Membranes and Proteins

A key difference between prokaryotic and eukaryotic

cells is in the elaboration of internal membranes
 Membranes define organelles

• Each membrane-bound compartment in a eukaryotic cell

has its own unique set of soluble proteins, and its own
unique set of membrane proteins.
• A major focus in the next few lectures will be how this is
accomplished
 Membranes perform several important
cellular functions
• Separate one compartment from another
-membranes are selectively permeable barriers across which
solutes are transported
• Provide a scaffold for biochemical activities
-one key example is energy transduction in mitochondria and
chloroplasts.
• Mediate some kinds of cell-cell interactions
• Key element of many signal transduction pathways

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

• Different cellular membranes are composed of

different amounts of lipids and cholesterol, and
have very different protein composition
• The differences in composition relate to
differences in function
• Each biological membrane has different
properties based upon the different molecules
used to make it
Different membranes have different lipid compositions
Different membranes have different lipid compositions
Lipid composition affects bilayer thickness and
membrane curvature
Membranes are dynamic structures: summary of
movements of phospholipids

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

In addition to the other functions of lipids, the

bilayer acts as a scaffold for proteins.

Membrane proteins contribute to:

• selective permeability
• signal transduction
• biochemical reactions
• cell-cell interactions
• membrane properties, etc.
 A crash refresher on proteins
Proteins are the basic machinery of cells. They serve three
basic roles:
-enzymatic
-structural
-regulatory

There is a hierarchy of protein structure

Primary Structure- the sequence of amino acids,
determined directly from the RNA sequence.
Secondary Structure- local shape (beta-sheet, alpha-
helix).
Tertiary Structure- the 3D shape of the whole protein.
Quaternary Structure- multi-subunit assemblies.
 Amino acid side chain
properties drive protein
structure and function
Hydrogen bonds
are main contributor
to secondary structure.

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

Cytoplasm is not an oxidizing environment, so disulfide

bonds are uncommon in cytoplasmic proteins
 Most membrane proteins and secreted proteins are
glycosylated via processing in the ER and Golgi apparatus

• Oligosaccharides influence
protein structure/function.

• Used as tags to mark the

state of protein folding.

• 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

Lipid anchored proteins- covalent addition of a lipid to a protein targets

the protein to the membrane.
-Fatty acids are added to attach proteins to the inner leaflet
-Glycophosphatidylinositol (GPI) added to attach proteins to
outer leaflet.
-Some lipid anchored proteins can cycle between
membrane-bound and soluble forms

Peripheral membrane proteins- indirectly attached to the membrane via

interactions with other membrane proteins, not lipids
Integral Membrane Proteins
 Lipid anchored Peripheral membrane proteins

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

Note non-polar amino acids

(Karp 4-16)
A multi-pass transmembrane protein:
bacteriorhodopsin
 Amphipathic helices can also position integral
membrane proteins in one leaf of the membrane
Beta sheets can also interact with membranes

• The R groups from the sheet are organized so that the

non-polar are on one side and polar on the other
• The sheet is rolled into a tube (Beta-barrel)
• The hydrophobic are out toward the bilayer
• The polar are inside
• Forms a pore through the membrane that is a
hydrophilic environment
Beta-sheet structure
ß-barrel membrane proteins
 The Fluid Mosaic Model of Membranes
• The lipid bilayer is a flexible 2-dimensional fluid sheet
• Membrane proteins “float” in this sheet
• Proteins can move laterally in the plane of the membrane,
but…
– A protein cannot easily leave the membrane once inserted.
• Too much energy is required to tear the hydrophobic region out of
the hydrophobic bilayer.
– The topology of a protein cannot easily change once inserted in
the membrane.
• If it is made with 7 transmembrane regions, it will stay that way.
– The conformation can change
• Shape changes allow proteins to pass signals from outside to inside
Many proteins are constrained and cannot move
freely within the membrane
• Not all membrane proteins are mobile!
• Some membrane proteins are restricted in their location to a
particular region of the membrane
– This can define a membrane domain
• In a single cell, some proteins may be evenly distributed while
others are restricted
• The same protein may be restricted in one type of membrane
and not in another
• Cells employ a variety of mechanisms to non-randomly
distribute proteins:
– Link them to other membrane proteins
– Link them to outside molecules
– Link them to inside molecules
– Prevent their diffusion to parts of the membrane
Method 1 for measuring membrane
protein mobility: cell fusion
• Label proteins of one cell with red dye
• Label those of a second cell with green dye
• Fuse the membrane of the two cells to form a
heterokaryon
– Note: cell membranes normally do not fuse. You
have to force it to happen
• Watch what happens to the two dyes
• Result- over time they become mixed
Cell Fusion shows protein mobility in membranes

(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