Protein Structure Determination
Protein Folding
Molecular Chaperones
Prions
Tertiary Structure of Proteins
Two methods: 1. X-RAY diffraction crystal structure 2. NMR solution structure
Electron density map
6 Å 2.0 Å 1.5 Å 1.1 Å
From the diffraction pattern (spots and intensity) one can get a
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.
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
Protein folding is
“one of the great unsolved problems of science”
protein folding can be seen as a connection
between the genome (sequence) and what the
Protein folding problem
• Prediction of three dimensional structure from its
amino acid sequence
Why solve the folding problem?
• Acquisition of sequence data relatively quick
• Acquisition of experimental structural information
slow
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.
Random search and the
Levinthal paradox
• 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
Physical nature of protein folding
• Denatured protein makes many interactions with
the solvent water
What happens if proteins don't fold correctly?
• Diseases such as Alzheimer's disease, cystic
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
Protein denaturation
• Can be denatured depending on chemical
environment
– Heat
– Chemical denaturant – pH
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
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)
High ΔC
pchanges enthalpy significantly with
temperature
• For a two state reversible transition
ΔH
D-N(T2)= ΔH
D-N(T1)+ ΔC
p(T
2– T
1)
• As
ΔC
pis positive the enthalpy becomes more
positive
High ΔC
pchanges entropy with temperature
• For a two state reversible transition
ΔS
D-N(T2)= ΔS
D-N(T1)+ ΔC
pT
2/ T
1• As
ΔC
pis positive the entropy becomes more
positive
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)
Cold unfolding
• Due to the high value of ΔCp
• Lowering the temperature lowers the enthalpy decreases
Tc = T2m / (Tm + 2(ΔHD-N /ΔCp)
Solvent denaturation
• Guanidinium chloride (GdmCl) H2N+=C(NH2)2.Cl
-• Urea 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
Solvent denaturation continued
• Thus free energy is given by
Acid - Base denaturation
• Most protein’s denature at extremes of pH
• Primarily due to perturbed pKa’s of buried groups
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
Theories of protein folding
• N-terminal folding
• Hydrophobic collapse
• The framework model
• Directed folding
Molecular Chaperones
• Three dimensional structure encoded in sequence
•
in vivo
versus in
vitro folding
• Many obstacles to folding
D<---->N
Molecular Chaperone Function
• Disulfide isomerases
• Peptidyl-prolyl isomerases (cyclophilin, FK506)
• Bind the denatured state formed on ribozome
What happens if proteins don't fold correctly?
• Diseases such as Alzheimer's disease, cystic
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
GroEL is allosteric
• Weak and tight binding states
• Undergoes a series of conformation changes upon binding ligands
Sigmoidal Kinetics
GroEL changes affinity for denatured proteins
• GroEL binds tightly
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
GroEL slows down individual steps in folding
• GroEL14 slows barnase refolding 400 X slower
• GroEL14/GroES7 complex slows barnase refolding 4 fold
Active site of GroEL
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
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
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
Transthyretin structure
• tetrameric. Each monomer has two 4-stranded-sheets, and a short -helix.
Fibril structure
• 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 -helical
Formation of proto-filaments
• Four twisted -helices make up a proto-filament (50-60A)
