Membrane Protein Folding and Stability

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Membrane Protein Folding and Stability

• Physical Principles

Episode 1 Introduction, Partitioning into
membranes
http//www.biochem.oulu.fi/juffer/
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Membrane proteins (MPs) (1)

• What are the general features of MPs?
• How are they assembled?
• How can their thermodynamic stability be
  described?
• What is the nature of the lipid bilayer as a host
  phase for MPs?
• What are the dominant forces that determine
  structure and stability?
• What can the folding and stability of MPs tell
  us about the protein folding problem generally?

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Membrane proteins (MPs) (2)

• Two structural motifs
• a-helix bundles
• b-barrels

1.5 nm
3 nm
b-barrels
a-helix bundle
Trp, Tyr side chains located typically in
interfacial region.
HChydrocarbon
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How many MP structures?
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MP versus soluble proteins

• MP are generally not soluble in water.
• MP interior is similar to those of soluble
  proteins.
• MP interior comprised of internally H-bonded
  a-helices and b-sheets.
• MP have direction in space
• Lys, Arg are more abundant in cytoplasmic domain
  relative to periplasmic domain.
• Tyr, Trp display strong preference for
  interfacial region.

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Assembly of MPs (1)

• Translocation apparatus involving the transient
  attachment of a active ribosome to a translocon
  embedded in membrane.
• Spontaneous insertion of soluble protein into
  membrane.

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Assembly of MPs (2)

• Regardless of insertion/assembly process
• Stably folded MPs reside in a free energy
  minimum.
• Minimum is determined by net energies of the
  interaction of peptide chains with
• Water.
• Each other.
• Lipid bilayer (hydrocarbon core and interface).
• Co-factors.
• Predicting of structure and stability from
  sequence is fundamentally a problem of physical
  chemistry.

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Thermodynamics of membrane partitioning (1)
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Thermodynamics of membrane partitioning (2)

• Four step model
• Partitioning, folding, insertion, and
  association.
• Several pathways
• Interfacial path, water path, combination of
  interfacial and water path.
• Free energy of transfer or partitioning

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Energetic components of partitioning (1)
Conformational Change.
Non-polar hydrophobic effect (expulsion of
non-polar compounds from water
Changes in motional degrees of freedom.
Difference in dielectric properties between water
and hydrocarbon region (mutual polarization
effects).
Direct electrostatic interaction between basic
residues and Anionic lipids.
Changes inside membrane.
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Energetic components of partitioning (2)

• Favorable partitioning of peptides and proteins
  into membranes is primarily caused by
• Nonpolar interactions, e.g. hydrophobic effect.
• Direct electrostatic interaction between charged
  amino acids and anionic lipids.

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Energetic components of partitioning (3)
Solvation free energy
Non-classical hydrophobic effect, bilayer effects
Classical hydrophobic effect
for proteins
A is solvent accessible area
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Stability of MP secondary structural elements (1)

• Knowledge forces stabilizing soluble proteins
  comes from
• Thermal unfolding experiments
• Denaturation experiments
• Computer experiments
• MP display higher stability of their secondary
  elements in membranes
• Strong resistance against denaturation and
  thermal unfolding.
• The importance of Hydrogen bonding.

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Stability of MP secondary structural elements (2)
Transfer free energy
DGt
Disruption of H-bond is unfavorable in
membrane e.g. 20 Residues would costs about 400
kJ mol-1.
non-H-bonded
Trp partitioning is favorable, but is not enough
to overcome non-H-bonded peptide bond DGt ? whole
residues can enter the HC core only if their
peptide bonds are H-bonded.
POPC interface more hydrophobic than water
Most hydrophobic side chain?
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Molecular interpretation of DGt

• Interpretation of DGt requires knowledge of
• Structure of bilayer.
• Transbilayer location of bound peptides.
• Structure of peptides adopt.
• Changes in bilayer as a result of partitioning.
• Cellular membranes are in a fluid state
  (La-phase) for normal cell function.
• High thermal disorder of membranes precludes
  high-resolution three-dimensional X-ray images.

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Structure of fluid lipid bilayers

• High thermal disorder reveals itself in
• Width of atoms or atom groups distributions.
• Combined thickness of interfacial regions is
  about the same as the HC core region
• One interface can easily accommodate an
  (un)folded peptide.
• Interfaces are extremely heterogeneous.

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Distributions across bilayers
Distribution of atom groups experimental based
upon combination X-ray and neutron diffraction
methods (liquid crystallography).
Polarity, charge distribution peptide coming
from solvent undergoes dramatic variation in
environments polarity
(Un)folded peptide fits easily in interfacial
region.