Three Dimensional Protein Structures

Protein Structure & Folding
(9 / 25 / 2008)
•Secondary Structure
•Tertiary Structure
•Quaternary Structure and Symmetry
•Protein Folding
Example of each level of protein structure
Fibrous Proteins
 Keratin - A Coiled Coil
Nails, hair, horns and feathers
 or -forms
30 variants, tissue specific
type I
type II
acidic negative charge
basic positive charge
 keratin
• hair- 20 M diameter
• macrofibril 2000 Å parallel to hair
• microfibril 80 Å and high sulfur content protein
• can break -S-S- with mercaptans and reconnect (i.e. can
give hair a “permanent” wave)
 keratin proteins are helical
but spacing differs from a regular -helix
a 5.1 Å vs. 5.4 Å pitch.
This change in pitch forms closely associated pairs of
helices. Each pair consists of a type I and type II
Left-handed coil
310 AA residues 7-residue pseudo repeat.
Helical wheel - Look down an  helix and residues
stick out from center of helix 3.6 residues/turn 360 =
100 per residue
repeat on side of helix
View down the coil axis
Helical wheel diagram
a and d residues are
antiparallel strands
A coiled coil
a & d are non-polar and face the same side of helix.
3.6 residues/turn
3.5 residues hydrophobic repeat
The hydrophobic strip aligns between two helices with 18
inclination from one to another.
Dimer  protofilament  microfibril  macrofibril  hair
 keratin rich in cys and form disulfides
hard keratin cys content is high
soft keratin cys content is low
Perms reduce R-S--S-R bonds to 2R-SH
Curly hair has more Cys residues.
Higher order  Keratin structure
Collagen -A Triple Helix
Bones, teeth, cartilage, tendon, ligament, blood vessels and skin
Strong, flexible, stretchy
Several types
vessels, fetal skin
Type I 285 kDA
skin, bone tendon, cornea vessels
14Å wide
3000 Å long 30 distinct peptide types 16 variants
1/3 gly; 15-30% 4-hydroxyproline (Hyp); some 3-hydroxyproline (3Hyp), and some 5-hydroxylysine (Hyl)
Gly-X-Y X often Pro Y often Hyp
like a poly Gly or poly Pro helix
Left-handed 3.0 residues/turn pitch 9.4 extended
conformation the prolines avoid each other.
3 left handed helices combine in a triple right
handed coil.
Rope twist or metal cable
longitudinal force (pulling) is
supported by lateral compression
opposite twisted strands prevents
twists from pulling out.
Vitamin C is required for hydroxyproline formation
Hydroxyproline gives collagen stability and strength by H-bonding.
Without prolyl hydroxylase, collagen denatures at 24C instead of 39
to form gelatin.
Scurvy-skin lesions, broken blood vessels, wounds don’t heal, teeth
fall out, cannot stand.
Crosslinking requires lysine oxidase
N  C - CH2 - CH2 - NH2
sweet pea
-Aminopropionitrile, from
Inhibits lysine oxidase i.e. no crosslinking several diseases:
Lathyrism (abnormalities in bones, joints, etc.)
Osteogenesis imperfecta, brittle bone, A single amino acid
change could be lethal
Ehlers-Danlos syndromes, hyperextensible joints and skin,
Indian rubberman
Osteoarthritis - cartilage.
Tertiary protein structure
• Describes the folding of its secondary structural elements
• Determined via nuclear magnetic resonance (NMR) spectroscopy and
X-ray crystallography
• 3D structures of many proteins are available at
The Protein Data Bank (PDB)
Nuclear Magnetic Resonance (NMR)
The calcyclin dimer. Potts et al.,
NSB 2, 790-796 (1995)
“Most atoms in the biological molecules have a little magnet
inside them. If we put any of these molecules in a big magnet,
all the little magnet in the molecule will orient themselves to line
up with the big magnet”, allowing to scientist to probe various
properties of the molecule…
NMR at the UH
• The UH Keck/IMD NMR
Center has the first 800
MHz NMR spectrometer
installed within Texas.
• The latest facility
enhancement, in January
2006, is the installation of
a Bruker 5mm TXI
CryoProbe for the 800
MHz instrument.
– This NMR probehead is
cooled by cyrogenic helium
gas to reduce thermal noise
and improve the signal to
noise ratio by as much as
three times a conventional
UH 800 MHz NMR Spectrometer
X-ray crystallography
X-rays are bounced off of the
X-rays are diffracted by electrons
in the various atoms/bonds
The diffraction pattern of the
X-rays is measured and an
electron density map is created
(cyan in the figure to the left)
Attempts are made to fit amino
acids into the electron density
Most Protein Crystal Structures exhibit less than atomic
How the quality of (degree of focus) of an electron density
map varies with its resolution limits?
Visualizing Proteins
The course of the polypeptide chain can be followed by tracing the
positions of its C atoms or by representing helices as helical
ribbons or cylinders and  sheets as sets of flat arrows pointing
from the N- to the C-termini.
Motifs and Domains
from whale hemoglobin
has non-polar residues
(yellow) and polar residues
(purple) on opposite sides
of the helix.
with protein binding
domain on the side
with non-polar
residues (orange)
leaving the polar ones
(purple) facing
solvent water
Supersecondary structural motifs
Nucleotide binding
Rossman Fold
Most common Common
Greek key
Examples of Globular Proteins (1)
Jack Bean Concanavalin A
Triose phosphate isomerase
Orange spheres are
metal ions
A glycolysis pathway enzyme
Examples of Globular Proteins (2)
cytochrome b562 (E. Coli) Fab (human)
Helix bundle
Immunoglobulin fold
Lactate dehydrogenase (dogfish)
6-stranded parallel  sheet
Retinol binding protein Peptide Asn amidase F
(F. meningosepticum)
(chicken muscle)
Large polypeptides form domains
2 4
Many single polypeptide proteins
fold into multiple structural domains,
each with their own function
This is glyceraldehyde-3-phosphate
(GAP) dehydrogenase
The red domain binds NAD+
The green domain binds GAP
A glycolysis pathway enzyme
Quaternary protein structure
4o structure is the relative
placement of different polypeptide segments
Hemoglobin is shown to the
left (1-yellow, 2-green,
1-cyan, 2-blue), heme
groups are in red - bind O2
Subunits usually associate noncovalently
Subunits are symmetrically arranged
Sidechain locations in proteins
• Non-polar sidechains (Val, Leu, Ile, Met, and
Phe) occur mostly in the interior of a protein
keeping them out of the water (hydro-phobic
• Charged polar residues (Arg, His, Lys, Asp, and
Glu) are normally located on the surface of the
protein in contact with water.
• Uncharged polar residues (Ser, Thr, Asn, Gln, and
Tyr) are usually on the protein surface but also
occur in the interior of the protein.
Protein Stability
Forces that stabilize protein structure: 1, 2, 3
1. The Hydrophobic Effect
2. Electrostatic Interactions
Ion pair (salt bridge) of
3. Chemical Cross-links
Zinc finger:
Nucleic acid-binding proteins
Protein Folding
Protein folding problem
• Levinthal paradox
• Prediction of three dimensional
structure from its amino acid
• Translate “Linear” DNA
Sequence data to spatial
Protein Folding Pathways
Proteins can be
Denatured proteins can be
refolded, sometimes requiring
helper proteins, and this
refolding takes place via
preferred pathways.
Common thought is that
secondary structures form first,
eventually collapsing due to the
formation of hydrophobic cores.
Folding funnel
relationship for
protein folding
Molecular chaperons
Molecular chaperones:
(1) Hsp70 proteins function as monomer
(2) Chaperonins, large multisubunit proteins
(3) Hsp90 proteins for the folding of proteins involved with signal
Reaction cycle of the GroEL/ES cycle
1. GroEL ring binding 7 ATP and a
substrate (improperly folded protein).
Then it binds a GroES cap to become
the cis ring.
2. The cis ring catalyzes the
hydrolysis of its 7 ATP.
3. A 2nd substrate binds to the trans
ring followed by 7 ATP.
4. The binding of substrate and ATP
to the trabs ring conformationally
induces the cis ring to release its
bound GroES, 7 ADP, and the better
folded substrate.The trans ring
becomes the cis ring.
Protein disulfide Isomerase
Diseases Caused by Protein Misfolding
Alzheimer’s disease
Transmissible spongiform encephalopathies (TSE)
Prion protein conformation
Once it has formed, an amyloid fibril
is virtually indestructible (interchain
H- bonds).
It seems likely that protein folding
pathways have evolved not only to
allow polypeptides to assume stable
native structures but also to avoid
forming interchain H-bonds that would
lead to fibril formation .
A model of an amyloid fibril
The factors that trigger amyloid
formation remain obscure, even when
mutation (hereditary amyloidoses) or
infection (TSEs) appear to be the
Lecture 11
Tuesday 9/30/08
Protein Structure and Purification

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