I. Functions of biological membranes

a. Selective permeability barrier with channels and carriers

b. Demarcate cell boundaries and compartments

c. Contain specific receptors for external stimuli

d. Generate signals--electrical and chemical

e. Act as 2D platform for enzymatic reactions

II. Common features despite diversity of biological membranes

a. Membranes are sheet-like structures 6-10 nm (60-100 A) thick

b. Membrane lipids are amphipathic, i.e., they posses both hydrophobic and hydrophilic components, arranged as lipid bilayers

c. Membranes are not covalently assembled but are linked by many noncovalent interactions

d. Membranes are fluid structures

e. Membranes can fuse and self seal

f. Membranes consist of polar lipids and proteins to which carbohydrates are often attached

g. Proteins serve as pumps, channels, receptors, energy transducers and enzymes

h. Membranes are selectively permeable

i. Membranes are asymmetric

j. Membranes are usually polarized electrically

k. They are in constant motion, very fluid, held together by weak forces

l. Membranes are asymmetric; essential for their function 

I.                   The Composition and Architecture of Membranes

a.       Each type of membrane has characteristic lipids and proteins.  The relative proportions of protein and lipid vary with the type of membrane, reflecting the diversity of biological roles

b.      Plasma membranes are enriched in cholesterol and contain no detectable cardiolipin; in the inner mitochondrial membrane of the hepatocyte, this distribution is reversed; very low cholesterol and high cardiolipin

c.       Cardiolipin is essential to the function of certain proteins of the inner mitochondrial membrane.  Cells clearly have mechanisms to control the kinds and amounts of membrane lipids they synthesize and to target specific lipids to particular organelles

Book Notes

(Function, structure and dynamics of membranes) 

I.                   Properties of Membranes-

a.       Membranes are flexible, self-sealing and selectively permeable to polar solutes.  Their flexibility permits the shape changes that accompany cell growth and movement

b.      Membranes are selectively permeable and retain certain compounds and ions within cell sand within specific cellular compartments, while excluding others

c.       Membranes include an array of proteins specialized for promoting or catalyzing various cellular processes: at the cell surface, transporters move specific organic solutes and inorganic ions across the membrane; receptors sense extracellular signals and trigger molecular changes in the cell; adhesion molecules hold neighboring cells together.  Within the cell membranes organize cellular processes such as the synthesis of lipids and certain proteins and the energy transduction in mitochondria and chloroplasts

I.                   All Biological Membranes Share Some Fundamental Properties

a.       Membranes are impermeable to most polar or charged solutes, but permeable to nonpolar compounds; they are 5-8 nm (50-80 A) thick and appear trilaminar when viewed in cross section with the electron microscope

b.      Phospholipids form a bilayer in which the nonpolar regions of the lipid molecules in each layer face the core of the bilayer and their polar head groups face outward, interacting with the aqueous phase on either side.  Proteins are embedded in this bilayer sheet, held by hydrophobic interactions between the membrane lipids and hydrophobic domains in the proteins

c.       Some proteins protrude from only one side of the membrane; others have domains exposed on both sides.  The orientation of proteins in the bilayer is asymmetric, giving the membrane “sidedness”: the protein domains exposed on one side of the bilayer are different from those exposed on the other side, reflecting functional asymmetry.  The individual lipid and protein units in a membrane form a fluid mosaic with a pattern that, unlike a mosaic of ceramic tile and mortar, is free to change constantly.

d.      The membrane mosaic is fluid because most of the interactions among its components are noncovalent, leaving individual lipid and protein molecules free to move laterally in the plane of the membrane

I.                   A Lipid Bilayer Is the Basic Structural Element of Membranes

a.       Glycerophospholipids, sphingolipids and sterols are virtually insoluble in water.  When mixed with water, they spontaneously form microscopic lipid aggregates in a phase separate from their aqueous surroundings, clustering together, with their hydrophobic moieties in contact with each other and their hydrophilic groups interacting with the surrounding water

b.      Lipid clustering reduces the hydrophobic surface exposed to water and thus minimizes the number of molecules in the shell of ordered water at the lipid-water interface, resulting in an increase in entropy

c.       Hydrophobic interactions among lipid molecules provide the thermodynamic driving force for the formation and maintenance of these clusters

d.      Depending on the precise condition and the nature or the lipids, three types of lipid aggregates can form when amphipathic lipids are mixed with water

e.       Micelles are spherical structures that contain anywhere form a few dozen to a few thousand amphipathic molecules.  These molecules are arranged with their hydrophobic regions aggregated in the interior, where water is excluded, and their hydrophilic head groups at the surface, in contact with water. 

f.       Micelle formation is favored when the cross-sectional area of the head group is greater than that of the acyl side chain(s) as in free fatty acids, lysophospholipids and detergents such as socium dodecyl sulfate

g.      A second type of lipid aggregate in water is the bilayer, in which two lipid monolayers (leaflets) form a two-dimensional sheet.  Bilayer formation occurs most readily when the cross-sectional areas of the head group and acyl side chains are similar, as in glycerophospholipids and sphingolipids…

h.      Bilayers become liposomes

III. Lipid Composition

a. Inner and outer leaflets: Two monolayers (leaflets) form a two-dimension sheet.  Occurs more readily when cross-sectional areas of teh head group and acyl side chains are similar (ex: glycerophospholipids and sphingolipids)

b. The hydrophobic portions in each monolayer, excluded from water interact with each other

c. The hydrophillic head groups interact with water at each surface of the bilayer

d. The bilayer sheet folds back on itself to form a hollow sphere, a vesicle or liposome.  By forming  vesicles, bilayers lose their hydrophobic edge regions, achieving maximal stability in their aqueous environment 

Fluid Mosaic Model

(Question 5) 

I. The fluid mosaic model

a. Phospholipids form a bilayer in which the nonpolar regions of the lipid molecules in each layer face the core of the bilayer and their polar head groups face outward, interacting with the aqueous phase on either side

b. Proteins are embedded in this bilyer sheet, held by hydrophobic interactions between the membrane lipids and hydrophobic domains in the proteins

c. The orientation of proteins in the bilayer is asemmetric, giving the membrane "sidedness": the protein domains exposed on one side of the bilayer are different from those exposed on the other side, reflecting fucntional asymmetry 

d. The individual lipids and proteins units in a membrane form a fluid mosaic with a pattern that is free to change constantly.  Lipid and protein molecules are free to move laterally in the plane of the membrane 

Fluid Mosaic Model 

(Question 7, Part I)

I. Three types of membrane proteins

a. Peripheral-

i. Held in place by hydrogen bonds and electrostatic interactions

ii. Removed by disrupting the hydrogen bonds and electrostatic interactions 

iii. Still active 

b. Integral-

i. Defined: interact with the hydrophobic core of the membrane

1) removeable only by treatments that disrupt hydrophobic interations (urea, detergents)

ii.Transmembrane alpha-helices 

 1) Membrane spanning alpha-helix

c. Hydropathy Plots-

i. 7-20 amino acids

ii. Measure hydrophobicity across the protein with 7-20 amino acid window 

Peripheral Proteins

(Question 7, Part II) 

I.                   Peripheral Membrane Proteins Are Easily Solubilized

a.       Membrane proteins may be divided operationally into two groups, integral proteins are very firmly associated with the membrane, removable only by agents that interfere with hydrophobic interactions, such as detergents, organic solvents, or denaturants

b.      Peripheral proteins associate with the membrane through electro static interactions and hydrogen bonding with the hydrophilic domains of integral proteins and with the polar head groups of membrane lipids.  They can be released by relatively mild treatments that interfere with electrostatic interactions or break hydrogen bonds

c.       Peripheral proteins may serve as regulators of membrane-bound enzymes or may limit the mobility of integral proteins by tethering them to intracellular structures

d.      Experiments with such topology-specific reagents show that the erythrocyte glycoprotein glycophorin spans the plasma membrane.  Its amino-terminal domain (bearing the carbohydrate chains) is on the outer surface and is cleaved by trypsin.  The carboxyl terminus protrudes on the inside of the cell, where it cannot react with impermeant reagents

e.       Both the amino-terminal and carboxyl-terminal domains contain many polar or charged amino acid residues and are therefore quite hydrophilic

f.       However, a segment in the center of the protein contains mainly hydrophobic amino acid residues.  One further fact may be decided from the results of experiments with glycophorin: its disposition in the membrane is asymmetric

g.      Similar studies of other membrane proteins show that each has specific orientation in the bilayer; one domain of a transmembrane protein always faces out, the other always faces in.  Furthermore, glycoproteins of the plasma membrane are invariably situated with their sugar residues on the outer surface of the cell

-held together by lysines and phosphates

phos...serine, phos..eth..amine=peripheral 

Integral Proteins

(Question 7, Part III) 

I.                   Integral proteins Are Held in the Membrane by Hydrophobic Interactions with Lipids

a.       The firm attachment of integral proteins to membranes is the result of hydrophobic interactions between membrane lipids and hydrophobic domains of the protein

b.      Some proteins have a single hydrophobic sequence in the middle (as in glycophorin) or at the amino or carboxyl terminus.  Others have multiple hydrophobic sequences, each of which, when in the α-helical conformation, is long enough to span the lipid bilayer

c.       One of the best-studied membrane-spanning proteins, bacteriorhodopsin, has seven very hydrophobic internal sequences and crosses the lipid bilayer seven times.  Bacteriorhodopsin is a light-driven proton pump densely packed in regular arrays in the purple membrane of the bacterium

d.      Halobacterium salinarum X-ray crystallography reveals a structure with seven α-helical segments, each traversing the lipid bilayer, connected by nonhelical loops at the inner and outer face of the membrane.  In the amino acid sequence of bacteriorhodopsin, seven segments of about 20 hydrophobic residues can be identified, each segment just long enough to form an α helix that spans the bilayer

e.       Hydrophobic interactions between the nonpolar amino acids and the fatty acyl groups of the membrane lipids firmly anchor the protein in the membrane.  The seven helices are clustered together and oriented not quite perpendicular to the bilayer plane, providing a transmembrane pathway for proton movement

f.       The reaction center of bacteriorhodopsin has four protein subunits, three of which contain alpha-helical segments that span the membrane.  These segments are rich in nonpolar amino acids, their hydrophobic side chains oriented toward the outside of the molecule where they interact with membrane lipids.  The architecture of the reaction center protein is therefore the inverse of that seen in most water-soluble proteins, in which hydrophobic residues are buried within the protein core and hydrophilic residues are on the surface

Lipids Mosaic Model

Three Types of Membrane Proteins 

I. Integral

a. Beta-barrel:

i. 20 or more transmembrane segments form beta-sheets that line a cylinder (each beta sheet 7-9 amino acids in length)

ii. Alternating amino acid are hydrophobic/hydrophillic: face bilayer/ line the cylinder

b. Asymmetry-

i. Only orientation in membranes  

ii. Cannot be predicted by hydropathy

c. Lipid anchored-

i. covalently linked to various lipids

ii. Defined: orientation specific (inner/outer membrane face)

iii. Functions: released by treatment with lipases

d. Fatty Acid Linkages- thioester linkages (on inner face of plasma membrane)

e. Isoprenoid linkages-(thioether)farnesyl and geranylgeranyl groups attached to carboxyl-terminal Cys residues are isoprenoids of 15 and 20 carbons.  (on inner face of plasma membrane)

f. GPI linked (glycosylated deriatives of phosphatidylinositol in which inositol bears a short oligosaccharide covalently joined to the carboxylterminal residue of a protein through phosphoethanolamine.  GPI linked proteins are always on the extracellular face of the plasma membrane 

Fluid Mosaic Model

Gel vs Fluid State (Dynamics)

(Question 9) 

I. Gel vs fluid state-

a. Gel state is thicker; can't rotate as much as fluid state

b. Fluid state is thinner

c. You can have microdomains of gel stage in fluid stage; not all or nothing

d. The structure and flexibility of the lipid bilayer depend on temperature and on the kinds of lipids present.

i. At relatively low temperatures, the lipids in the bilayer form a semisolid gel phase, in which all types of motion of individual lipid molecules are strongly constrained

ii. At relatively high tempeartures, individual hydrocarbon chains of fatty acids are in constant motion produced by rotation about the carbon-carbon bonds of the long axyl side chains.  iii. The lipids exist in a liquid-ordered state; there is less thermal motion in the acyl chains of the lipid bilayer, but lateral movement in the plane of the bilayer stillt akes place

II. Regulation of membrane fluidity

a. Cells regulate their lipid composition to achieve a constant membrane fluidity under various growth conditions

b. For example: bacteria synthesize more unsaturated fatty acis and fewer saturated ones when cultured at low temperature than when cultered at higher temperatures.  As a result of this adjustment in lipid composition, membranes of bacteria culture dat high or low temperatures have about the same degree of fluidity 

a.       GPI-Some membrane proteins contain one or more covalently linked lipids of several types: long-chain fatty acids, isoprenoids, sterols or glycosylated derivatives of phosphatidylinositol (GPI)

b.      The attached lipid provides a hydrophobic anchor that inserts into the lipid bilayer and holds the protein at the membrane surface. The strength of the hydrophobic interaction between a bilayer and a single hydrocarbon chain linked to a protein is barely enough to anchor the protein securely, but many proteins have more than one attached lipid moiety.

c.       Other interactions, such as ionic attractions between positively charged Lys residues in the protein and negatively charged lipid head groups, probably contribute to the stability of the attachments. 

d.      The association of these lipid-linked proteins with the membrane is certainly weaker than that for integral membrane proteins and is, in at least some cases, reversible.  Treatment with alkaline carbonate does not release GPI-linked proteins, which are therefore, by the working definition, integral proteins

e.       Beyond merely anchoring a protein to the membrane, the attached lipid may have a specific role.  Attachment of a specific lipid to a newly synthesized membrane protein therefore has a targeting function, directing the protein to its correct membrane location

Hydropathy Plots

(Question 8) 

I. Hydropathy Plots-

a. 7-20 amino acids

b. Measure hydrophobicity across the protein with 7-20 amino acid window

c. Hydropathy index is plotted against residue number for two integral membrane proteins.  The hydropathy index for each amino acid residue in a sequence of defined length (called a window) is iused to calculate the average hydropathy for the residues in that window.  

d. The relative polarity of each amino acid has been determined experiemntally by measuring the free-energy change accompanying the movement of that amino acid side chain from a hydrophobic solvent into water.  this free energy of transfer ranges from very exergonic for charged or polar residues to very endergonic for amino acids with aromatic or aliphatic hydrocarbon side chains

e. The overall hydrophobicity of a sequence of amino acids is estimated by summing the free energies of transfer for the residues in the sequence which yields the hydropathy index for that region

f. hydropathy index predicts number of hydrophobic helices for molecules 

Transverse and Lateral Diffusion

(Book notes, Q 10) 

I.                   Transbilayer Movement of Lipids Requires Catalysis

a.       At physiological temperature, transbilayer-or “flip-flop”-diffusion of a lipid molecules from one leaflet of the bilayer to the other occurs very slowly if at all in most membranes

b.      Transbilayer movement requires that a polar or charged head group leave its aqueous environment an dmove into the hydrophobic interior of the bilayer, a process with a large, free energy change.

c.       A family of proteins called flippases facilitate flip-flip diffusion, providing a transmembrane path that is energetically more favorable and much faster than the uncatalyzed movement

 d.Flippases are enzymes driven by ATP-

i. Transport specific lipids from one leaf of membrane to another

ii. Move lipids in transverse direction

II.                Lipids and Proteins Diffuse Laterally in the Bilayer

a.       Individual lipid molecules can move laterally in the plane of the membrane by changing places with neighboring lipid molecules. 

b.      A molecule in one monolayer, or leaflet, of the lipid bilayer can diffuse laterally so fast that it circumnavigates the erythrocyte in seconds.  This rapid lateral diffusion within the plane of the bilayer tends to randomize the positions of individual molecules in a few seconds

c.       Single particle tracking, allows one to follow the movement of a single lipid molecule in the plasma membrane on a much shorter time scale.  Results from these studies confirm the rapid lateral diffusion within small, discrete regions of the cell surface and show that movement from one such region to a nearby region is inhibited; lipids behave as though corralled by fences that they can occasionally jump

d. One possible explanation for the pattern of lateral diffusion of lipid molecules is that membrane proteins immobilized by their association with spectrin are the "fences" that define the regions of relatively unrestricted lipid motion 

Microdomains

(Question 11) 

I. Sphingolipid/Cholesterol Microdomains (Rafts)

a. Proteins partition into microdomains (have multiple acyl groups attached)

b. The cholesterol-sphingolipid microdomains in the outer monolayer of the plasma membrane, visible with atomic-force microscopy, are slightly thicker and more ordered (less fluid) than neighboring microdomains rich in phospholipids and are more difficult to dissolve with nonionic detergents; they behave like liquid-ordered sphingolipid rafts adrift on an ocean of liquid-disordered phospholipids

c. These lipid rafts are remarkably enriched in two classes of integral membrane proteins: those anchored to the membrane by two covalently attached long-chain saturated fatty acids and GPI-anchored proteins.

d. The "raft" and "sea" domains of the plasma membrane are not ridigly separated; membrane proteins can move into and out of lipid rafts on a time scale of seconds.  But in the shorter time scale (microseconds) more relevant to many membrane-mediated biochemical processes, many of these proteins reside primarity in a raft

e. stable associations of sphingolipids and cholesterol in the outer leaflet produce a microdomain of slightly thicker than other membrane regions, that is enriched with specific types of membrane proteins.

f. GPI-linked proteins are commonly found in the outer leaflet of such rafts and proteins with one or several covalently attached long-chain acyl groups are common in the inner leaflet 

II. Caveolin-

a. Has three acyl groups linked to it

b. Integral membrane protein

c. Structure-forms a dimer in the membrane; causes an invagination

d. Function-often have membrane receptors associated with regions

e. Caveolin is an integral membrane protein with two globular domains connected by a hairpin-shaped hydrophobic domain, which binds the protein to the cytoplasmic leaflet of the membrane

f. Caveolin binds cholesterol in teh membrane, and the presence of caveolin forces the associated lipid bilayer to curve inward, forming caveolae ("little caves") in the surface of the cell

g. Caveolae are unusual rafts: they involve both leaflets of the bilayer-the cytoplasmic leaflet, from which the caveolin glocular domains project, and the exoplasmic leaflet, a typical sphingolipid/cholesterol raft with associated GPI-anchored proteins

h. Caveolae are implicated in a variety of cellular functions, including membrane frafficking within cells and the transduction of external signals into cellular responses

i. The receptors for insulin and other grown facts, as well as certain GTP-binding proteins and protein kinases associated with transmembrane signaling, appear to be localized in rafts and perhaps in caveolae 

Membrane Fusion

(Question 12) 

I. Membrane Fusion-

a. Exocytosis, endocytosis, cell division, fusion of egg and sperm cells and entry of a membrane-enveloped virus into its host cell all invovled membrane reorganization in which teh fundamental operation is fusion of two membrane segments without loss of continuity

b. Specific fusion of two membranes requires that:

i. They recognize each other

ii. Their surfaces become closely apposed, which requires the removal of water molecules normally associated with the polar head groups of lipids

iii. Their bilayer structures become locally disrupted, resulting in fusion of the outer leaflet of each membrane

iv. Their bilayers fuse to form a single continuous bilayer.  AReceptor-mediated indocytosis, or regulated secretion, aslo requires that-

v. the fusion process is triggered at the appropriate time or in response to a specific signal

 c. Integral proteins called fusion proteins mediate these events, bringing about specific recognition and a transient local distortion of the bilayer structure that favors membrane fusion