Enzyme Kinetics

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Enzyme Kinetics Protein Folding9/7/2004
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Protein folding is one of the great unsolved
problems of science Alan Fersht
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protein folding can be seen as a connection
between the genome (sequence) and what the
proteins actually do (their function).
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Protein folding problem

• Prediction of three dimensional structure from
  its amino acid sequence
• Translate Linear DNA Sequence data to spatial
  information

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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

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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.
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The sequence contains all the information to
specify 3-D structure
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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.

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Energy profiles during Protein Folding
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Physical nature of protein folding

• Denatured protein makes many interactions with
  the solvent water
• During folding transition exchanges these
  non-covalent interactions with others it makes
  with itself

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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

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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

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Protein denaturation

• Can be denatured depending on chemical
  environment
• Heat
• Chemical denaturant
• pH
• High pressure

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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

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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 (?Cp)
• icebergs of solvent around exposed hydrophobics
• Weakly structured regions in the denatured state

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The hydrophobic effect
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High ?Cp changes enthalpy significantly with
temperature

• For a two state reversible transition
• ?HD-N(T2) ?HD-N(T1) ?Cp(T2 T1)
• As ?Cp is positive the enthalpy becomes more
  positive
• i.e. favors the native state

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High ?Cp changes entropy with temperature

• For a two state reversible transition
• ?SD-N(T2) ?SD-N(T1) ?CpT2 / T1
• As ?Cp is positive the entropy becomes more
  positive
• i.e. favors the denatured state

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Free energy of unfolding

• For
• ?GD-N ?HD-N - T?SD-N
• Gives
• ?GD-N(T2) ?HD-N(T1) ?Cp(T2 T1)-
  T2(?SD-N(T1) ?CpT2 / T1)
• As temperature increases T?SD-N increases and
  causes the protein to unfold

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Cold unfolding

• Due to the high value of ?Cp
• Lowering the temperature lowers the enthalpy
  decreases
• Tc T2m / (Tm 2(?HD-N / ?Cp)
• i.e. Tm 2 (?HD-N ) / ?Cp

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Measuring thermal denaturation
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Solvent denaturation

• Guanidinium chloride (GdmCl) H2NC(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
• ?GD-N ?HD-N - T?SD-N

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Solvent denaturation continued

• Thus free energy is given by
• ?GD-N ?GH2OD-N - mD-N denaturant

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Acid - Base denaturation

• Most proteins denature at extremes of pH
• Primarily due to perturbed pKas of buried
  groups
• e.g. buried salt bridges

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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 ltgt N

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Folding of a 20-mer poly Ala
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Unfolding of the DNA Binding Domain of HIV
Integrase
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Two state transitions in multi-state reactions
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Rate determining steps
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Theories of protein folding

• N-terminal folding
• Hydrophobic collapse
• The framework model
• Directed folding
• Proline cis-trans isomerisation
• Nucleation condensation

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Molecular Chaperones

• Three dimensional structure encoded in sequence
• in vivo versus in vitro folding
• Many obstacles to folding
• Dlt----gtN
• ?
• Ag

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Molecular Chaperone Function

• Disulfide isomerases
• Peptidyl-prolyl isomerases (cyclophilin, FK506)
• Bind the denatured state formed on ribozome
• Heat shock proteins Hsp (DnaK)
• Protein export delivery SecB

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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

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GroEL
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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

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GroEL is allosteric

• Weak and tight binding states
• Undergoes a series of conformation changes upon
  binding ligands
• Hydrolysis of ATP follows classic sigmoidal
  kinetics

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Sigmoidal Kinetics

• Positive cooperativity
• Multiple binding sites

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Allosteric nature of GroEL
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GroEL changes affinity for denatured proteins

• GroEL binds tightly
• GroEL/GroES complex much more weakly

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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

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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

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Active site of GroEL

• Residues 191-345 form a mini chaperone
• Flexible hydrophobic patch

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Role of ATP hydrolysis
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The GroEL Cycle
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A real folding funnel
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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

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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

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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

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Transthyretin structure

• tetrameric. Each monomer has two 4-stranded
  b-sheets, and a short a-helix. Anti-parallel
  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.

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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 b-helical structure, with
  24 b-strands per turn of the b-helix.

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Formation of proto-filaments

• 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)