose Receptor
Fibroblast Growth Factor
51
Domains can be shared among proteins
52
(No Transcript)
53
Quaternary Structure (Hemoglobin)
54
Folding Energetics
Favoring Folding Favoring Unfolding
-DH from formation of intrachain H-bonds and salt links High DS from going from folded ? unfolded state
DS from disulfide formation High -DH from making H-bonds with solvent
Enormous DS from burial of hydrophobic side chains in the interior
55
Denaturation
56
Denaturants

• Heat (increases negative TDS contribution)
• Cold (H2O becomes less disordered)
• Pressure
• High and low pH (electrostatic effects)
• Low-polarity and non-polar solvents (e.g. EtOH)
• Chaotropes (urea, guanidinium chloride)

57
Protein Folding

• Milliseconds to seconds
• Rapid nucleation and hydrophobic collapse to
  molten globule
• Slower compaction into the native state
• Disulfides lessen negative DS
• 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.

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 2o structure formation
Collapse to molten globule
Reshuffle to final state
60
(No Transcript)
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
63
(No Transcript)
64
Simpler Picture of GroEL Action
65
A Problem in FoldingCreutzfeldt-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

• Scrapie

• Kuru

• BSE

68
Spongioform Encephalopathy your brain on CJD
Normal
Moderate
Severe
69
Brain atrophy in CJD youre usually dead before
it reaches this stage
70
Prion Proteins
Normal cellular prion protein (PrPc) mostly
a-helical C-terminal domain
PrPc
71
Prion Proteins C terminal region
PrPc
PrPsc
72
Infectious ProteinsThe presence of one
misfolded PrPsc causes adjacent PrPc to toggle
into the misfolded state.
73
Various Mutations in CJD Prion Proteins
Codon Amino acid change Reference
178 aspartate to asparagine Goldfarb 1991b
180 valine to isoleucine Kitamoto 1993a
188 threonine to alanine Collins 2000
196 glutamate to lysine Peoch 2000
200 glutamate to lysine Goldgaber 1989
203 valine to isoleucine Peoch 2000
208 arginine to histidine Mastrianni 1996
210 valine to isoleucine Pocchiari 1993
211 glutamate to glutamine Peoch 2000
232 methionine to arginine Kitamoto 1993a
74
(No Transcript)