Lecture 3
Lecture 3
Lecture 3
Membranes are fluid lipid bilayers studded with proteins and often
contain areas of differing composition called rafts that float around
like icebergs on the ocean
Three primary components of
biological membranes
• Lipids
– These form the membrane bilayer itself
• Cholesterol (a special type of lipid)
– Affects bilayer properties
– Found only in eukaryotes
• Proteins
– These are associated with the bilayer or inserted into
it to add function
Three classes of membrane lipids
Phospholipids, Glycolipids and Sterols
• These are all amphipathic molecules. Hydrophilic end and hydrophobic end.
1. Phospholipids
• All have a phosphate linkage to a “head” group, and 2 fatty acid chain “tails”.
• Phosphoglycerides
– Major component of most membranes
– Consist of two fatty acids linked to glycerol, with differing chemical groups added
to glycerol phosphate in the head group.
– One fatty acid chain is saturated, one unsaturated*
– Many different types with different structures
• Sphingomyelin
– Sphingosine amino group, instead of glycerol, links to phosphate in head
– Two saturated fatty acid chains*
2. Glycolipids
– Sphingosine amino group links to sugars instead of phosphate in head
– Have two saturated fatty acid chains*
3. Sterols are amphipathic, four-ring hydrocarbons. Cholesterol can increase or decrease
membrane fluidity depending on conditions.
*Saturated fatty acid chains give rise to thicker and less fluid bilayers.
Phospholipids Glycolipids
Sterols
Wikipedia
Lipids in water can form two types of structures,
micelles and bilayers
• You need to get the
hydrophobic tails of
phospholipids away from
water- There are two common
ways of accomplishing this
– Micelles
• Small sphere with
tails pointed in
– Bilayers
• Two layers of lipids
with tails pointed
toward each other
• Which structure is formed is
dependent on the type
(chemistry- charge on head,
tail length, shape, number)
and concentration of the lipid
• Biological phospholipids can
form bilayers! Two “leaflets”
Properties of Lipid Bilayers
• Bilayers close upon themselves to
make a continuous surface
interacting with water.
• No “edges” are left exposed;
water interacting with
hydrophobic tails unfavorable
• Membranes try to reseal if
broken or punctured
– A cell will die if the seal does
not reform fast enough
Biological Membrane Composition
transverse
Using microscopy to determine the lateral
mobility of lipids in the plane of the membrane
Fluorescence recovery after
photobleaching (FRAP)
– Label phospholipids
with a fluorescent probe
– Shine a bright laser on a
small spot of membrane
to bleach & destroy the
fluorescence on those
lipids
– Measure how long it
takes for other
fluorescent lipids to
diffuse into the
bleached region until it
is as bright as the rest of
the membrane
Membranes are not uniform seas of phospholipids with
proteins floating in them, they are asymmetric!
• The two different leaflets of the membrane have different
compositions
– The lipids are synthesized in the ER and inserted into one or the
other faces of the bilayer
– Membrane proteins (flipases) flip-flop the lipids back to their
normal sides to maintain the asymmetry!
• Lipids are not distributed randomly in the plane of the
membrane
– Some lipids, especially sphingolipids, like to cluster relative to
other membrane lipids
– This can create microdomains with locally high concentrations of
certain lipids and proteins, such as lipid rafts
Proteins are the other major component of membranes
Hydrogen bonding
pattern is determined
by primary amino
acid sequence
Tertiary structure is determined by:
– Hydrogen bonds
– Hydrophobic interactions
– Ionic interactions
– Polar interactions
– Van der Waals forces
– Covalent-disulfide bonds
Protein Folding
• Protein folding can occur co-translationally.
• There are many possible ways to fold a protein, but only one is
“right” for proper function.
• Sometimes, an N-terminal hydrophobic domain should pair
with one at the C-terminus.
• Because protein synthesis occurs over time, the right C-
terminal may not even exist for a while, creating a situation
where the N-terminal pairs incorrectly.
• Some proteins can fold properly on their own, others do not.
• Chaperones and Chaperonins are proteins that bind to
proteins during or after synthesis and help them fold properly.
Hsp70 family proteins are the major
chaperones in all organisms
Some proteins need even more help from protein
complexes called chaperonins
Disulfide bonds stabilize protein structures
in oxidizing environments
• Oligosaccharides influence
protein structure/function.
• Membrane glycoproteins
are oriented so that the
carbohydrate chains face
the extracellular domain.
Proteins are comprised of functional domains
• A combination of helices and sheets with turns and connecting
regions can fold into a functional domain that acts as a unit but is still
only part of a protein. Functions can include things like:
– ATP binding sites (myosin motor)
– Ca++ binding sites
– Enzyme activity of a particular sort
– Regulation via interactions with another protein
• A protein normally consists of more than one domain. Each domain
performs a specific function.
• Evolutionarily, new proteins can be formed by putting together new
combinations of domains (domain shuffling)
• Some of the same domains get used over and over by different
proteins in different cells
• It is increasingly possible to analyze primary sequences and deduce
the function of a protein by analyzing its domain structure.
What determines the way proteins bind to
other proteins or DNA?
What determines the way proteins bind to
other proteins or DNA?
What determines the way proteins bind to
other proteins or DNA?
affinity
Three Classes of Membrane Proteins
Integral membrane proteins- tightly associated with the lipid bilayer.
-Amino acids interact directly with lipid portion.
-Transmembrane proteins span the bilayer one or more
times while others associate with only one leaflet
GPI-link
Fatty Acid-link
How do you show a protein is associated
with the membrane?
• Use immunofluorescence or immuno-EM to immunolocalize the
protein to the membrane
• Purify the membrane and determine which proteins are present
– You can break cells by homogenization
– The membranes have a different density than other molecules allowing
them to be separated via sucrose gradient centrifugation
– Different membranes of the cell have different compositions and so can
be separated from each other
– Special detergents can be used to dissolve the membrane but keep the
membrane protein active for immunoblotting or biochemical assay
• Purify the protein and show that association with membrane
lipids is required for its function
Proteases cleave or digest accessible protein regions and can be
used to deduce the topology of a protein in the membrane
A A B
Some proteases such as trypsin are digestive; they break the protein into many
small, often non-functional, peptide fragments or amino acids. Others are highly
specific and only cleave protein substrates at a certain specific sequence, thus
generating intact, potentially functional fragments.
Karp Figure 4.17
What is the structure of the transmembrane
region of proteins?
• Typically hydrophobic or amphipathic alpha helix, or amphipathic
beta sheet
• If alpha helix-based, can span one or more times
– Single Pass Transmembrane- crosses the bilayer once; 1 helix.
– Multipass Transmembrane- crosses 2 or more times; 2+ helices.
• The length of an alpha helix needed to cross the membrane is
about 20-30 amino acids (~4 nm). Remember though that
different lipid composition gives rise to different bilayer
thickness, so the length of a membrane protein’s hydrophobic
alpha helix will influence what kind of lipid composition that
protein “wants” to be in.
• Most amino acids in single pass alpha helical domain of a
transmembrane protein are non-polar (hydrophobic) to associate
with the inner hydrophobic region of the membrane
Integral Membrane Proteins
A single alpha helix in a membrane
(Karp)
Method 2 for measuring membrane protein
mobility: FRAP of fluorescently labeled protein
Method 2 for measuring protein mobility: FRAP of
fluorescently labeled membrane protein
Lipid rafts
Raft components include
cholesterol, sphingolipids, and
proteins. Rafts tend to accumulate
different proteins than the non-raft
areas.
sphingomyelin
GPI-anchored protein
cholesterol