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

• H bonds between
• carbonyl O of residue n
• amide H of residue n4

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
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

16
(No Transcript)
17
(No Transcript)
18
(No Transcript)
19
(No Transcript)
20
CO of residue n with N-H of residue n3
21
Gamma Turns
CO of residue n with N-H of residue n2
22
F-y Angles for Secondary Structures
NOTE Left-handed a-helix has f 57, y 47
23
Ramachandran Plot Blue areas are permitted F and
Y 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

a-keratin hair, horns, and hoofs of
mammals b-keratin scales, claws and shells of
reptiles, beaks and claws of birds, porcupine
quills
27

• a-keratin
• Lots of Ala, Gly, Cys
• All a-helix

Right handed
Left handed
28
(No Transcript)
29
Disulfides in the Barber Shop
Sodium thioglycolate
Various peroxides
30
Fibrous Proteins - Fibroin
75-80 Ala/Gly 15 Ser
31
(No Transcript)
32
(No Transcript)
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.
35
(No Transcript)
36
Hydroxyproline participates in H-bonding between
tropocollagen chains
37
(1)
(2)
In the absence of vitamin C, reaction 2 oxidizes
Fe2 to Fe3.
38
Lack of hydroxyls causes serious destabilization
of the triple helix
39
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

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
a-helix
Antiparallel b-sheet
42
Globular Proteins (examples)
43
(No Transcript)
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

• Domains
• Common patterns found in different proteins
• Typically have similar function
• Caused by evolution (gene recombination /
  duplication)

• 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

47
C-rich Domain of Earthworm Mannose Receptor
Fibroblast Growth Factor
48
Domains can be shared among proteins
49
(No Transcript)
50
Quaternary Structure (Hemoglobin)
51
Folding Energetics
Favoring Folding Favoring Unfolding
-DH from formation of interchain H-bonds and salt links High DS from going from unfolded ? folded state
DS from disulfide formation High DH from breaking H-bonds with solvent
Enormous DS from burial of hydrophobic side chains in the interior
52
Denaturation
53
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)

54
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.

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 2o structure formation
Collapse to molten globule
Reshuffle to final state
57
(No Transcript)
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
60
(No Transcript)
61
Simpler Picture of GroEL Action
62
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)

63

• Scrapie

• Kuru

• BSE

64
Spongioform Encephalopathy your brain on CJD
Normal
Moderate
Severe
65
Brain atrophy in CJD youre usually dead before
it reaches this stage
66
Prion Proteins
Normal cellular prion protein (PrPc) mostly
a-helical C-terminal domain
PrPc
67
Prion Proteins C terminal region
PrPc
PrPsc
68
Vrious 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
69
(No Transcript)