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Section 5
Junaid Malek, M.D.
Membrane Proteins
• Most membrane functions carried out by
proteins, which comprise 50% of the mass of
most membranes
• Functions: receptors, transporters, anchors
or enzymes
• Associations: transmembrane, membraneassociated, lipid-linked, protein attached
• We can use different tests to determine
protein-membrane associations
• Adding detergent - Using amphipathic
molecules
• Adding lipase - an enzyme that can
degrade protein/lipid covalent bonds
• Salt wash - washing with a high
concentration of salt in water disrupts
ionic and H-bond interactions
Effects on MembraneAssociated Proteins
Technique
Result
Detergent added
Liberated
Lipase
No effect
Salt wash
No effect
Effects on Lipid-Linked
Proteins
Technique
Result
Detergent added
Liberated
Lipase
Liberates fragment
Salt wash
No effect
Effects on ProteinAttached Proteins
Technique
Result
Detergent added
Liberated
Lipase
No effect
Salt wash
Liberates fragment
Effects on Transmembrane Proteins
Technique
Result
Detergent added
Liberated
Lipase
No effect
Salt wash
No effect
Transmembrane
Proteins
• Utilize specific secondary structures to
bridge hydrophobic span of lipid bilayer
• α-helix
• β-sheet
In these secondary
structures, where are the
side-chains located?
• α-helix
• Side-chains stick out from the helix
• β-sheet
• Side-chains alternate facing inside of the
barrel and outside of the barrel
Lipid Rafts
• Slightly thicker regions of the cell membrane
(longer FA chains)
• Region of membrane specialization
• Different proteins can segregate there with
different function
• Q: Why might selective segregation and
concentration be important for membrane
function?
• A: They help sort proteins to the correct
destination. They could concentrate signaling
proteins in the same area so once activation
occurs the signal can be rapidly transmitted.
Membranes as a barrier to
restrict molecular movement
Which molecules can freely
diffuse across a lipid
membrane?
Glycine?
No
Glutamate?
No
Methanol?
Yes
Water?
Yes
O2?
Yes
H+?
No
Cellular Transport
• In order to survive and grow, cells must
exchange molecules with their environment
• These molecules moved across the
membrane by special transport proteins
• Transport can be active (requiring energy)
or passive (not requiring energy)
Cellular Homeostasis
• Cells keep an internal ion concentration
different from the external environment
• This difference is crucial to cellular function,
including function of nerve cells
Intracellular
Extracellular
Na+
+
Na
Cl-
Cl
+
K
K+
Key Points
• Sodium is the most plentiful extracellular ion. Potassium
is the most plentiful intracellular ion.
• Too much electrical charge cannot build up inside or
outside of the cell so the amount of positive charge
inside (or outside) of the cell must be balanced with an
almost equal number of negatively charge
• Outside the cell, the high concentration of sodium is
balanced by chloride
• Inside the cell, the high concentration of potassium is
balanced by negatively charged organic ions (anions)
Key Points
• Tiny excesses of negative or positive charge can build
up near the plasma membrane - this has important
consequences
• Uncharged molecules - concentration gradient drives
passive transport
The Electrochemical
Gradient
• Influences transport of charged molecules
• Combination of the concentration gradient
and the membrane potential (charge
difference across the membrane)
• Active transport of ions against the
electrochemical gradient is crucial for
maintaining the internal ion composition of
cells
Electrochemical
Gradient
• Two factors can influence ion movement:
concentration of the ion and concentration
of charges
• If an ion like Na+ is trying to enter a cell, it
is moving down it’s concentration gradient,
and also from a region that is more positive
to a region that is more negative. The
energy released is greater than that of either
concentration or charge alone.
Electrochemical
Gradient
• If an ion like K+ is trying to exit a cell, it is
moving down it’s concentration gradient, but
up the charge gradient. Thus the energy
released when K+ exits the cell is less than
one would expect relying on concentration
alone.
Ion Channels
• Exhibit tremendous selectivity and efficiency
• Remember, structure affects function!
Potassium Channel
•
4 subunits positioned
precisely against one
another
•
Has a pore of defined
diameter that will only
allow one ion (stripped of
water) to go through at a
time
Potassium Channel
•
Remember
thermodynamics: Na+ or
K+ can interact with 4
water molecules
(favorable enthalpy).
When in the pore, Na+ is
smaller and can only form
bonds with 2 carbonyl
oxygens (enthalpically
unfavorable) while K+
can form bonds with 4
carbonyl oxygens
(enthalpically neutral)
Where can the carbonyl oxygens in
the pore come from?
carbonyl oxygen can also come from peptide backbone
Membrane Potential
• Originates from a charge imbalance due to
the movement of potassium ions
• Created due to the flow of K+ through leak
channels and the Na+/K+ ATPase pump,
though mainly due to the K+ leak channels
Membrane Potential
• A charge imbalance is created and then K+
is allowed to leak in to try to balance the
charge imbalance. K+ enters attracted by
the negative charge inside the cell, but the
concentration has a limit based on the
disfavorable process of moving the ions from
a region of low concentration to high.
• Thus a point is reached where there is a
balance between the electrical gradient and
the concentration gradient of K+ so the
electrochemical gradient for K+ is 0
HIV Entry Into The Cell:
Membrane Insertion
• gp120 binds to the chemokine receptor
• This induces a change in the shape of gp120
that allows gp41 to unfold so the 3 N- and 3
C- alpha helices come apart
• gp41 springs out and spears the plasma
membrane of the host cell with its tip (called
the fusion peptide) anchoring the HIV virus
to the host cell
What type of amino
acids would you expect
to be present in this tip?
• Hydrophobic amino acids
HIV Entry Into The Cell:
Membrane Fusion
• The pre-hairpin intermediate (gp41
“stretched out”) spontaneously rearranges
back into a hairpin so that its alpha helices
bundles are now close
• The energy released by this favorable
structural rearrangement is used to pull the
two membranes together
Fuzeon: HIV Fusion
Inhibitor
• 36AA peptide corresponding to a region
from the C-terminus of gp41
• Binds to the N-terminal α-helical bundles
and prevents them from binding to the real
C-terminal bundles
• This causes the virus-host cell complex to
get stuck in the pre-hairpin intermediate
• In the absence of fusion, the virus will fall off
the cell fairly rapidly
Prokaryotic Cell
• plasma membrane, cell wall, periplasmic
space, no compartments, genetic material is
DNA-organized into nucleoid
Eukaryotic Cell
• plasma membrane surrounds cell, eukaryotic
cells much more complex, organized into
membrane-bound compartments called
organelles, genetic material is DNA –
contained in nucleus
The Organelles
• Mitochondria - energy generators of the cell
that make ATP; surrounded by a double
membrane
• Endoplasmic Reticulum (ER) - a maze of
interconnected spaces surrounded by a
membrane serves as the site of synthesis of
proteins destined for membranes
The Organelles
• Golgi Apparatus - stack of flattened disks of
membrane that receives proteins from the
ER, modifies them and directs them to other
organelles, the plasma membrane or to the
exterior of the cell
• Lysosomes - site of degradation of
macromolecules
• Peroxisomes - contained environment for
reactions involving hydrogen peroxide, a
highly reactive molecule
Virus Anatomy
Cellular Transport
Cellular Transport
• Nucleus - transport through nuclear pores
• Large enough that ions and small
molecules (e.g. metabolites) can freely
diffuse through them, but proteins and
nucleic acids cannot
• Chloroplast, ER, mitochondrion - no pore
• Proteins, ions, and small molecules must be
transported across the membranes
Cellular Transport
• Proteins move within the secretory pathway
between the ER, Golgi, plasma membrane,
and lysosomes
• Movement between these compartments
occurs inside lipid vesicles
• Vesicles are loaded with cargo proteins from
the lumen, or interior space, of one
compartment, and discharge their cargo into
a second compartment
Protein Targeting
• Signal sequences are portions of proteins that
act as a zip code to tell the cellular machinery
where the protein’s correct destination is
• Signal sequences are recognized by transport
receptors, which help target proteins to the
correct compartment
• Typical signal sequences can either be
contiguous stretches of AAs or they can consist
of AAs distributed throughout the protein
sequence, which are close together in the
folded structure of the protein
Targeting Proteins to ER
• ER is entrypoint for proteins going to
lysosome, GA and cell membrane
• Entry directed by signal sequence
• Most proteins enter the ER before they are
fully translated
• Both water soluble and transmembrane
proteins can be transferred from cytosol to
ER
• Once in ER, though, these proteins rarely
return to cytosol
Targeting of gp160 to the ER
• gp160 is an integral membrane protein in
that it is the precursor to gp120 and gp41
• gp160 has an N-terminal signal sequence
that directs it to the ER
Processing of gp160
• During protein translocation into the ER, the
signal peptide is cleaved by the signal
peptidase
• The growing polypeptide is modified by the
addition of N-linked oligosaccharides within
the lumen of the ER
• Proteins inside the ER help gp160 fold and
promote the formation of the correct
disulfide bonds
Processing of gp160
• gp160 possesses ~30 potential N-linked
glycosylation sites and 10 disulfide bonds
• Without proper glycosylation and disulfide
bond formation, the newly synthesized
gp160 proteins aggregate and remain in the
ER