Biological Membranes BMMB597a, Fall 2004 - PowerPoint PPT Presentation

Title: Biological Membranes BMMB597a, Fall 2004


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Biological MembranesBMMB597a, Fall 2004
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• Importance of membranes
• Plasma membrane separates cellular components
  from the environment.
• Allows organelles to execute specialized
  functions by keeping the contents of the
  organelle separate from the rest of the cell.
• Provides boundaries that establish
  electrochemical gradients. These are used to make
  ATP and to generate nerve impulses.

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Biological membranes are thin films composed
mainly of amphipathic lipids and proteins. Most
of the molecules are held together by noncovalent
interactions.
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• Lipids
• Lipid molecules organized into a bilayer
  approximately 5 nm thick.
• Each layer of the bilayer is called a leaflet.
• Lipids diffuse rapidly in the plane of the
  bilayer, but with the exception of cholesterol,
  lipids do no flip from one leaflet to the other
  without assistance from specific proteins.
• Self sealing - when one microinjects something
  into a cell, the membrane seals automatically
  when the needle is withdrawn.
• Purified phospholipids spontaneously form
  bilayers in water.

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• Membrane Proteins
• Proteins embedded in the membrane span from one
  side to the other.
• Proteins diffuse in the plane of the bilayer
  unless they are anchored to something.
• Proteins will not flip-flop.

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• Lipids provide the basis for the spontaneous
  formation of membranes.
• Lipids are amphipathic meaning they have a
  hydrophobic part (nonpolar tail) and a
  hydrophilic part (polar head).
• This example is a phospholipid which is the most
  abundant type of lipid found in animal cell
  membranes.

Polar head
Hydrocarbon tail derived from fatty acid.
Nonpolar tail
Unsaturated hydrocarbon tail
saturated hydrocarbon tail
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Amphipathic molecules pack so as to minimize the
interaction between water and the nonpolar part
of the molecule. The two hydrocarbon tails give
phospholipids a cylindrical shape that causes the
molecules to pack as a bilayer in water.
Minimum contact between water and the hydrocarbon
chains is achieved by forming the bilayer into a
closed compartment.
liposome
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Four major phospholipids are found in mammalian
plasma membranes.
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I want you to know the structure of
phosphatidylserine and its constituent parts.
Serine
phosphate
glycerol
fatty acid
fatty acid
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• Another type of lipid called cholesterol
  decreases fluidity because it restricts the
  movement of the hydrocarbon chains.
• Only found in animals cells.

• Important chemical characteristics of
  cholesterol
• Hydroxyl group constitutes the polar head group.
• OH is attached to rigid steroid ring.
• One hydrocarbon tail.

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In addition to phospholipids and cholesterol,
animal membranes contain glycolipids. Sugars
constitute the polar head group. Gangliosides
are common in nerve cells where they influence
the electrical properties of cell membranes.
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• Some lipids are asymmetrically distributed.
• glycolipids are restricted to the extracellular
  leaflet.
• phosphatidylserine is restricted to the
  cytoplasmic leaflet in a healthy cell.

• An example of a function for the asymmetry
• Dead cells are distinguished from live cells
  because phosphatidylserine becomes exposed on the
  outer leaflet in dead cells. The asymmetry is
  maintained by a phospholipid translocator that
  transports PS to the inner leaflet. This is
  inactivated in dying cells. A second protein
  called scramblase becomes active and transfers
  phospholipids nonspecifically in both directions
  resulting in exposure of PS on the outside of the
  cell. Macrophages detect the PS and destroy the
  dead cell.

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Lipid rafts provide an example of an uneven
distribution of lipids in the plane of the
membrane
Sphingomyelin and glycolipids attract each other
and coalesce into lipid rafts. Certain
proteins involved in cell signaling are grouped
together in these rafts. The grouping of the
lipids and proteins into a lipid raft occurs
because of weak noncovalent interactions between
the molecules and because the molecules are free
to diffuse laterally in the plane of the membrane
so they can contact each other.
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• Membrane proteins
• As with most aspects of a cell, proteins endow
  membranes with special functions. These special
  functions include transport, cell-cell
  communication, and many enzymatic activities.

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• How proteins associate with membranes
• Direct contact between hydrophobic amino acid
  side chains and the hydrophobic core of the lipid
  bilayer.
• Covalent attachment to a lipid.
• Association with part of another membrane protein
  that is exposed at the surface of the membrane.

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Most proteins that contact the hydrophobic core
of a lipid bilayer have alpha helical
transmembrane domains.
This covalently attached lipid is not essential

• Proteins 1 and 2 are examples of transmembrane
  domains composed of alpha helices. 1 is a
  single pass and 2 is a multipass.
• Regions of the polypeptide chain that penetrate
  the lipid bilayer are enriched in nonpolar amino
  acids.
• The peptide bond is intrinsically polar (it forms
  hydrogen bonds), but by forming hydrogen bonds
  between every fourth amino acid, interaction with
  the nonpolar lipid core is avoided.
• Transmembrane domain complete span the bilayer
  because breaks in the alpha helix expose polar
  groups in the peptide backbone.

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Recall that the amino acid side chains (R-groups)
project outward from the alpha helix generated by
the peptide backbone.
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The hydrophobic core of the bilayer is 3 nm
thick. One turn of the helix spans 0.54 nm and
there are 3.5 a.a./turn. Hence, an transmembrane
domain composed of an alpha-helix is around 20
amino acids long.
the 5 nm seen in many pictures refers is the
distance from one polar head group to another
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It is possible to predict alpha helical
transmembrane domains by analyzing the primary
amino acid sequence for stretches of 10 to 20
hydrophobic amino acids. This analysis, however,
does not reveal the orientation in the membrane.
Hydropathy plots
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N-terminal part of Cystic Fibrosis Transmembrane
Regulator (CFTR)
MQRSPLEKAS VVSKLFFSWT RPILRKGYRQ RLELSDIYQI
PSVDSADNLS EKLEREWDRE LASKKNPKLI NALRRCFFWR
FMFYGIFLYL GEVTKAVQPL LLGRIIASYD PDNKEERSIA
IYLGIGLCLL FIVRTLLLHP AIFGLHHIGM QMRIAMFSLI
YKKTLKLSSR VLDKISIGQ LVSLLSNNLNK FDEGLALAHF
Underlined regions are predicted by a hydropathy
plot to be transmembrane domains.
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• Additional characteristics of many alpha helical
  transmembrane proteins
• SH groups facing the cytoplasm are reduced and
  those facing outside the cell are oxidized to
  S-S. Reduction is due to glutathione in
  eucaryotes and thioredoxin in bacteria.
• Sugars are covalently attached to certain amino
  acid side chains in regions that face outside the
  cell.

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The beta-barrel structure emphasizes the
importance of the organization of hydrophobic
residues in membrane proteins.

• Protein 3 is known as a beta barrel and is
  composed of a beta sheet curved into a barrel
• Much rarer than the alpha helix transmembrane
  domain - limited to the outer membranes of
  bacteria, chloroplasts, and mitochondria.
• One well-known representative is a protein called
  Porin. The inside of the barrel is lined with
  polar amino acid side chains and the outside of
  the barrel is lined with nonpolar amino acid side
  chains.
• The polar groups in the peptide backbone are
  hidden by hydrogen bonding with antiparallel
  strands.

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Recall how the side chains of alternating amino
acids project on the same side of the beta sheet.
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Keeping the previous structure of the beta sheet
in mind, it should be clear why hydrophobic amino
acids should alternate with hydrophilic amino
acids in a transmembrane beta barrel.
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• 5 and 6 are examples of proteins associated with
  membranes by the covalent attachment to lipids.
• Note that the polypeptide chain does not
  penetrate the lipid bilayer.
• 7 and 8 are examples of proteins associated with
  other membrane proteins.
• Note that the polypeptide chain does not
  penetrate the lipid bilayer.
• Although these examples show association with
  alpha helical transmembrane proteins, the
  association could have been with proteins like 2,
  3, 4, 5, 6

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• Membrane proteins are often classified as
  peripheral membrane proteins or integral membrane
  proteins based on what is required to extract the
  protein from a membrane.
• Peripheral membrane proteins - dissociate from
  membranes when exposed to high (sometimes low)
  salt or extreme pH. These treatments disrupt
  noncovalent interactions and indicate that the
  association is like that of proteins 7 and 8.
• Integral membrane proteins - these are not
  released from the membrane unless the membrane is
  destroyed with detergent. This implies a
  membrane association like proteins 1 to 6.

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Detergents are very important for the study of
membrane proteins.
Mild nonionic detergent that dissolves membranes
without unfolding proteins.
Both are amphipathic
Ionic detergent that dissolves membranes and
unfolds proteins.
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A major advance in the study of membrane proteins
involved in transport was the discovery that the
proteins would spontaneously assemble with
purified lipids in vesicles sometimes called
proteoliposomes. Liposome indicates a
vesicle made of lipids and proteo indicates
that proteins are present.
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Fluorescence recovery after photobleaching
provides evidence that many proteins undergo
lateral diffusion.
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Not all proteins undergo lateral diffusion. For
example, attachment to the cytoskeleton
immobilizes some proteins.
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Tight junctions between cells of epithelia lining
many organs restrict proteins to one side of a
cell.
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Transmembrane protein molecules of the same kind
are all oriented in the same direction.
Moreover, they do not flip-flop.
Hypothetical possibilities.
Flip-flopping
Treat with protease that can cut off both ends of
the protein, if the protease has access to the
ends.
These possibilities could be distinguished by
SDS-PAGE (Polyacrylamide Gel Electrophoresis)
followed by western blotting to identify the
protein (note that the antibody used for the
western blot would have to bind the part of the
protein that remains in each case after
proteolysis). The right two results would never
be observed because all of the protein molecules
are always oriented in the same direction.