Lecture:April 5, 2004

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TOPICS IN BIOCHEM. MOL. BIOL. SPRING 2004
Lecture April 5, 2004 Topic Peripheral
Membrane Proteins Catalysis in Two-Phase
Systems References See Links
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Peripheral Membrane Protein
Different types of membrane-protein interactions.
Interactions of peripheral proteins (amphitropic)
with bulk lipids include coulombic interactions
between cationic residues and anionic lipids
(red) (a), interactions between aromatic residues
(green) and zwitterionic lipid (blue) (b), and
hydrophobic interactions involving aliphatic
residues (and Phe) that are, in general, exposed
by conformational changes of protein (c). d,
peripheral proteins can also interact with a
lipid second messenger (magenta) such as DAG. Not
shown is amphipathic helix (charged on one face
and hydrophobic on the opposite face). Cho
(2001) J. Biol. Chem. 276 32407
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Phospholipases
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Model of membrane interaction of cPLA2-C2. Also
shown are Ca2 ions (magenta) bound to the
domains. Hydrophobic (red) residues involved in
membrane interactions are shown in space-filling
representation.
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PIx's are regulators in themselves as are their
breakdown products Phospholipase C's activated
by external signals via G-Proteins and are
subject to substrate dilution kinetics
DAG, PtdSer and Ca2 are activators of Protein
Kinase Cs
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Schematic representation of the primary structure
of conventional, novel, and atypical protein
kinase Cs. Indicated are the pseudosubstrate
domain (green), C1 domain comprising one or two
Cys-rich motifs (red), C2 domain (yellow) in the
regulatory half, and the ATP-binding lobe (C3,
pink) and substrate-binding lobe (C4, blue) of
the catalytic region. The C2 domain of novel
protein kinase Cs1 lacks amino acids involved in
binding calcium but has key conserved residues
involved in maintaining the C2 fold (hence its
description as C2-like''). Atypical protein
kinase Cs have only one Cys-rich motif, and
phorbol ester binding has not been
detected. Newton (1995) J. Biol. Chem. 270 28495
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Model of membrane interaction of PKC-C1B domain.
Phorbol ester (white), Zn2 ions (purple) bound
to the domains. Cationic (blue) and hydrophobic
(red) residues involved in membrane interactions
are shown in space-filling representation.
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Model of membrane interaction of PKC-C2. Also
shown are Ca2 ions (magenta) bound to the
domain. Cationic (blue) interactions are shown in
space-filling representation.
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Model for the regulation of protein kinase C.
Newly synthesized protein with the
detergent-insoluble fraction of cells. Three
functionally distinct phosphorylations render the
kinase catalytically competent, stabilize the
catalytically competent conformation, and release
the protein into the cytosol. Membrane
translocation is mediated by diacylglycerol
binding to the C1 domain and phosphatidylserine
(PS) binding to the C2 domain. The high affinity
binding mediated by both domains results in
pseudosubstrate release and maximal activation.
Asterisks indicate the exposed hinge, which
becomes proteolytically labile upon membrane
binding and the exposed pseudosubstrate, which
becomes proteolytically labile upon activation.
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MinD is an Amphitropic Protein Regulating Septum
Position

• ATP binding exposes a C-terminal amphipathic
  helix.
• Helix has higher affinity for anionic lipid (red)
  enriched domains at cell poles (E. coli).
• Membrane binding induces MinD polymerization.
• MinD polymerization restricts septum formation to
  cell center where MinD is low.
• MinE hydrolyzes ATP on MinD causes dissociation.

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The binding of MinD-ATP to liposomes of different
phospholipid composition
E. Mileykovskaya, et al. (2003) J. Biol. Chem.
278 22193-22198
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Illustration of the application of "surface
dilution kinetics Carmen et al. JBC Minireview
27018711-18714, 1995.
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where KsA k-1/k1 and KmB (k-2 k3)/k2. A is
the bulk concentration of surface available for
binding either as total membrane surface or total
specific phospholipid. B is the concentration of
substrate in mole fraction terms. The Vmax is the
true Vmax at infinite mole fraction of
phospholipid substrate and at infinite bulk
concentration of surface for binding, KmB is the
interfacial Michaelis constant (expressed in
surface concentration units), and KsA is the
dissociation constant (expressed in bulk
concentration terms) for the mixed micelle
binding site.
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Dowhan et al.(1974) JBC 2493079-3084
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A PL
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Peripheral MP Summary

• Many proteins bind to membranes through surface
  interactions
• Peripheral MPs interaction in 4 modes (5 if
  consider amphipathic helix)
• Membrane association causes large changes in
  activity
• Surface dilution kinetics can be used to analyze
  catalysis at a membrane surface
• Activity is dependent on mole fraction within a
  surface not on bulk concentration in solution

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Summary of Membranes Section I. Hydrophobic
Effect- Driving force for expelling hydrocarbons
from water. A. Water structure and DS drive and
not DH. B. Short range and additive for
hydrocarbon. C. Cooperative effects in water
structure. II. Amphipathic Molecules A. Can
treat hydrophobic and hydrophilic domains
separately and in additive manner. B. Treat
detergents, phospholipids and proteins in similar
manner. C. Understand the forces and principles
driving membrane protein assembly into membranes
and their folding into their final structures.
III. Detergents, micelles, and vesicles A.
Principle of Opposing Forces. B. Structure
dependent on Hydrophobic Effect and steric
considerations. C. Net ionic, zwitterionic, and
nonionic detergents. D. Effect of salt,
ethanol, and chaotropic agents on micelle
properties. E. Determinants of cmc and m
(monomers per micelle). F. Protein
hydrophobic domains similar to membrane core or
micelle core. IV. Protein-SDS interactions A.
As denaturant-Monomer versus micelle effects,
salt effect on cmc and m. B. SDS causes
cooperative changes in protein structure. C.
Principles of SDS gel electrophoresis and reasons
for anomalies.
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V. Protein-Non-Denaturing Detergent
Interactions A. Binding isotherms and their
explanation. B. Cooperative changes are in
detergent and not in protein. C. Principles
to consider in solubilizing membranes and
membrane proteins. D. Considerations of mixed
micelles of phospholipid, detergent and protein.
E. Non-denaturing detergents affect hydrodynamic
properties of membrane but not soluble proteins.
VI. Enzyme kinetics in 2-phase systems A.
Understand mixed micelle substrate structures and
physical properties B. Enzyme-micelle versus
enzyme-substrate interactions C. Substrate
concentrations expressed in mole fraction or
surface concentration D. Implication of surface
dilution kinetics on classical Vmax and Km VII.
Lipids and lipid polymorphism A. Be familiar
with the structures of the lipids listed in the
syllabus (focus on differences and
similarities). B. Know the different physical
organizations that lipids can adopt and
understand the physical bases for such
organization. D. Why might cells require
lipids that can assume multiple physical states?
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VIII. Membrane Protein Structure A.
Differentiate between integral and peripheral
membrane proteins. B. Describe 4 (5) modes of
peripheral membrane association with membranes.
C. Understand how ß-sheets and a-helices form
transmembrane domains. D. What factors govern
partitioning of protein domains into the
bilayer. E. How do lipids affect membrane
protein organization? F. What are lipids rafts
and how do they govern association of proteins
with membrane?