Transcript Lecture 9

Lecture 11
– Test next week in class
– Protein structure
Collagen
• Most abundant protein of vertebrates.
• Extracellular protein-insoluble fibers, great tensile strength
• Major component of connective tissues (bone, teeth, cartilage,
tendon, ligament, etc.)
• Type I collagen-3 polypeptide chains, 285 kD.
– 3000 Å long and 14 Å diameter.
• Distinct amino acid composition; 1/3 are Gly and 15-30% are Pro
and 4-hydroxyprolyl (Hyp) residues.
Collagen
• Collagen has a triple-helical structure
• Amino acid sequence has repeating triplets of Gly-X-Y with X=Pro
and Y= Hyp over 1011 residues out of 1042 residue polypeptide.
• Forms a right-handed triple helical structure.
The triple helix of collagen.
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• Shows how left-handed
polypeptide helices are twisted
together to form a right-handed
superhelical structure.
• Individual polypeptide has 3.3
residues per turn and pitch of
10 Å.
• The collagen triple helix has 10
Gly-X-Y units per turn and a
pitch of 86.1 Å.
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Figure 8-30b X-Ray structure of the triple helical collagen model
peptide (Pro-Hyp-Gly)10 in which the fifth Gly is replaced by Ala. (b)
View along helix axis.
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Figure 8-31
Electron micrograph of collagen fibrils from skin.
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Figure 8-32
Banded appearance of collagen fibrils.
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Figure 8-30c
X-Ray structure of the triple helical collagen model
peptide (Pro-Hyp-Gly)10 in which the fifth Gly is replaced by Ala. (c)
A schematic diagram.
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Figure 8-33
A biosynthetic pathway for
cross-linking Lys, Hyl, and His side chains
in collagen.
• Collagen fibrils are covalently
cross-linked.
• Collagen almost no cysteine.
• It is cross linked y Lys and His.
• Lysyl oxidase coverts lysine to
allysine.
• Allysine are condesed to
allysine aldol.
• This reacts with His to form
Aldol-His.
• Aldol-His reacts with 5-hydroxyLys crosss linking the four side
chains.
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Table 8-3 The Arrangement
of Collagen Fibrils in Various
Tissues.
Globular proteins
• Diverse group of proteins that exist as compact spherical
molecules.
• Enzymes, transport, and receptor proteins.
• Most structural information from X-ray crystal structure
and NMR.
• X-ray crystallography directly images molecules.
• X-ray wavelengths are small 1.5 Å (visible light is 4000 Å)
• X-rays generated by synchrotrons, a type of particle
accelerator to make X-rays of high intensity.
Crystalline proteins
• Molecules in protein crystals are arranged in regularly
repeating 3-D lattices.
• Unlike other small organic or inorganic molecules,
proteins are highly hydrated (40-60% H2O)
• Water is required for the native structure of the proteins.
• Generally disordered by >1 Å.
• Typical resolution is 1.5 to 3.0 Å.
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Figure 8-36a Electron
density maps of proteins.
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Figure 8-36b Electron
density maps of proteins.
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Figure 8-36c
Electron density maps of
proteins.
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Figure 8-37
Sections through the electron
density map of diketopiperazine calculated at
the indicated resolution levels.
Crystalline proteins
• Crystalline proteins assume the same structure they have
in solution
• Crystals have 40-60% water content (similar to most cells)
• Proteins may crystallize in of several forms depending on
conditions. Different crystal forms of the same protein
have identical conformations.
• Many enzymes are catalytically active in the crystalline
state.
NMR for protein structure determination
• Use of 2D NMR
• Yields interaatomic distances between specific protons
that are <5 Å apart.
• Interproton distances through space can be determined
by nuclear Overhauser effect spectroscopy (NOESY)
• Interproton distance through bonds as determined by
correlated spectroscopy (COSY).
• Present methods are good only with molecular masses up
to 40 kD.
• Usually well correlated with X-ray data, but sometimes
differs.
• NMR can probe motions over time scales of 10 orders of
magnitude so can be used to study protein folding and
dynamics.
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Figure 8-38a The 2D proton NMR structures of proteins.
(a) A NOESY spectrum of a protein presented as a contour plot
with two frequency axes w1 and w2.
Off diagonal peaks
(cross peaks) occur
from interaction of 2
protons that are <5 Å
apart in space and
whose 1D-NMR peaks
are located where the
horizontal and vertical
lines cross through the
cross peak intersect the
diagonal.
Nuclear Overhauser
Effect (NOE)
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a
a
d
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d
b
a
c
d
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Figure 8-38b The 2D proton NMR structures of proteins.
(b) The NMR structure of a 64-residue polypeptide comprising the
Src protein SH3 domain.
Tertiary structure
• Tertiary structure is the three dimensional arrangement of
a protein.
• Includes the folding of secondary structural elements and
spatial dispositions of the side chains.
• Determined by X-ray crystallography and NMR
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Figure 8-39a Representations of the X-ray structure of sperm
whale myoglobin. (a) The protein and its bound heme are drawn in
stick form.
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Figure 8-39b Representations of the X-ray structure of sperm
whale myoglobin. (b) A diagram in which the protein is represented
by its computer-generated Ca backbone.
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Figure 8-39c Representations of the X-ray structure of sperm
whale myoglobin. (c) A computer-generated cartoon drawing in an
orientation similar to that of Part b.
Globular proteins have both a helices
and  sheets
• Most proteins have a significant amount of secondary
structure
• On average 31% a helix, 28%  sheet, and a total content
of helices, sheets, turns and  loops comprising 90% of
the structure of a protein.
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Figure 8-40
The X-ray structure of jack bean protein
concanavalin A.
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Figure 8-41
Human carbonic anhydrase.
Side chain location varies with polarity
• Globular proteins lack the repeating sequences responsiblee for the
regular conformations of fibrous proteins.
• The amino acid side chains in globular proteins are distributed
according to polarities.
• Nonpolar residues (Val, Leu, Ile, Met, and Phe) occur in the interior of
a protein.
• Charged polar residues (Arg, Lys, His, Asp, Glu) are mostly located
on the surface of a protein.
• Uncharged polar residues (Ser, Thr, Asn, Gln, Tyr, and Trp) are
usually on the surface but can occur in the interior of the protein.
– If in the interior, they are H-bonded to neutralize their polarity.
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Figure 8-42a
The X-Ray structure of horse
heart cytochrome.
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Figure 8-43a The H helix of sperm whale myoglobin. (a) A helical
wheel representation in which the side chain positions about the a
helix are projected down the helix axis onto a plane.20
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Figure 8-43b
The H helix of sperm whale myoglobin.
(b) A skeletal model, viewed as in Part a.
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Figure 8-43c
The H helix of sperm whale myoglobin.
(c) A space-filling model, viewed from the bottom of the page in
Parts a and b and colored as in Part b.
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Figure 8-44
A space-filling model of an antiparallel  sheet from
concanavalin A.
Energy diagram of the protein folding process.
The completely unfolded protein is thought to be in the least stable form.
For most proteins, the native conformation is the most thermodynamically
stable and the only form that is biologically active.
Denaturation and renaturation of a protein
The complete loss of organized
structure in a protein is
called “denaturation”.
Denaturation results in loss of
biological activity!
Denaturation process occurs
during cooking an egg.
Denaturants include:
• Large evil fire ants??
• Heat
• Organic solvents
• Urea
• Detergents
• Acid or base
• Shear stress
• Hydrophobic interfaces
Structural Motifs in Proteins.
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Individual units of 2ndary structure combine into stable,
geometrical arrangements.
Called supersecondary structure or motifs.
Are often repeated in same protein, different proteins.
Certain motifs have associated biological functions:
a-Helix-loop-a-helix motif binds DNA, sequesters
calcium ion.
Secondary structures often depicted as ribbon diagrams
Ribbons invented by Jane Richardson, originally drawn by
hand, now done by computer programs.
Some common structural motifs of folded proteins
a) The aa motif
(helix-turn helix)
Some common structural motifs of folded proteins
b) The  motif;
antiparallel
Some common structural motifs of folded proteins
c) The 
“Greek
Key” motif
Some common structural motifs of folded proteins
d) The a
motif
Several a motifs combine to form a superbarrel in the
glycolysis enzyme triose phosphate isomerase (TIM barrel)
Quaternary structure
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Spatial arrangement of protein subunits.
Polypeptide subunits associate in a geometrically specific manner.
Why subunits?
Easier to repair self-assembling single subunit vs. a large
polypeptide.
• Increasing a protein’s size through subunits is more efficient for
specifying the active site.
• Provides a structural basis for regulating activity.
Domains in proteins.
• Common sequence regions in native proteins can
fold up to form compact structures called
“domains”.
• Domains can range in size from 50-400 amino
acids, have upper limit in forming compact
hydrophobic core.
• Domains are a type of folding motif, typically have
separate hydrophobic core.
• Larger proteins are composed of multiple
domains, often connected by flexible linker
peptide regions.
• Classic example: antibodies
Antibody Immunoglobulin Domains
Structural elements of IgGs:
Naturally occurring immunoglobulins (IgG molecules) have identical heavy chains and light
chains giving rise to multiple binding sites with identical specificities for antigen.
Antibody Immunoglobulin Domains
Antibodies are composed of:
V (for variable) regions - encodes the
antigen binding activity
C (for constant) regions - encodes
immune response signal/effector
functions:
1.
Complement fixation (activation
of complement cascade)
2.
Binding and activation of Ig
receptors (transport from
maternal source, activate
immune system T cells to engulf,
destroy foreign cells, particles,
proteins)
3.
Also binds bacterial Protein A,
Protein G (used in purification)
Note: dashed lines indicate
interchain disulfide bonds
Antibody Immunoglobulin Domains
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There is a conserved glycosylation site in the CH2 domain of IgG (purple
region).
A carbohydrate is covalently attached here by postranslational
modification.
Antibody Immunoglobulin Domains
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IgG secondary/tertiary structure: multiple beta-sheet domains.
Termed “immunoglobulin domain”.
Repeated motif in many immune and receptor proteins.
Antibody Immunoglobulin Domains
Modes of Flexibility of IgG structure
Subunit interactions
• Identical subunits in a protein are called protomers
• Proteins with identical subunits are oligomers.
• Hemoglobin is a dimer (oligomer of two protomers) of
aprotomers.