Transcript Structural Genomics

Protein Structure Determination
Protein Folding
Molecular Chaperones
Prions
Alzyheimer’s
Tertiary Structure of Proteins
Two methods: 1. X-RAY diffraction crystal structure
2. NMR solution structure
This is a crystal X-ray diffraction pattern of sperm whale myoglobin
Electron density map
6Å
2.0 Å
1.5 Å
1.1 Å
From the diffraction pattern (spots and intensity) one can get a
mathematical description of the electron density of a molecule. With
proper model construction a 3-D image of the protein is constructed.
NMR
By using chemical shifts of backbone hydrogens and their
chemical splitting bond angles can be determined. COSY NMR
or Correlated Spectroscopy. By manipulating parameters
protons that are close to each other in space but not linked
through bonds can be determined by NOSY NMR or Nuclear
Overhauser spectroscopy. Growing the protein in bacteria where
the carbon source can be substituted by 13C and the nitrogen by
15N (stable isotope substitution) more restraints can be achieved.
The liquid structure(s) can be determined as a group that fit a
certain structure space.
Quaternary Structure and
Symmetry
Subunits can associate noncovalently, subunits are
protomers if identical.
Protomer subunits are symmetrically arranged
Only rotational symmetry allowed.
i.e. cyclic symmetry C2, C3, C6 etc.
Dihedral symmetry N-fold intersects a two-fold
rotational symmetry at right angles
Other higher order types, octahedral or tetrahedral
Protein folding is
“one of the great unsolved problems of science”
Alan Fersht
protein folding can be seen as a connection
between the genome (sequence) and what the
proteins actually do (their function).
Protein folding problem
• Prediction of three dimensional structure from its
amino acid sequence
• Translate “Linear” DNA Sequence data to spatial
information
Why solve the folding problem?
• Acquisition of sequence data relatively quick
• Acquisition of experimental structural information
slow
• Limited to proteins that crystallize or stable in
solution for NMR
Protein folding dynamics
Electrostatics, hydrogen bonds and van der Waals forces hold a
protein together.
Hydrophobic effects force global protein conformation.
Peptide chains can be cross-linked by disulfides, Zinc, heme or
other liganding compounds. Zinc has a complete d orbital , one
stable oxidation state and forms ligands with sulfur, nitrogen and
oxygen.
Proteins refold very rapidly and generally in only one stable
conformation.
The sequence contains all the information to
specify 3-D structure
Random search and the
Levinthal paradox
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The initial stages of folding must be nearly random, but if the entire process
was a random search it would require too much time. Consider a 100 residue
protein. If each residue is considered to have just 3 possible conformations the
total number of conformations of the protein is 3100. Conformational changes
occur on a time scale of 10-13 seconds i.e. the time required to sample all
possible conformations would be 3100 x 10-13 seconds which is about 1027
years. Even if a significant proportion of these conformations are sterically
disallowed the folding time would still be astronomical. Proteins are known to
fold on a time scale of seconds to minutes and hence energy barriers probably
cause the protein to fold along a definite pathway.
Physical nature of protein folding
• Denatured protein makes many interactions with
the solvent water
• During folding transition exchanges these noncovalent interactions with others it makes with
itself
What happens if proteins don't fold correctly?
• Diseases such as Alzheimer's disease, cystic
fibrosis, Mad Cow disease, an inherited form of
emphysema, and even many cancers are believed
to result from protein misfolding
Protein folding is a balance of forces
• Proteins are only marginally stable
• Free energies of unfolding ~5-15 kcal/mol
• The protein fold depends on the summation of all
interaction energies between any two individual
atoms in the native state
• Also depends on interactions that individual atoms
make with water in the denatured state
Protein denaturation
• Can be denatured depending on chemical
environment
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Heat
Chemical denaturant
pH
High pressure
Thermodynamics of unfolding
• Denatured state has a high configurational entropy
S = k ln W
Where W is the number of accessible states
K is the Boltzmann constant
• Native state confirmationally restricted
• Loss of entropy balanced by a gain in enthalpy
Entropy and enthaply of water must be added
• The contribution of water has two important
consequences
– Entropy of release of water upon folding
– The specific heat of unfolding (ΔCp)
• “icebergs” of solvent around exposed hydrophobics
• Weakly structured regions in the denatured state
The hydrophobic effect
High ΔCp changes enthalpy significantly with
temperature
• For a two state reversible transition
ΔHD-N(T2) = ΔHD-N(T1) + ΔCp(T2 – T1)
• As ΔCp is positive the enthalpy becomes more
positive
• i.e. favors the native state
High ΔCp changes entropy with temperature
• For a two state reversible transition
ΔSD-N(T2) = ΔSD-N(T1) + ΔCpT2 / T1
• As ΔCp is positive the entropy becomes more
positive
• i.e. favors the denatured state
Free energy of unfolding
• For
ΔGD-N = ΔHD-N - TΔSD-N
• Gives
ΔGD-N(T2) = ΔHD-N(T1) + ΔCp(T2 – T1)- T2(ΔSD-N(T1) + ΔCpT2 / T1)
• As temperature increases TΔSD-N increases and causes the
protein to unfold
Cold unfolding
• Due to the high value of ΔCp
• Lowering the temperature lowers the enthalpy decreases
Tc = T2m / (Tm + 2(ΔHD-N / ΔCp)
i.e. Tm ~ 2 (ΔHD-N ) / ΔCp
Measuring thermal denaturation
Solvent denaturation
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Guanidinium chloride (GdmCl) H2N+=C(NH2)2.ClUrea H2NCONH2
Solublize all constitutive parts of a protein
Free energy transfer from water to denaturant solutions is
linearly dependent on the concentration of the denaturant
• Thus free energy is given by
ΔGD-N = ΔHD-N - TΔSD-N
Solvent denaturation continued
• Thus free energy is given by
ΔGD-N = ΔGH2OD-N - mD-N [denaturant]
Acid - Base denaturation
• Most protein’s denature at extremes of pH
• Primarily due to perturbed pKa’s of buried groups
• e.g. buried salt bridges
Two state transitions
• Proteins have a folded (N) and unfolded (D) state
• May have an intermediate state (I)
• Many proteins undergo a simple two state transition
D <—> N
Folding of a 20-mer poly Ala
Unfolding of the DNA Binding Domain of
HIV Integrase
Two state transitions in multi-state reactions
Rate determining steps
Energy profiles during Protein Folding
Theories of protein folding
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N-terminal folding
Hydrophobic collapse
The framework model
Directed folding
Proline cis-trans isomerisation
Nucleation condensation
Molecular Chaperones
• Three dimensional structure encoded in sequence
• in vivo versus in vitro folding
• Many obstacles to folding
D<---->N
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Ag
Molecular Chaperone Function
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Disulfide isomerases
Peptidyl-prolyl isomerases (cyclophilin, FK506)
Bind the denatured state formed on ribozome
Heat shock proteins Hsp (DnaK)
Protein export & delivery SecB
What happens if proteins don't fold correctly?
• Diseases such as Alzheimer's disease, cystic
fibrosis, Mad Cow disease, an inherited form of
emphysema, and even many cancers are believed
to result from protein misfolding
GroEL
GroEL (HSP60 Cpn60)
• Member of the Hsp60 class of chaperones
• Essential for growth of E. Coli cells
• Successful folding coupled in vivo to ATP
hydrolysis
• Some substrates work without ATP in vitro
• 14 identical subunits each 57 kDa
• Forms a cylinder
• Binds GroES
GroEL is allosteric
• Weak and tight binding states
• Undergoes a series of conformation changes upon binding
ligands
• Hydrolysis of ATP follows classic sigmoidal kinetics
Sigmoidal Kinetics
• Positive cooperativity
• Multiple binding sites
Allosteric nature of GroEL
GroEL changes affinity for denatured proteins
• GroEL binds tightly
• GroEL/GroES complex much more weakly
GroEL has unfolding activity
• Annealing mechanism
• Every time the unfolded state reacts it partitions to give a proportion
kfold/(kmisfold + Kfold) of correctly folded state
• Successive rounds of annealing and refolding decrease the amount of
misfolded product
GroEL slows down individual steps in folding
• GroEL14 slows barnase refolding 400 X slower
• GroEL14/GroES7 complex slows barnase refolding 4 fold
• Truncation of hydrophobic sidechains leads to weaker
binding and less retardation of folding
Active site of GroEL
• Residues 191-345 form a mini chaperone
• Flexible hydrophobic patch
Role of ATP hydrolysis
The GroEL Cycle
A real folding funnel
Amyloids
• A last type of effect of misfolded protein
• protein deposits in the cells as fibrils
• A number of common diseases of old age, such as
Alzheimer's disease fit into this category, and in some
cases an inherited version occurs, which has enabled study
of the defective protein
Known amyloidogenic peptides
CJD spongiform encepalopathies prion protein fragments
APP Alzheimer beta protein fragment 1-40/43
HRA hemodialysis-related amyloidosis beta-2 microglobin*
PSA primary systmatic amyloidosis immunoglobulin light chain and fragments
SAA 1 secondary systmatic amyloidosis serum amyloid A 78 residue fragment
FAP I** familial amyloid polyneuropathy I transthyretin fragments, 50+ allels
FAP III familial amyloid polyneuropathy III apolipoprotein A-1 fragments
CAA cerebral amyloid angiopathy cystatin C minus 10 residues
FHSA Finnish hereditary systemic amyloidosis gelsolin 71 aa fragment
IAPP type II diabetes islet amyloid polypeptide fragment (amylin)
ILA injection-localized amyloidosis insulin
CAL medullary thyroid carcinoma calcitonin fragments
ANF atrial amyloidosis atrial natriuretic factor
NNSA non-neuropathic systemic amylodosis lysozyme and fragments
HRA hereditary renal amyloidosis fibrinogen fragments
Transthyretin
• transports thyroxin and retinol binding protein in the
bloodstream and cerebrospinal fluid
• senile systemic amyloidosis, which affects people over
80, transtherytin forms fibrillar deposits in the heart. which
leads to congestive heart failure
• Familial amyloid polyneuropathy (FAP) affects much
younger people; causing protein deposits in the heart, and
in many other tissues; deposits around nerves can lead to
paralysis
Transthyretin structure
• tetrameric. Each monomer has two 4-stranded b-sheets, and a short a-helix. Antiparallel beta-sheet interactions link monomers into dimers and a short loop from
each monomer forms the main dimer-dimer interaction. These pairs of loops keep the
two halves of the structure apart forming an internal channel.
Fibril structure
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Study of the fibrils is difficult because of its insolubility making NMR solution
studies impossible and they do not make good crystals
X-ray diffraction, indicates a pattern consistent with a long b-helical structure,
with 24 b-strands per turn of the b-helix.
Formation of proto-filaments
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Four twisted b-helices make up a proto-filament (50-60A)
Four of these associate to form a fibril as seen in electron microscopy (130A)