CH339K
Proteins: Higher Order Structure
Higher Levels of Protein Structure
Side chains hang off the backbone
Repetitive background: -N-C-C-N-C-C-
The shape of the peptide chain can be defined by the
three consecutive bond torsional angles
Bond
NH to Ca
Ca to C=O
C=O to NH
Rotation
free
free
rigid planar
Torsion angle defined
phi
psi
omega
Since w is
constrained,
only f and y
can vary
There are steric
restrictions on
what values
they can
assume
Permissable F-Y Angles
(Ramachandran Plot)
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
a-helix
Pauling, Corey, and
Branson (1951)
H bonds between
• carbonyl O of residue n
• amide H of residue n+4
Each amino acid is rotated
100o from the previous
one.
3.6 amino acids per turn
R/V Alpha Helix
Woods Hole Oceanographic Institute 1966-2011
Helical parameters – Pitch and Rise
Backbone forms helix
Side chains extend
outwards
f ≈ -57o
y≈ -47o
3.6 residues/turn
Helix Types
a-helix: C=O H-bonded to NH of residue
n+4 (aka 3.613 helix)
310 helix: C=O H-bonded to NH of
residue n+3
– (f ≈ -49o y ≈ -26o)
p-helix: C=O H-bonded to NH of residue
n+5 (aka 4.116 helix)
(f ≈ -57o y ≈ -80o)
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 a -helix)
(b) Nbr of atoms in the loop (e.g. 13 for a -helix)
3.613
310
4.116 or 
O H
O H
O H
O H
O H
O
HN HC C N
HC C N
HC C N
HC C N
HC C N
HC C
H
R
R
R
R
R
R
etc
Idealized Helices
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
C=O of residue n with N-H of residue n+3
Gamma Turns:
C=O of residue n with N-H of residue n+2
F-y Angles for Secondary
Structures
NOTE: Left-handed a-helix has f = +57, y = +47
Ramachandran Plot: Blue areas are permitted F and Y
angles
Ramachandran plot for pyruvate kinase
Tertiary Structures
• Three dimensional folding
• Determined by side chain interactions
– Salt links
– H-Bonds
– Disulfides
– Hydrophobic interactions
• Fibrous Proteins
• Globular Proteins
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
a-keratin
• Lots of Ala, Gly, Cys
• All a-helix (well, almost)
Right handed
Left handed
Disulfides in the Barber Shop
Sodium thioglycolate
Various peroxides
Fibrous Proteins - Fibroin
75-80% Ala/Gly
15% Ser
Within a fiber:
crystalline regions
are separated by
amorphous regions.
Fibrous Proteins - Collagen
Left handed helix of
tropocollagen forms right
handed triple helix of collagen.
Hydroxyproline participates in H-bonding
between tropocollagen chains
(1)
(2)
In the absence of vitamin C, reaction 2 oxidizes Fe2+ to Fe3+.
Lack of hydroxyls causes serious destabilization of
the triple helix
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…
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
British Empire at its Peak
• A healthy navy is a victorious navy (of course, my
ancestors were less than thrilled…)
Protein structure cartoons
a-helix
Antiparallel b-sheet
Globular Proteins (examples)
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
Greek Key Motif (antiparallel b-sheets)
a) Schematic of motif
b) Staphylococcus nuclease protein
More motifs
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
C-rich Domain of
Earthworm
Mannose Receptor
Fibroblast
Growth
Factor
Domains can be shared among proteins
Quaternary Structure (Hemoglobin)
Folding Energetics
Favoring Folding
Favoring Unfolding
-DH from formation of intrachain Hbonds 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
Denaturation
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)
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.
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.
Rapid 2o structure formation
Collapse to molten globule
Reshuffle to final state
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.
Chaperonins - GroEL
Simpler Picture of GroEL Action
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)
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
• Scrapie
• Kuru
• BSE
Spongioform Encephalopathy – your brain on CJD
Normal
Moderate
Severe
Brain atrophy in CJD – you’re usually dead
before it reaches this stage
Prion Proteins
Normal cellular prion protein (PrPc) – mostly ahelical C-terminal domain
PrPc
Prion Proteins – C terminal region
PrPc
PrPsc
Infectious Proteins
The presence of one
misfolded PrPsc
causes adjacent PrPc
to toggle into the
misfolded state.
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
Peoc’h 2000
200**
glutamate to lysine
Goldgaber 1989
203
valine to isoleucine
Peoc’h 2000
208
arginine to histidine
Mastrianni 1996
210
valine to isoleucine
Pocchiari 1993
211
glutamate to glutamine
Peoc’h 2000
232
methionine to arginine
Kitamoto 1993a