Multiple Pathways on a ProteinFolding Energy Landscape: Kinetic Evidence by Goldbeck, et al'

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Multiple Pathways on a Protein-Folding Energy
Landscape Kinetic Evidenceby Goldbeck, et al.
(PNAS, Vol. 96, pp. 2782-2787, March 1999)

• Presented by Jean Rockford
• Biophysics 607
• March 28, 2005

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Protein Folding

• Proteins normally want to fold into their
  native, or lowest-energy, conformation the
  most stable
• Christian B. Anfinsen won the Nobel Prize in 1973
  for finding that this folding is based on
  directions from the amino acid sequence and
  from its hydrophobic/phillic nature
• He started by unfolding proteins into random
  coils, and found the proteins folded back into
  their native shape spontaneously, without any
  other catalysts

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When Folding Goes Wrong

• Protein Misfolding Proteins fold into
  conformations which are local energy minima, but
  not the global minimum
• States reaching local, but not global, minima are
  sometimes called intermediate states
• Misfolding is responsible for many diseases due
  to the accumulation of plaque of misfolded
  proteins Alzheimers, Parkinsons, Mad Cow
  Disease, etc.
• Often other chaperone proteins are present to
  help the target protein reach the desired
  functional (native) conformation

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Energy Landscapes in 2-D
Transition states

• In the process of folding from a random coil to
  the native state (at the global minimum),
  proteins pass through intermediate states (at
  local minima)
• Sometimes the proteins can get stuck in these
  intermediate states and cannot get over the
  energy hump needed (at transition states) to
  get to the final native conformation
• The study of protein folding from the denatured
  to the native state of vice versa, over time, is
  kinetics

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The Funnel (3-D) Landscape Model

• Protein Folding actually proceeds (as down a
  funnel) through multiple kinetic pathways
• Unstable (High Energy) unfolded conformations at
  rim can follow many energy gradients to get to
  the single global minimum (Lowest Energy)
• The steepest path is the fastest folding
  trajectory, but other slower trajectories to the
  minimum exist
• Note the diverse multitude of unfolded
  conformations, but only one conformation for the
  native state

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The Energy Landscape
E Energy P Available conformational
space Q Proportion of native contacts
formed Yellow Fast
folding Green Slow Folding that crosses the
high energy barrier Red Slow folding pathway
which returns to a less folded state before
following the pathway for fast folding
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The Goldbeck Paper

• Showed through direct and modeling experimental
  evidence that cytochrome c (a globular protein)
  has at least 2 folding pathways
• An ultrafast (folds in microseconds) pathway
• And a fast (folds in milliseconds) pathway
• The fast pathway is heterogenous during early
  folding events, and then joins the ultrafast
  pathway later in the trajectory towards the
  native state (Like yellow and red pathways)

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CO Photolysis in Cytochrome c

• First done by Jones et al (PNAS, Vol 90, pp.
  11860-11864, 1993)
• Adding CO, and guanidine HCl partially denatures
  cytochrome c, CO covalently binds to a heme
  group, makes a stable intermediate
• Flashing light at this complex breaks the CO-heme
  bond (photolysis!) and initiates folding back to
  the native state
• Laser light is pulsed in nanosecond pulses as
  sample flows through cell, conformational changes
  detected using Absorption (Jones) or Time
  Resolved Circular Dichroism (Goldbeck)

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Time Resolved Circular Dichroism (TRCD)

• Circular Dicroism is a special form of
  absorption spectroscopy
• It sends circularly polarized light through a
  sample
• Light absorption differs as the direction of
  light is modulated, while the magnitude of light
  remains constant
• Both left and right circularly polarized light
  are sent through a sample
• Proteins in solution will show detectable optical
  activity when there are differences in absorption
  of left right circularly polarized light
• 2 transient structural changes can be detected
  on the nanosecond range through time resolution,
  thus TRCD is a kind of real-time probe of folding
  in action!

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TRCD vs. Absorption

• By monitoring changes in beam polarization rather
  than direct absorbance, time resolution of
  nanosecond pulses and sensitivity to structural
  change is improved
• Structural changes in the early nanosecond range,
  after photolysis, can thusly be resolved
  absorption methods alone cannot do this

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TRMCD Time-Resolved Magnetic Circular Dichroism
TRCD TRMCD

• Natural TRCD can only probe structures that are
  optically active (I.E. they are chiral)
• TRMCD applies a magnetic field to the samples
  that perturbs the electronic states (magnetic
  moments) of the protein, thusly creating
  detectable difference when circularly polarized
  light is flashed through
• 1st magnet sample solvent, 2nd magnet just
  solvent (this removes magnetization effects that
  may be in the solvent alone)

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Heterogeneous vs. Homogeneous Model

• Homogenous kinetic model suggests that proteins
  all fold to their native state along the same
  pathway
• Heterogenous kinetic model suggests that
  proteins fold to the native structure along
  different pathways that may pass through
  different local minima, and take different
  amounts of time
• (Goldbeck proposes this one for cytochrome c)

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Findings of Goldbeck, et al

• TRMCD finds that distinct structural changes
  occur at times later than the microsecond range
  found with TRCD
• TRMCD also suggests different species of heme
  binding corresponding to the changes detected at
  different times

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Findings of Goldbeck et al, continued

• The fast pathway immediately has the ferrous heme
  bind with Met in cytochrome c, to form the final
  native state
• The slower pathway finds that heme first binds
  with His, then binds with Met later to also
  arrive at native state
• There are also some heme groups who are
  photolyzed but yet do not bind with the protein
  at all

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Summary of Goldbeck Findings

• TRCD spectroscopy directly found secondary
  structure change of cytochrome c after photolysis
  of CO that indicated folding to the native state
  in the ultrafast, microsecond range
• This ultrafast pathway is concomitant with heme
  binding to methionine
• Kinetic modeling of TRMCD data suggests a second
  slower pathway after photolysis, in the
  millisecond range
• This fast pathway indicates heme binding to
  histidine
• This slower pathway creates a local minimum in
  the energy landscape, corresponding to the
  histidine ligation trap
• These two pathways proceed independently, but in
  parallel