Download Structural Genomics - University of Houston

Enzyme Kinetics & Protein Folding
9/7/2004
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
•
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.
Energy profiles during Protein Folding
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
•
•
•
•
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