Download Structure and Function of Membrane Proteins: Overview

CAPÍTULO 4 (8 na 6ª Edição Internacional)
A ESTRUTURA E FUNÇÃO DA MEMBRANA PLASMÁTICA – PARTE 1
OBJECTIVOS
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Descrever as funções das membranas celulares.
Enumerar os principais componentes moleculares das membranas biológicas e descrever a sua organização
espacial.
Relacionar as propriedades das membranas com a sua composição química
Identificar a localização dos açúcares na estrutura da membrana e relacionar com funções possíveis.
Descrever diferentes tipos de proteínas presentes nas membranas e relacionar com as funções desempenhadas por
essas proteínas.
Compreender a importância da fluidez da membrana para a manutenção de todos os mecanismos moleculares da
célula.
Descrever os mecanismos utilizados pelas células para manter a fluidez das membranas.
Descrever a assimetria da membrana e a natureza dinâmica da sua estrutura e função.
Descrever os diferentes mecanismos utilizados pelas células para o transporte de materiais através da membrana,
como a difusão simples e facilitada, proteínas canal e transporta activo.
Enumerar as principais propriedades da membrana dos eritrócitos, como um exemplo em que a composição
molecular se encontra bem determinada.
LECTURE OUTLINE
First Detection of Cell Membrane
I. Cells are separated from the world by a thin, fragile structure, the plasma membrane – 5 – 10 nm thick
A. ~10,000 plasma membranes stacked one on top of another would equal the thickness of a book's page
B. No hint of a plasma membrane is detected in a thin section under a light microscope since it is so thin
C. Finally, in the late 1950s, techniques for preparing & staining tissues had progressed to the point where they could be
visualized in the electron microscope
1. J. D. Robertson (Duke Univ.) – portrayed plasma membrane as three-layered structure, consisting of darkly
staining inner & outer layers & a lightly staining middle layer
2. All membranes examined closely (plasma, nuclear or cytoplasmic) or from plants, animals or microorganisms had
the same ultrastructure
3. The pictures touched off a vigorous debate as to the molecular composition of the various layers of the membrane
4. The 2 dark-staining layers in the electron micrographs correspond primarily to the inner & outer polar surfaces of
the bilayer
Summary of Membrane Functions and Overview of Membrane Structure
I. Compartmentalization - membranes enclose entire cell or diverse intracellular spaces in which occur specialized
activities that proceed with little outside interference & are regulated independently
A. They are continuous, unbroken sheets
B. A cell's various membrane-bound compartments have markedly different contents
II. Scaffold for biochemical activities – membranes are also distinct compartments themselves
A. They provide cell with extensive framework (scaffolding) within which components can be ordered for effective
interaction
III. Provide selectively permeable barrier – membrane can be compared to moat around a castle; a general barrier that has
gated "bridges" that allow desirable things to enter & leave space they surround
A. They prevent the unrestricted exchange of molecules from one side to the other - control what gets into & out of
cell; H2O moves easily
B. They also provide the means of communication between the compartments they separate
IV. Transporting solutes – they have transport machinery to move substances from one side to the other
A. Can transport substances (ions, sugars, amino acids, etc.) up or down concentration gradient; sugars & amino acids
taken up since they are needed to fuel metabolism & build macromolecules
B. Can establish ionic gradients across itself (critical for nerves, muscles, maybe helps all cells respond to their
environment) by transporting specific ions
V. Response to external signals (signal transduction) – plays critical role in response to external stimuli (hormones, growth
factors, neurotransmitters)
A. Receptors in membrane combine with specific molecules (ligands) having complementary structure & then initiate
response
1. Different cells have different receptors; can therefore recognize & respond to different ligands in environment
B. Interaction of receptor with external ligand causes generation of new signal (second messenger); stimulates or
inhibits internal cell activities like:
1. Making more glycogen, preparing for cell division, concentrating particular compounds, releasing calcium from
internal stores, even committing suicide
VI. Intercellular interactions - allows cells to recognize & signal one another, adhere when appropriate & exchange
materials & information; mediates interactions between cells of multicellular organisms
VII. Energy transduction – intimately involved in processes by which one type of energy is converted to another type
(energy transduction); done by membranes of chloroplasts & mitochondria
A. Photosynthesis & electron transport site in chloroplasts & mitochondria
1. Chloroplasts absorb sunlight energy in membrane-bound pigments & convert it into the chemical energy of
carbohydrates
2. Mitochondrial membranes transfer chemical energy from carbohydrates & fats to ATP
B. Allows storage of energy in gradients
A Brief History of Studies on the Structure of the Plasma Membrane
I. Ernest Overton, University of Zurich (1890s) - knew that nonpolar solutes dissolve more readily in nonpolar solvents than
in polar ones & that polar solutes are most soluble in polar solvents
A. Since he realized that to enter a cell, a solute must pass first through the membrane, he used root hairs with hundreds of
different solutes & found that more lipid-soluble solutes enter root hair cells faster
B. Concluded dissolving power of outer cell boundary matched that of fatty oil
II. Gorter & Grendel (Dutch scientists, 1925) first proposed lipid bilayer; extracted lipids from red blood cell (RBC)
membrane, measured their surface area on H2O & compared it to estimated RBC surface area
A. Found surface area covered about twice (ratio was between 1.8 & 2.2) the surface area of RBCs
1. Used mammal RBCs since they lack both nuclei & cytoplasmic organelles; no other membranes
2. Plasma membrane is the only lipid-containing structure in cell; all lipids extracted from cells can be assumed to
have resided in plasma membrane
B. Propose lipid bilayer (bimolecular layer of lipids) with hydrophilic heads pointed out on both sides
1. Thermodynamically favored arrangement; polar lipid head groups interact with surrounding H2O molecules &
hydrophobic fatty acid (acyl) tails protected from aqueous environment
2. Polar heads face cytoplasm on one side & blood plasma on the other; polar heads of each leaflet were directed
outward toward aqueous environment
C. Got right answer, but used several miscalculations; however, mistakes compensated for each other
III. 1920s & 1930s - evidence accrued that there must be more to membranes than lipid bilayer
A. Lipid solubility was not the sole determinant of what can pass through membrane
B. Surface tensions of membranes were much lower than those of pure lipid structures
1. Protein film over artificial lipid membrane lowered its surface tension
2. Presence of protein could explain difference in surface tension
C. Artificial membranes can be formed spontaneously & studied
1. Natural & artificial membranes very similar (even in EM in more recent years)
2. Differences between natural & artificial membranes are due to proteins (especially permeability & electrical
resistance); cell membranes are about 5 – 10 nm wide
IV. Hugh Davson & James Danielli (1935) - proposed that membrane was composed of lipid bilayer lined on inner &
outer surfaces by layer of globular proteins
A. Revised version (1954) added penetration of bilayer by protein-lined pores; allow polar solutes/ions through
membrane to account for selective permeability
1. Provide conduits for that allow these solutes to enter & exit the cell
B. In mid to late 1950s, with techniques for preparing & staining tissue, J. D. Robertson & others were able to resolve cell
membranes in the electron microscope (see above)
V. S. Jonathan Singer & Garth Nicolson (UC-San Diego, 1972) - proposed the Fluid-Mosaic Model, the central dogma
of membrane biology for >3 decades
A. Lipid bilayer remains core of membrane, but it is not frozen & immobile, but fluid; individual lipids move
laterally within plane of membrane
B. Proteins distributed differently - mosaic of discontinuous particles that penetrate into or through membrane or
contact its polar heads without penetrating the membrane
C. Membranes are dynamic structures whose components are mobile & able to engage in a variety of transient or
semipermanent interactions
The Chemical Composition of Membranes
I. Membranes - lipid-protein assemblies held together in thin sheet by noncovalent bonds
A. Lipid bilayer is structural backbone of membrane & barrier preventing random movements of water-soluble
materials into & out of cell
B. Proteins of membrane carry out most of its specific functions
C. Often include carbohydrates attached to membrane lipids & proteins
II. Lipid:protein ratio varies greatly depending on membrane type (cell membrane vs. ER vs. Golgi),
organism
(prokaryote vs. plant vs. animal) & cell (cartilage vs. muscle vs. liver)
A. These differences largely relate to the particular functions of the membranes, e.g., inner mitochondrial vs. myelin
sheath
B. Example: inner mitochondrial membrane – very high protein:lipid ratio relative to RBCs that are high relative to
myelin sheath (multilayered wrapping around nerve cell axon)
1. Inner mitochondrial membrane has protein carriers of electron transport chain; lipid reduced
2. Myelin sheath is primary electrical insulation for nerve cell it encloses; this function is best carried out by a thick
lipid layer of high electrical resistance with a minimal protein content
III. Membrane lipids – wide diversity of amphipathic lipids with both hydrophobic & hydrophilic portions
A. Most have phosphate groups & are phospholipids (except cholesterol, glycolipids)
B. 3 types of membrane lipids: phosphoglycerides, sphingolipids, cholesterol
IV. Phosphoglycerides (see drawing in Ch. 2) - most membrane lipids contain phosphate groups so they are called
phospholipids
A. Most membrane phospholipids are built on a glycerol backbone & are thus called phosphoglycerides
1. In phosphoglycerides, glycerol is esterified to 1 phosphate group & 2 fatty acids (diglycerides)
2. With just phosphate & 2 fatty acids it is phosphatidic acid (virtually absent in most membranes)
B. Extra polar group usually added to phosphate (usually choline, ethanolamine, serine, inositol) to form polar head
group; called phosphatidylcholine (PC), phosphatidylethanolamine (PE), etc.
1. These groups are small & hydrophilic & combined with phosphate form hydrophilic domain at one end (polar
head group)
2. At physiological pH, phosphatidylserine (PS) & phosphatidylinositol (PI) head groups have an overall negative
charge; those of phosphatidylcholine & phosphatidylethanolamine are neutral
C. Fatty (acyl) acid chains – hydrophobic, unbranched hydrocarbons ~16 – 22 Cs long
1. Fatty acid tails may be polyunsaturated (>1 double bond), monounsaturated (1 double bond), saturated (no double
bonds)
2. Phosphoglycerides often contain 1 saturated + 1 unsaturated fatty acyl chains
D. Recent interest has focused on the apparent health benefits of 2 highly unsaturated fatty acids (EPA & DHA) found at
high concentration in fish oil
1. EPA & DHA contain 5 & 6 double bonds, respectively, & are incorporated primarily into PE & PC molecules of
certain membranes, most notably in the brain & retina
2. EPA & DHA are described as omega-3 fatty acids, because their last double bond is situated 3 carbons from the
omega (CH3) end of the fatty acyl chain
E. With fatty acid chains at one end & a polar head group at the other end, all of the phosphoglycerides have amphipathic
character
V. Sphingolipids - less abundant class of membrane lipids; derivatives of sphingosine
A. Sphingosine (an amino alcohol with long hydrocarbon chain) + fatty acid (attached to amino group; R in figure) = a
ceramide; all are amphipathic & similar in structure to phosphoglycerides
B. The various sphingosine-based lipids have additional groups esterified to the terminal alcohol of sphingosine moiety
1. Attach phosphorylcholine to terminal alcohol of sphingosine = sphingomyelin (the only membrane phospholipid
not built with a glycerol backbone)
2. Attach carbohydrate at terminal alcohol = glycolipid; if carbohydrate is simple sugar = cerebroside; if
carbohydrate is a small cluster of sugars = ganglioside
3. Since all sphingolipids have 2 long hydrophobic chains on one end & hydrophilic region on the other end, they
are amphipathic & similar in structure to phosphoglycerides
C. Glycolipids – relatively little known about them, but evidence suggests that they play crucial roles in cell function;
particularly prominent in membranes of nervous system
1. Ex.: galactocerebroside (galactose + ceramide) - mice without the enzyme that adds galactose to ceramide have
severe muscular tremors & eventual paralysis
2. Similarly, humans who are unable to synthesize a particular ganglioside (GM3) suffer from a serious
neurological disease characterized by severe seizures & blindness
D. Glycolipids also play role in certain infectious diseases; cholera & botulism toxins enter their target cell by first
binding to cell-surface gangliosides, as does influenza virus
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VI. Cholesterol – a sterol that can be up to 50% of animal membrane lipids; it is missing from most plant & all bacteria cell
membranes
A. Small hydrophilic hydroxyl group is oriented toward membrane surface; the rest is embedded in the lipid bilayer
B. Cholesterol rings are flat & rigid; interfere with movement of phospholipid fatty acid tails
The Nature and Importance of the Lipid Bilayer
I. Each type of cell membrane has its own characteristic lipid composition
A. Differ from each other in types of lipids, nature of head groups & particular species of fatty acyl chain(s)
B. Some biological membranes may contain hundreds of chemically distinct species of phospholipids; the role of this
remarkable diversity of lipid species remains the subject of interest & speculations
C. The percentages of some major types of lipids vary from membrane to membrane
II. Lipid composition can influence biological properties of membrane; not just structural elements
A. Can influence activity of particular membrane proteins
B. Can determine physical state of membrane
C. Can play role in health & disease - Tay-Sachs disease (fatal inherited condition caused by build-up of particular
lipid, a ganglioside, in brain cells)
D. Provide precursors for highly active chemical messengers that regulate cellular function
III. Combined fatty acyl chains of lipid bilayer span width of ~30 Å & each row of head groups (with its adjacent shell of
water molecules) adds another 15 Å; thus entire bilayer is only ~60 Å (6 nm) thick
IV. Presence in membranes of this thin film of amphipathic lipid molecules has remarkable consequences for cell
structure & function
A. Due to thermodynamic considerations, the hydrocarbon chains of the lipid bilayer are never exposed to the
surrounding aqueous solution
1. Thus, membranes never seen to have a free edge due to cohesion & spontaneous formation (they are closed
bimolecular sheets)
2. They are always continuous, unbroken structures & thus form extensive interconnected networks within cell
B. Due to flexibility of lipid bilayer, membranes are deformable & can change their overall shape (as in locomotion &
cell division)
C. Bilayer facilitates regulated fusion or budding of membranes – events of secretion (cytoplasmic vesicles fuse to plasma
membrane; exocytosis), endocytosis or fertilization (2 cells fuse to form single cell)
1. Both involve processes in which 2 separate membranes come together to become a continuous sheet
D. Membrane is also important in maintaining proper internal composition of cell & in separating electric charges
across plasma membranes & many other cell activities
V. Membrane can self-assemble in aqueous solutions (demonstrated easily within test tube)
A. If a small amount of phosphatidylcholine is dispersed in aqueous solution, the phospholipid molecules assemble
spontaneously to form the walls of liposomes (fluid-filled spherical vesicles)
1. Their walls made of single continuous lipid bilayer organized in same way as natural membrane
2. Valuable in membrane studies - insert membrane proteins, study their function in simpler environment than that of
a natural membrane
B. Liposomes have been developed as vehicles to deliver drugs or DNA to specific target cells in body; can be linked to
liposome walls or placed at high concentrations in liposome central cavity (lumen)
1. Build liposome walls to contain specific proteins (antibodies, hormones)
2. The proteins allow liposomes to bind selectively to surfaces of particular target cells where drug or DNA is
supposed to go
3. When first tried, immune system phagocytes removed them - now stealth liposomes (e.g., Caelyx) are given
synthetic polymer outer coating that protects them from immune destruction
VI. Asymmetry of membrane lipids – lipid bilayer consists of 2 distinct leaflets that have distinctly different lipid
composition
A. Experiments that have led to this conclusion take advantage of fact that lipid-digesting enzymes cannot penetrate
plasma membrane &, thus, can only digest lipids residing in membrane outer leaflet
1. If treat intact human RBCs with lipid-digesting phospholipases (only affect outer leaflet lipids since they cannot
penetrate membrane)……..
a. ~80% of the membrane phosphatidylcholine (PC) is hydrolyzed, but only ~20% of membrane
phosphatidylethanolamine (PE) & <10% of its phosphatidylserine (PS) are attacked
b. Thus, relative to inner leaflet, outer one has relatively high [PC] (& [sphingomyelin]), low [PS] & [PE]
2. Thus, one can think of bilayer as 2 more-or-less stable, independent monolayers having different physical &
chemical properties; different classes of lipids in membrane also have different properties
B. Biological role of lipid asymmetry - not generally well understood; may lead to different charges on each side of
membrane
C. All glycolipids of plasma membrane are in outer leaflet, where they probably serve as receptors for extracellular
ligands
1. Phosphatidylethanolamine, which is concentrated in inner leaflet, tends to promote the curvature of the membrane,
which is important in membrane budding & fusion
2. PS is also concentrated in inner leaflet – it has a net"-" charge at physiological pH, which makes it a candidate for
binding positively-charged lysine & arginine residues
a. Such arginine & lysine residues are adjacent to the membrane-spanning -helix of glycophorin A & PS may
bind them
b. PS appears on the outer surface of aging lymphocytes & thus marks them for destruction by macrophages
c. PS's appearance on outer surface of platelets leads to blood coagulation
3. Phosphatidylinositol, which is concentrated in inner leaflet, plays a key role in the transfer of stimuli from the
plasma membrane to the cytoplasm
Membrane Carbohydrates
I. Eukaryotic cell plasma membranes also contain carbohydrate
A. Depending on species & cell type, carbohydrate content of plasma membrane ranges between 2 - 10% by weight,
e.g., RBC membrane - ~52% protein, 40% lipid, 8% carbohydrate
B. <10% of membrane carbohydrate is covalently linked to lipids to form glycolipids & >90% of membrane
carbohydrates are covalently linked to protein to form glycoprotein
II. All membrane carbohydrates face toward outside of cells into extracellular space or toward organelle interior
(carbohydrates of internal cellular membranes); in both cases, they face away from cytosol
A. Phosphatidylinositol of membrane does not count even though it contains sugar group
B. Composition & structure of oligosaccharides attached to membrane proteins & lipids vary considerably
C. Provides for their specificity in interactions with each other & other molecules
III. Glycoproteins - carbohydrates are short, branched oligosaccharides with < ~15 sugars per chain; addition of these
sugars or glycosylation is most complex of protein modifications occurring in cell
A. Oligosaccharides vary considerably in composition & structure; sialic acid usually on end giving negative charge
B. Attach to several different amino acids by two major types of linkages
C. Play an important role in mediating interactions of cell with other cells & nonliving environment & sorting of
membrane proteins to different cell compartments
IV. Glycolipids - short, branched oligosaccharide chains
A. On RBCs, glycolipids determine ABO blood type (have different enzymes that add sugars to ends of carbohydrate
chains)
1. Person with blood type A has enzyme that adds N-acetylgalactosamine to end of chain
2. Person with blood type B has enzyme that adds galactose to chain terminus
3. The 2 enzymes are encoded by alternate versions of the same gene, yet they recognize different substrates
4. AB people possess both enzymes; people with type O blood lack enzymes capable of attaching either terminal
sugar
5. The function of the ABO blood-group antigens remains a mystery
B. May play roles in certain infectious diseases (cholera toxin & influenza virus bind to gangliosides); this suggests that
they probably serve as some kind of receptor in normal cell function
Structure and Function of Membrane Proteins: Overview
I. Membranes may contain hundreds of different proteins depending on cell type or particular organelle
A. Each membrane protein has defined orientation relative to cytoplasm; thus, the properties of 1 membrane surface are very
different from those of other surface - this asymmetry referred to as membrane sidedness
B. All proteins are asymmetrically situated according to function - properties of one membrane surface are very different
from those of other surface
C. Parts of proteins interacting with extracellular substances, other cells, extracellular matrix elements face out; those
interacting with cytoplasmic molecules face inward
II. Three classes of membrane proteins distinguished by intimacy of their relationship to lipid bilayer
A. Integral proteins - penetrate into lipid bilayer; they pass entirely through bilayer (transmembrane)
1. Have domains that protrude from both sides of membrane (extracellular & cytoplasmic)
2. Some have only one membrane-spanning segment; others are multispanning
3. Genome-sequencing studies suggest that integral membrane proteins constitute 20 -30% of all encoded proteins
B. Peripheral proteins – located entirely outside of bilayer on either the extracellular or cytoplasmic side; associated
with membrane surface by noncovalent bonds
C. Lipid-anchored proteins – located outside bilayer on either extracellular or cytoplasmic side, but they are covalently
linked to membrane lipid situated within bilayer
Structure and Function of Membrane Proteins: Integral Membrane Proteins
I. Most integral membrane proteins function in the following capacities:
A. As receptors that bind specific substances at the membrane surface
B. As channels or transporters involved in the movement of ions & solutes across the membrane or
C. As agents that transfer electrons during the processes of photosynthesis & respiration
II. Integral membrane proteins - amphipathic; hydrophobic parts contact fatty acids in bilayer & seal proteins into
membrane lipid "wall"; hydrophilic parts on outside or coating aqueous channel through it
A. Amino acid residues in transmembrane domains for van der Waals interactions with fatty acyl chains of bilayer
1. Intimate contact of membrane & integral proteins preserves permeability barrier & protein is brought into direct
contact with surrounding lipid molecules
2. Lipid molecules that are closely associated with a membrane protein might play an important role in the protein's
activity
3. However, the degree to which a particular protein requires specific interactions with particular lipid molecules
remains unclear
B. Those portions of an integral membrane protein that project into cytoplasm or extracellular space tend to be more like
globular proteins
1. These nonembedded domains tend to have hydrophilic surfaces that interact with water-soluble substances (low
MW substrates, hormones, other proteins) at the edge of the membrane
C. Several large families of membrane proteins have an interior channel that provides an aqueous passageway through
the lipid bilayer
1. The linings of these channels typically contain key hydrophilic residues at strategic locations
D. Integral proteins need not be fixed structures but may move laterally within membrane
II. Freeze-fracture replication analysis – showed that proteins can penetrate through membranes
A. Procedure – tissue is frozen solid & then struck with knife blade, fracturing the block into 2 pieces
1. A favored path of the fracture plane is between the 2 leaflets of bilayer so the membrane is split
2. Metals are deposited on exposed membrane surfaces to form shadowed replica & viewed in EM
3. Looks like road strewn with pebbles (called membrane-associated particles)
4. Since fracture plane passes through bilayer center, particles correspond to integral proteins that extend at least
halfway through lipid core of bilayer
5. When fracture plane reaches a given particle, it goes around it rather than cracking it in half so each protein
(particle) separates with one half of membrane leaving a pit in the other half
B. Allows an investigation of the microheterogeneity of membrane so one can see localized differences in parts of
membrane
1. Biochemical observations average out such individualities; microscopic observations do not & thus allow such
individualities to be appreciated
III. Studying structure & properties of integral membrane proteins – difficult to isolate in soluble form due to their
hydrophobic transmembrane domains
A. Extraction from membrane normally requires the use of detergents
1. Ionic (charged) detergents like SDS, which denatures proteins
2. Nonionic (uncharged) like Triton X-100, which usually does not alter protein tertiary structure
B. These detergents are amphipathic (polar end, nonpolar hydrocarbon chain) so they can substitute for phospholipids in
stabilizing the proteins, while solubilizing them in aqueous solution
1. Once solubilized, various analytical procedures can be carried out to determine protein's amino acid composition,
molecular mass, amino acid sequence, etc.
C. Hard to get crystals of most integral proteins for X-ray crystallography
1. In fact, <1% of known high-resolution protein structures represent integral membrane proteins
2. Furthermore, most of these structures represent prokaryotic versions of a particular protein, which are often
smaller than their eukaryotic homologues & easier to obtain in large quantity
3. Once the structure of one member of membrane protein family is determined, researchers can usually apply
strategy called homology modeling to learn about structure & activity of other family members
a. Example – solution of bacterial K+ channel KcsA structure provided a wealth of data that could be applied to
structure & mechanism of action of the much more complex eukaryotic K+ channels
D. One of the first membrane proteins whose entire 3D structure was determined by X-ray crystallography: bacterial
photosynthetic reaction center; it has 3 subunits with 11-membrane-spanning -helices
E. Some technical difficulties in preparing membrane protein crystals have been overcome with new methodologies &
laborious efforts
1. Researchers were able to get high-quality crystals of a bacterial transporter after testing & refining >95,000
different conditions for crystallization
2. Despite such success, researchers still rely largely on indirect approaches for determining 3D organization of most
membrane proteins
F. Many integral membrane proteins have substantial portion present in cytoplasm or extracellular space – sometimes
this soluble portion has been cleaved from the transmembrane domain
1. It is then crystallized & its tertiary structure determined
2. Provides valuable data about protein, but fails to provide information about the protein's orientation within the
membrane
IV. Identifying transmembrane domains – which segments are embedded in membrane?
A. A great deal can be learned about the structure of a membrane protein & its orientation within the lipid bilayer
from a computer-based (computational) analysis of its amino acid sequence
1. This is readily deduced from the nucleotide sequence of an isolated gene
2. Segments embedded within membrane (called transmembrane domains) have a simple structure & consist of
string of 20–30 predominantly nonpolar amino acids that span bilayer as an -helix
a. Ex.: glycophorin A, major erythrocyte cell membrane integral protein – of the 20 amino acids of its lone -helix
(amino acids 73 – 92)
b. All but 3 have hydrophobic R groups (or an H atom in the case of glycine residues); the exceptions are serine &
threonine, which are non-charged, polar residues
c. Hydroxyl groups of threonine residue side chains can form H bond with one of the oxygen atoms of the peptide
backbone
d. Fully charged residues may also appear in transmembrane helices, but they tend to be accommodated in ways
that allow them to fit into their hydrophobic environment
e. As example, if helix contains a pair of charged residues, the side chains can reach out & interact with the
innermost polar regions of membrane, even if it requires distorting the helix to do so
f. Aromatic ring on tyrosine can be oriented parallel with hydrocarbon chains with which it has become integrated
3. The maximum number of H bonds between neighboring amino acids allowed by -helix creates a highly stable
(low-energy) configuration
4. This is important for a membrane-spanning polypeptide that is surrounded by fatty acyl chains and is thus unable
to form H bonds with an aqueous solvent anyhow
5. Since each amino acid occupies 1.5 Å of polypeptide length & the hydrophobic core of bilayer is 30 Å wide, it
takes at least 20 amino acids to span the hydrophobic part of membrane
6. A few integral membrane proteins have been found to contain loops or helices that penetrate but do not span the
bilayer
B. Transmembrane segments usually identified using hydropathy plot; each site along polypeptide is assigned value
giving a measure of the hydrophobicity of amino acid at that site & its neighbors
1. Gives a running average of hydrophobicity of short sections of polypeptide & guarantees that one or a few polar
amino acids in a sequence do not alter the profile of the whole stretch
2. Hydrophobicity is determined by various criteria: lipid solubility or energy required to transfer them from an
aqueous into a lipid medium
3. Transmembrane segments usually identified as a jagged peak extending well into hydrophobic side of spectrum
4. Reliable predictions concerning transmembrane segment orientation within bilayer can usually be made by
examining flanking amino acid residues
5. Those parts of the polypeptide at the cytoplasmic flank of a transmembrane segment tend to be more positively
charged than those at the extracellular flank
C. Not all integral membrane proteins have hydrophobic transmembrane -helices
1. A number of membrane proteins contain a relatively large channel positioned within circle of membrane-spanning
-strands organized into a barrel
2. To date, aqueous channels constructed of -barrels have only been found in the outer membranes of bacteria,
mitochondria & chloroplasts
V. Determining spatial relationships between amino acids within integral membrane proteins
A. Use of site-directed mutagenesis - you have isolated a gene for an integral membrane protein, which based on its
sequence, predicts protein has 4 apparent membrane-spanning -helices
1. How are they oriented & which amino acids face lipids? – start by introducing specific changes into gene that
codes for protein
2. Replace aminos in neighboring helices with cysteine residues that may then form disulfide bond
3. If disulfide bond forms, then the helices must reside in very close proximity
4. Helix VII of bacterial lactose permease, a sugar-transporting protein in bacterial cell membranes, was found to be
close to both helices I & II using this method
B. Can also clarify dynamic events occurring as protein functions – introduce chemical groups whose properties are
sensitive to distance between them; shows distance between selected protein residues
1. Nitroxides are chemical groups that contain unpaired electron (produces characteristic spectrum when monitored
by technique called electron paramagnetic resonance [EPR] spectroscopy)
2. Can introduce nitroxide at any site in protein by first mutating that site to cysteine & then attaching nitroxide to —
SH group of cysteine
C. Example: used to detect conformational changes in protein as its channel is activated in response to changes in
medium pH; the bacterial K+ channel (tetramer made of 4 identical subunits)
1. Cytoplasmic opening to channel is bounded by 4 transmembrane helices, one from each subunit
2. Introduce nitroxide near cytoplasmic end of each transmembrane helix —> EPR spectra change when pH is 6.5
(channel closed) & pH is 3.5 (channel open)
3. Shape of each line depends on proximity of nitroxides to one another – spectrum broader at pH 6.5 since nitroxide
groups on 4 subunits closer together at this pH (lowers EPR signal intensity)
4. Suggests that channel activation is accompanied by increased separation between the labeled residues of the 4
subunits
5. Increase in channel opening diameter allows cytoplasmic ions to reach actual permeation pathway in channel
allowing only the passage of K+ ions
Structure and Function of Membrane Proteins: Peripheral Membrane Proteins
I. Peripheral membrane proteins - attach by noncovalent (weak electrostatic) bonds to hydrophilic head groups of lipids or
to hydrophilic portions of integral proteins protruding from bilayer
A. Can usually be solubilized by extraction with aqueous salt solutions
B. In multisubunit proteins, some subunits may be peripheral & others integral (blurs distinction between integral &
peripheral proteins)
II. Best-studied peripheral proteins are located on cytosolic membrane surface where they form fibrillar network that acts
as membrane skeleton
A. These proteins give mechanical support to membrane & function as an anchor for integral proteins
B. Other peripheral proteins on internal membrane surface function as enzymes, specialized coats or factors that
transmit transmembrane signals
III. Typically have dynamic relationship with membrane, being recruited to or released from membrane depending on
prevailing conditions
Structure and Function of Membrane Proteins: Lipid-Anchored Membrane Proteins
I. Lipid-anchored proteins - 2 kinds marked by lipid anchor types & surface on which they are exposed
A. GPI-anchored proteins - on external face of plasma membrane; bound to membrane by short oligosaccharide linked
to molecule of glycophosphatidylinositol (GPI) in membrane outer leaflet;
1. Discovered when certain membrane proteins were found that are released by a phospholipase that specifically
recognized & cleaved inositol-containing phospholipids
2. Include various receptors, enzymes, cell-adhesion proteins, PrPC (normal cellular scrapie protein)
3. Rare anemia (paroxysmal nocturnal hemoglobinuria) – results from GPI synthesis deficiency that makes RBCs
susceptible to lysis
B. Another group on cytoplasmic side of membrane is anchored to membrane by long hydrocarbon chains embedded in
bilayer inner leaflet
1. At least two, Src & Ras, are implicated in transformation of a normal cell to a malignant state
Membrane Lipids and Membrane Fluidity
I. Physical state of membrane lipids described by fluidity (or viscosity) – fluidity (measure of ease of flow) & viscosity
(measure of resistance to flow) are inversely related
II. Lipids exist in 2 states (solid & liquid phase of varying viscosity depending on temperature); ex. – artificial bilayer with
phosphatidylcholine & phosphatidylethanolamine (largely unsaturated fatty acids)
A. At warm temperatures (37°C), lipid in relatively fluid, liquidlike state; a 2 dimensional liquid crystal
1. Molecules retain a specified orientation as in crystal; molecule long axes stay essentially parallel
2. Yet individual molecules can rotate around axis or move laterally within bilayer plane
B. At colder temperatures, forms frozen crystalline gel in which phospholipid fatty acid chain movement is greatly
restricted
1. If temperature is lowered slowly, a point is reached where bilayer distinctly changes
2. Temperature at which this change occurs is called transition temperature
III. Transition temperature - temperature at which membrane goes from fluid state to crystalline gel
A. Transition temperature depends on ability of lipid molecules to be packed together which depends in turn on the
particular lipids of which the membrane is constructed
1. Saturated chains pack more closely & are less fluid since they have shape of straight, flexible rod
2. Cis-unsaturated fatty acids have crooks in chain since carbons sharing double bond cannot rotate; crooks in chains
cause unsaturated lipids to pack together less tightly
3. Thus, phospholipids with saturated chains pack together more tightly than those with unsaturated chains
4. Higher degree of bilayer fatty acid unsaturation —> lower temperature before bilayer gels
5. Introduce one double bond into stearic acid —> lowers melting temperature almost 60°C
6. Plant oils highly unsaturated (polyunsaturated) & liquid; animal fats highly saturated & solid
B. Fatty acid chain length can affect membrane fluidity & transition temperature: the shorter the fatty acyl chains the
lower its melting temperature
C. Different lipids undergo their phase change over a very wide temperature range
1. Various phosphatidylcholines can be made & used to build bilayers whose transition temperatures range from
below 0°C to >60°C
D. Cholesterol – also affects membrane physical state; interacts with membrane phospholipid fatty acid chains &
alters the way the fatty acids pack together
1. Because of their orientation within the bilayer, cholesterol disrupts the close packing of fatty acyl chains &
interferes with their mobility
2. It tends to abolish sharp transition temperatures & creates a condition of intermediate fluidity
3. In physiological terms, it tends to raise membrane durability & lower membrane permeability
V. Importance of membrane fluidity
A. Membrane fluidity provides a perfect compromise between rigid, ordered structure & a totally fluid, nonviscous liquid
1. In the rigid structure - mobility would be absent
2. In the totally fluid structure - components could not be oriented; structural organization & mechanical support
would be lacking
B. Moderate fluidity also allows interactions to take place within membrane; clusters of membrane proteins can assemble
at particular sites within membrane & form specialized structures
1. Among the specialized structures: intercellular junctions, light-capturing photosynthetic complexes, synapses
2. Molecules that interact can come together, carry out necessary reaction & move apart
C. Membranes arise only from preexisting membranes – their growth is accomplished by the insertion of lipid & protein
components into the fluid matrix of the membranous sheet
D. Many of the most basic cell processes (cell movement, growth & division; intercellular junction formation, secretion,
endocytosis/exocytosis) depend on membrane component movement
1. These processes would probably not be possible if membranes were rigid, nonfluid structures
VI. Maintaining membrane fluidity - cells respond to environmental changes in temperature by regulating membrane
fluidity, except for birds & mammals, which are warm-blooded; homeostasis at cellular level
A. Since correct degree of fluidity is essential for many activities, cells alter membrane phospholipids to maintain
fluidity when temperature changes
1. Cell membrane physical properties are matched to prevailing environment
B. Lower cell culture temperature —> cells respond metabolically with initial response handled by enzymes that
remodel membranes to make them more cold resistant
1. Fatty acyl chain single bonds are desaturated forming double bonds; catalyzed by desaturases
2. Chains are reshuffled between different phospholipids to produce those with 2 unsaturated chains; this greatly
lowers the bilayer's melting temperature
a. Reshuffling done by phospholipases (split fatty acids from glycerol backbone)
b. Acyltransferases transfer the fatty acid chains to a different phospholipid
3. Cell also changes the types of phospholipids synthesized to those containing more unsaturated fatty acids
C. The above strategies are seen in hibernating mammals, pond-dwelling fish (temperature changes markedly from
day to night), cold-resistant plants, bacteria living in hot water springs
VII. Lipid rafts – community of cell biologists is split into believers & nonbelievers
A. When membrane lipids are extracted from cells & used to prepare artificial lipid bilayers, cholesterol &
sphingolipids tend to self-assemble into microdomains
1. These microdomains are more gelated & highly ordered than surrounding regions consisting primarily of
phosphoglycerides
2. Due to distinctive physical properties, microdomains tend to float within more fluid, disordered artificial
membrane environment; thus, these cholesterol/sphingolipid patches are called lipid rafts
3. When added to artificial bilayers, certain proteins tend to become concentrated in lipid rafts, whereas others tend
to remain outside their boundaries
4. GPI-anchored proteins show a particular fondness for the ordered regions of the bilayer
B. Controversy arises over whether similar types of cholesterol-rich lipid rafts exist within living cells
1. Most evidence in favor of lipid rafts is derived from studies that employ detergent extraction or cholesterol
depletion, which makes the results difficult to interpret
2. Attempts to demonstrate presence of lipid rafts in living cells have generally been unsuccessful,
3. This either means that lipid rafts do not exist or that they are so small (5 – 25 nm in dia) & short-lived as to be
difficult to detect with current techniques
C. The concept of lipid rafts is very appealing because it provides a means to introduce order into a seemingly random
sea of lipid molecules
D. Lipid rafts are postulated to serve as floating platforms that concentrate particular proteins, thereby organizing the
membrane into functional compartments
1. They may provide a favorable local environment for cell-surface receptors to interact with other membrane
proteins that transmit signals from the extracellular space to the cell interior
The Dynamic Nature of the Plasma Membrane: Lipid and Protein Mobility
I. Lipid bilayer is relatively fluid - polar lipid heads linked to gold particles or fluorescent compounds are seen to move
in the microscope
A. Phospholipids move laterally within the same leaflet with considerable ease - it has been estimated that a
phospholipid can diffuse from one end of bacterium to the other end in 1 – 2 sec
B. Lipids do not flip-flop to other leaflet very often (takes a matter of hours to days), since polar heads moving through
hydrophobic membrane is thermodynamically unfavorable; most restricted movement
1. However, cells contain enzymes (flippases) that move certain phospholipids between leaflets
2. Flippases may play role in establishing lipid asymmetry or reverse the slow rate of passive transmembrane
movement
C. Lipids provide the matrix in which integral proteins of membrane are embedded; thus the physical state of lipids is an
important determinant of integral protein mobility
1. Demonstrated movement of integral membrane proteins was a cornerstone in the formulation of the fluid-mosaic
model
II. Diffusion of membrane proteins after cell fusion – Larry Frye & Michael Edidin, Johns Hopkins (1970)
A. Cell fusion is a technique in which 2 different cell types or cells from 2 different species can be fused to produce
one cell with a common cytoplasm & a single continuous membrane
1. Make cell membranes sticky to ease fusion by adding some inactivated viruses that attach to the membrane
surface, by a mild electric shock or by adding polyethylene glycol
2. These treatments make the plasma membranes adhere to one another
3. Cell fusion has played important role in cell biology & is now used as part of a technique to prepare specific
antibodies
B. Fused mouse & human cells —> follow lateral movement of proteins with specific antibodies covalently linked to
fluorescent dyes (mouse proteins – green dye; human proteins – red dye)
1. Mouse & human protein location seen by viewing cells under a fluorescence light microscope
2. Right after fusion, cell is half green & half red; later, proteins move laterally into opposite halves
3. By 40 minutes, all proteins were uniformly distributed around the entire hybrid cell membrane
4. At lower temperatures, the mobility of the membrane proteins decreased because of the increased viscosity
(decreased fluidity) of the lipid bilayer
5. These early experiments suggested that integral membrane protein movements were virtually unrestricted; later,
membrane dynamics were found to be much more complex than first thought
III. Protein mobility patterns in living cell membranes are shown by 2 light microscopy methods; they are excellent for
measuring protein movement extent & rate
A. FRAP (fluorescence recovery after photobleaching) - fluorescently label integral membrane components in cultured
cells in a general or specific manner
1. Treat with fluorescein isothiocyanate, a nonspecific dye, [reacts with all exposed proteins] or label with
fluorescent antibodies or other specific probes
2. Place under microscope & individually irradiate cells by sharply focused laser beam —> irreversibly bleaches
small circular area (~1 µm dia) on cell —> follow fluorescence recovery rate
3. If labeled proteins are mobile, their random movements cause fluorescence to reappear in circle
4. Rate of fluorescence recovery is measure of diffusion rate (D - diffusion coefficient); recovery extent
(percentage of original intensity) reflects percentage of labeled molecules free to diffuse
5. Early studies – proteins move much more slowly in cell membranes than in artificial bilayers & a significant
fraction of membrane proteins (30-70%) was not free to diffuse back into circle
6. Drawbacks to technique - only follows large population of labeled molecules diffusing over a relatively large
distance (1µm); cannot see individual protein paths
7. Thus, it is hard to tell immobile proteins from those that diffuse only a limited distance in the time allowed, so
other techniques have been developed to compensate for these deficiencies
B. Single-particle tracking (SPT) - label individual membrane proteins with antibody-coated gold particles (~40 nm in
diameter) & track them with computer-enhanced video microscopy
1. Solves problems of FRAP with individual protein tracking & results often depend on the particular protein
being studied
2. Some proteins move randomly through membrane at rates lower than in artificial bilayers
a. If mobility is based strictly on physical parameters (lipid viscosity, protein size), one would expect proteins
to migrate with diffusion coefficients of ~10-8 – 10-9 cm2/sec
b. The rates actually observed for these molecules are 10-10 – 10-12cm2/sec
c. The reasons for the reduced diffusion coefficient have been debated
3. Some proteins fail to move & are considered to be immobilized
4. Some proteins move in highly directed (nonrandom) manner toward one part of cell or other, e.g., one might
move toward leading or trailing edge of a moving cell
5. Most proteins exhibit random (Brownian) movement in membrane at rates like free diffusion (diffusion
coefficients ~5 x 10-9 cm2/sec
a. But these protein molecules are unable to move more than a few tenths of a micron
b. The membrane appears to contain barriers that prevent extended movement
IV. Restraints on protein mobility (factors affecting membrane protein diffusion) – in summary, lipid matrix viscosity &
protein mass are partially responsible, but other forces also restrain protein mobility
The Dynamic Nature of the Plasma Membrane: Control of Membrane Motility
I. Interactions occurring within membrane itself & materials on outer surface control membrane motility
A. Some membranes are crowded with proteins; thus, a protein's movement is impeded by its neighbors
B. The strongest influences on an integral membrane protein are thought to be exerted from just beneath the membrane
on its cytoplasmic face
1. Membranes of many cells possess a fibrillar network, a membrane skeleton, consisting of peripheral proteins
situated on the cytoplasmic surface
2. A certain proportion of integral membrane proteins are tethered to membrane skeleton or otherwise restricted by it;
if not firmly anchored, skeleton may limit distance they can freely migrate
C. Optical tweezers have been used to trap integral proteins & drag them through the membrane with a known force;
yields information about the presence of membrane barriers
1. Takes advantage of the tiny optical forces generated by a focused laser beam
2. Tag subject integral proteins with antibody-coated beads (serve as handles gripped by laser field)
3. Usually, optical tweezers drag integral proteins a limited distance before they encounter a barrier that causes their
release; upon release, they typically spring backward suggesting elastic barriers
D. Genetic modification of cells so that they produce altered membrane proteins can be used to study factors that
affect membrane protein mobility
1. Genetic deletion of the cytoplasmic portions of these proteins allows them to move much greater distances than
their intact counterparts
2. This indicates that barriers reside on the cytoplasmic side of the membrane, suggesting that the membrane's
underlying skeleton forms a network of "fences" around portions of the membrane
3. This creates compartments that restrict the distance an integral protein can travel
E. At times, proteins move across boundaries to different microdomain through breaks in fences
1. Breaks may appear & disappear along with dynamic disassembly/reassembly of meshwork portions
2. Membrane compartments may function primarily to keep specific combinations of proteins in close enough
proximity to facilitate their interaction
F. External materials restrict movement - proteins engineered to lack the portion that normally projects into the
extracellular space move at much faster rate than the wild type version of protein
1. Suggests that extracellular materials entangle external parts of transmembrane proteins, slowing them
II. Membrane lipid mobility – phospholipids are small molecules that make up the very fabric of the lipid bilayer & one
would expect their movement to be unfettered, but they also seem to be restricted
A. Tag individual phospholipids, follow them by under microscope using ultra-high-speed cameras —> they are
confined for very brief periods & then hop from one confined area to another
1. Individual phospholipid followed over period of 56 µsec – it diffuses freely within one compartment before it
jumps "fence" into neighboring compartment
2. It then jumps over another fence into an adjacent compartment, etc.
B. Treatment of membrane with agents that disrupt the underlying membrane skeleton also removes the fences that
restrict phospholipid diffusion
C. How does membrane skeleton interfere with phospholipid movement if it lies beneath bilayer?
1. Some conclude that the fences are made of rows of integral membrane proteins whose cytoplasmic domains are
attached to membrane skeleton (like cows confined by picket fence)
III. Membrane domains & cell polarity – most studies of membrane dynamics were initially of the relatively homogeneous
upper or lower surface of cell on culture dish
A. Most membranes are not like this; they exhibit distinct variations in protein composition & mobility, especially in
cells whose various surfaces display distinct functions & must maintain order
B. Example: epithelial cells lining intestinal wall & microscopic kidney tubules (both highly polarized); their different
surfaces carry out different functions
1. Apical plasma membrane of epithelial cells (intestinal, kidney tubules) selectively absorbs lumenal materials &
has enzymes different from lateral surface
2. Lateral surfaces of epithelial cells interact with neighboring cells
3. Basal membrane sticks to underlying extracellular substrate (basement membrane)
4. Neurotransmitter receptors are concentrated in regions of membrane within synapses
5. Low density lipoprotein receptors are concentrated in patches of membrane specialized to facilitate their
internalization
The Plasma Membrane of the Red Blood Cell (RBC)
I. Why is RBC membrane so well studied & the best-understood membrane?
A. The cells are inexpensive to obtain and readily available in huge numbers from whole blood
B. The cells are already present as single cells and need not be dissociated from a complex tissue
C. RBCs are simple in comparison to other cells; they have no contaminating internal cell membranes
D. One can obtain pure, intact RBC membranes (ghosts) by hypotonic lysis of cell (hemolysis)
1. One can do this by placing the cells in a dilute (hypotonic) salt solution
2. In response to this osmotic shock, cells expand, their cell membranes become leaky & contents (mostly
hemoglobin) flow out
II. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) used to separate (fractionate) membrane proteins (see Ch. 18)
after they have been solubilized; gives one an idea of membrane protein diversity
A. Extract proteins & denature with ionic detergent SDS (sodium dodecyl sulfate) that solubilizes &
coats
integral proteins with negative charges allowing their migration in an electric field
B. The charge distribution of the proteins is equal despite protein size, since number of SDS molecules per unit weight
of protein is constant; thus, proteins separate on basis of molecular weight
C. The largest proteins move slowest & the smallest move fastest through molecular sieve of gel
D. The major erythrocyte membrane proteins are separated into ~12 bands by SDS-PAGE; they include:
1. A variety of enzymes: glyceraldehyde 3-phosphate (a glycolytic enzyme), transport proteins(for ions & sugars), &
skeletal proteins (like spectrin)
2. Various proteins can be correlated with the transport of oxygen & carbon dioxide & with the physical stresses that
the cells encounter as they circulate through the body
E. At the same time, the erythrocyte membrane is presumed to be simpler than that of most other cells because it lacks
proteins involved in cell signaling & cell-cell or cell-matrix interactions
III. Erythrocyte membrane integral proteins – its most abundant integral proteins are 2 CHO-containing, membranespanning proteins (band 3 & glycophorin A); freeze-fracture shows them at high density
A. Band 3 (third band on electrophoresis gel) - has a relatively small amount of carbohydrate (6-8% of molecule's
weight); each subunit spans the membrane at least 12 times
1. It is a homodimer meaning that it is a dimer composed of 2 identical subunits; each band 3 dimer has a channel in
the center that serves as a channel for passive anion exchange across membrane
2. As blood circulates through tissues, CO2 dissolves in blood plasma (fluid of bloodstream) & undergoes the
following reaction: H2O + CO2 —> H2CO3 —> HCO3- + H+
3. Exchange HCO3- (bicarbonate) & Cl- ions; HCO3- ions move in & Cl- ions move out of RBCs
4. In lungs where CO2 is released, HCO3- ions leave RBCs & Cl- ions go into cells
B. Glycophorin A - its amino acid sequence was the first of a membrane protein to be determined; other related
glycophorins (B, C, D, & E) are also present at much lower concentrations
1. Like band 3, glycophorin A is also present in the membrane as a dimer
2. Unlike band 3, each glycophorin A subunit spans membrane only once; it contains a bushy carbohydrate cover (16
oligosaccharide chains; together they make up ~60% of molecule's MW)
3. Primary function of the glycophorins may derive from the large number of "-" charges on the sugar residue (sialic
acid) on end of each carbohydrate chain
4. The "-" charges may allow RBCs to repel each other & prevent clumping as they circulate through the body's tiny
blood vessels
5. If people lack glycophorins A & B —> no ill effects; band 3 proteins are more heavily glycosylated to
compensate for missing negative charges needed to prevent cell-cell interactions
6. Glycophorin also is the receptor used by the protozoan that causes malaria; provides a path for entry into RBC;
without glycophorins A & B, a person may be protected against malaria
7. Differences in glycophorin amino acid sequence determines MM, MN or NN blood type
IV. Erythrocyte fibrillar membrane skeleton – made of peripheral membrane proteins on inner surface; maintains biconcave
RBC shape under punishment of circulation
A. The membrane skeleton can also establish domains within the membrane that enclose particular groups of membrane
proteins & may greatly restrict the movement of these proteins
B. Spectrin – major component of the skeleton; long, flexible fibrous protein, 2 subunits ( & ) that curl around one
another; a heterodimer (~100 Å long)
1. Two such dimeric molecules are linked at head ends to form 200 Å-long filament; it is both elastic & flexible
2. It is attached to the internal membrane surface by noncovalent bonds to ankyrin (also a peripheral protein), which
is linked noncovalently to the cytoplasmic domain of band 3 molecule
3. Spectrin filaments are organized into hexagonal or pentagonal arrays; this 2-dimensional network is constructed
by linking both ends of each spectrin filament to a cluster of proteins
4. The cluster of proteins includes a short filament of actin & tropomyosin, proteins typically involved in contractile
activities
C. Gene mutations that alter the structure & function of ankyrin & spectrin yield fragile, abnormally shaped RBCs
that lead to genetic diseases, specifically the hemolytic anemias
D. Remove peripheral proteins from RBC ghosts —> the membrane fragments into small vesicles, indicating that the
inner protein network is required to maintain the membrane's integrity
V. Erythrocytes are circulating cells that are squeezed under pressure through microscopic capillaries whose diameter is
considerably less than that of the erythrocytes themselves
A. To traverse these narrow passageways day after day, RBCs must be highly deformable, durable & capable of
withstanding shearing forces that tend to pull them apart
B. The spectrin-actin network gives RBCs the strength, elasticity & pliability needed to carry out its function
C. When first discovered, the erythrocyte membrane skeleton was thought to be a unique structure suited to the unique &
mechanical needs of this cell type
1. However, as other cell types were examined, similar types of membrane skeletons containing members of the
spectrin & ankyrin families have been revealed
2. This indicates that inner membrane skeletons are widespread
D. Dystrophin, for example, is a member of the spectrin family that is found in the membrane skeleton of muscle cells
1. Mutations in dystrophin are responsible for causing muscular dystrophy, a devastating disease that cripples & kills
children
2. As in the case of cystic fibrosis, the most debilitating mutations are ones that lead to a complete absence of the
protein in the cell
3. The plasma membranes of muscle cells lacking dystrophin are apparently destroyed as a consequence of the
mechanical stress exerted upon them as the muscle contracts
4. As a result, the muscle cells die & eventually are no longer replaced
QUESTÕES
1. Imaginem que são cientistas a bordo de uma nave espacial e que descobrem uma nova forma de vida,
aparentemente semelhante a outras existentes na Terra. Extraem uma série de moléculas dos organismos e
caracterizam-nas. De acordo com as características particulares de cada molécula, identifique-as e diga qual a sua
provável localização celular!
a) Molécula anfipática, com duas cadeias longas de hidrocarbonetos e um grupo polar de oligossacarídeo;
b) Molécula anfipática com duas cadeias de hidrocarbonetos e um grupo contendo fosfato;
c) Polímero constituído por aminoácidos com os resíduos hidrofóbicos expostos à superfície da molécula, na sua
parte média, e duas extremidades com resíduos hidrofílicos à superfície.
2. De que modo podemos preparar um lipossoma para que possa ser utilizado no tratamento de um tumor cujas
células possuam nas suas membranas uma proteína chamada “tumor cell antigen” (TAG)? Tenha em conta que
esta proteína não está presente nas células “normais”.
3. Imagine que determinou experimentalmente a temperatura de transição de duas membranas. A primeira tem uma
temperatura de transição de 28ºC e a segunda de 15ºC. O que pode concluir acerca da composição das duas
membranas?
4. Determinou-se a temperatura de transição de uma membrana e verificou-se que variava numa vasta gama de
temperaturas. Além disso, esta membrana apresentava elevada estabilidade e baixa permeabilidade, à semelhança
do que acontece na maioria das membranas de mamíferos. Qual o componente membranar responsável por estas
características e de que maneira ele as influencia?
5. A meio da noite analisou-se a composição em fosfolípidos de uma amostra de membranas. A meio do dia
recolheu-se nova amostra e procedeu-se da mesma maneira. Verificou-se que a composição lipídica sofreu
alterações da noite para o dia. De que modo ocorreram essas alterações e qual o seu propósito?
6. Por que razão a parte transmembranar da proteína membranar mostrada na figura 4.4c tem poucas probabilidades
de ser constituída por uma hélice-com aminoácidos hidrofílicos?
7. Qual é a doença causada pela destruição da estrutura descrita na figura 4.5?
8. De que modo as moléculas de colesterol apresentadas na figura 4.7 realçam a fluidez das membranas?
9. Relativamente à figura 4.9:
a) Por que razão o lipossoma está coberto com uma camada de “polyethylene glycol”?
b) O que direcciona o lipossoma para o seu tecido alvo?
c) Nestes lipossomas, onde estão localizadas as drogas solúveis em lípidos e as drogas solúveis em água?
10.
A figura 4.12 mostra oligossacarídeos ligados a lípidos membranares, formando um gangliósido que determina
os grupos sanguíneos A, B, O.
a) Que grupo sanguíneo teria uma pessoa cujos glicolípidos apresentariam gangliósidos com resíduos terminais
de fucose?
b) Que grupo sanguíneo teria uma pessoa cujos glicolípidos apresentariam gangliósidos com resíduos terminais
de fucose e de N-acetilgalactosamina?
c) Que grupo sanguíneo teria uma pessoa cujos glicolípidos apresentariam gangliósidos com resíduos terminais
de fucose e de galactose?
d) Que grupo sanguíneo teria uma pessoa cujos glicolípidos apresentariam gangliósidos com resíduos terminais
de fucose, de N-acetilgalactosamina e de galactose?
11.
De acordo com a figura 4.13, quais os tipos de proteínas que podemos encontrar numa membrana?
12.
Relativamente à figura 4.20:
a) Quantos domínios transmembranares são indicados no gráfico para a glicoforina A?
b) Qual é a posição aproximada da porção de aminoácidos que atravessa a membrana?
13.
14.
15.
16.
Na figura 4.23b a bicamada lipídica está abaixo da temperatura de transição e o movimento das moléculas
encontra-se altamente restringido. Como podemos definir o estado desta membrana?
Por que razão são tão raros os movimentos de flip-flop, observados na figura 4.25?
Na figura 4.26a, que cor deverá apresentar a célula resultante da fusão das células de rato e humana, após 40
minutos da fusão?
Relativamente a uma célula epitelial como a apresentada na figura 4.30:
a) Qual a superfície que mais provavelmente contacta com uma célula adjacente?
b) Qual a superfície que mais provavelmente interage com colagénio?
17.
Como provaria a existência de uma bicamada lipídica a revestir uma célula?