MEMBRANE LIPIDS,PROTEINS & CARBOHYDRATES

The nature of cell membranesLipids in membranes

Proteins in membranesLipid-protein interactions

Carbohydrate presence & roles

Cell membranes:

In the early part of the 20th century very littlewas known about cell membranes either atthe cell surface (plasma membranes) or within the “organs” of cells (sub-cellularorganelles).

Individuals who pioneered early work in this area were: Overton, Langmuir, Gorter, Grendel,Danielli and Davson.

Overton found (1895) that cell membranes were lipid in nature and not easily penetrated. Langmuir developed a device (1917) to study lipid layers spread out on thin films to examine their air-water interfaces. Gorter & Grendel (1925) first proposed that membranes were made up of lipid bilayers by using Langmuir’s device.

The bilipid model is often incorrectly attributed to Danielli & Davson. The 1925 model (above) failed to account for any proteins associated with the membranes. Danielli & Davson’s contributions will be shown later.

Hydrophilic part

Hydrophobic part

It is now confirmed that the 1925 biliipid layerhypothesis was correct.The lipid portion of thestructure is composedof two lipids facing foot-to-foot in which the “feet” (red arrow) are the hydrocarbon tails of the fatty acids and the hydrophilic“heads” (polar head groups)are exposed to an aqueousenvironment (blue arrows).

BILIPID LAYERS,IN BIOLOGICAL CELLS, ARE FORMED BY THEIRORIENTATION AGAINST AN AQUEOUS ENVIRONMENT. HOWEVER,BILIPID LAYERS CAN ARRANGE THEMSELVES IN A NONPOLAR ENVIRONMENT AS WELL. BELOW IS ANOTHER ORIENTATION.

THE ARRANGEMENT OF THE BILIPID LAYER IS DOMINATED BY THEPREVAILING NEED FOR THE MOST STABLE, LOW ENERGY ASSOCIATIONOF THE LIPIDS.

WALLOFSOAPBUBBLE

AIR OUTSIDE

AIR INSIDE

AMPHIPATHIC* LIPIDS SPONTANEOUSLY FORM STRUCTURES IN WATER (BY HYDROPHOBIC BOND FORMATION). THE ARRANGEMENT IS NOT

ALWAYS A BILIPID LAYER.

*LIPIDS WITH A CHARGE AT ONE END OF THE MOLECULE SUCH AS A PHOSPHOLIPID OR A FATTY ACID.

MICELLES..ARE STRUCTURES THAT FORM FROM FATTYACIDS. THEY ARE SMALL AND HAVE ONLY NON-POLARLIPID INSIDE. DETERGENTS CAN ALSO FORM MICELLES.

BILIPID LAYERS..ARE STRUCTURES THAT FORM FROMPHOSPHOLIPIDS. THEY ARE “LINEAR” AND REMAIN STABLEEXCEPT AT THE EDGES. BECAUSE OF THE “END PROBLEM”,THEY WILL READILY WRAP BACK ON THEMSELVES TO FORMCLOSED VESICLES.

LIPOSOMES..ARE SPERICAL VESSELS OF BILIPID LAYERS.THERE MAY BE ONE OR MORE BILIPID LAYERS. CELLS HAVEESSENTIALLY LIPOSOME COVERINGS WITH A SINGLE BILIPIDLAYER. ARTIFICIAL LIPOSOMES MAY BE USED TO DELIVERDRUGS AND REAGENTS FOR DIAGNOSTIC PURPOSES. HOW?

WHAT ABOUT SPHINGOLIPIDS AND CHOLESTEROL IN MEMBRANES?

AS PART OF A BILIPID LAYER,SPHINGOLIPIDS FIT IN A MEMBRANE JUST LIKEPHOSOPHOLIPIDS, BUT THEY ALSO CONTRIBUTE DIFFERENT POLAR HEAD GROUPS – ESPECIALLY THEGANGLIOSIDES.

THE CARBOHYDRATESACT AS CELL SURFACEIMMUNOIDENTIFIERS

USUAL PART OF THE BILIPID LAYEREXAMPLE: LACTOSYLCERAMIDE

membrane (outer) surface

CHOLESTEROL IS ACTUALLYA RATHER FLAT MOLECULEAND IS EASILY WEDGED INTOTHE ADJOINING PHOSPHOLIPIDSOF A BILIPID LAYER.

½ OF BILIPID LAYER

CYTOPLASMIC SIDE

PHOSPHOLIPID

WHEN THE LIPID COMPOSITION IS MODIFIED, THE APPEARANCE & STRUCTURE OF SUCH LAYERS MAY OR MAY NOT BE CHANGED, FOR EXAMPLE, WHEN CHOLESTEROL IS INCORPORATED INTO A BILIPID LAYER NO CHANGE ISOBVIOUS:

+ CHOLESTEROL

HOWEVER:THE BILIPID LAYER BECOMESMORE RIGID WITH GREATER CHOLESTEROL INCORPORATION.

CHOLESTEROL

THE LENGTH AND UNSATURATION OFFATTY ACIDS THAT MAKE UP MEMBRANEPHOSPHOLIPIDS MAY HAVE MARKEDEFFECTS ON MEMBRANE STRUCTURE AND PROPERTIES. HEREIT IS APPARENT THAT UNSATURATION OF FATTYACIDS CAUSES TWO EFFECTS:MEMBRANE THINNING AND DISORDER (=INCREASE INMEMBRANE FLUIDITY). THE EFFECTS PRODUCED ARE ALSOTHE SAME WITH AN INCREASE INMEMBRANE TEMPERATURE. ANINCREASE IN CHAIN LENGTH WOULDHAVE THE OPPOSITE EFFECTS. HOWEVER,ON THE AVERAGE, CHAIN LENGTHS INMEMBRANES ARE LESS VARIABLE THAN THEDEGREE OF UNSATURATION. ALSO MEMBRANETEMPERATURES FOR MAMMALS TEND TO BESTABLE. NOTE THAT MEMBRANE FLUIDITY IS AN IMPORTANT PROPERTY IN MEMBRANE FUNCTIONS SUCH AS TRANSPORT.

STATE TRANSITION. This is the property of change inthe structure of a membrane. As learned before, phospholipids/sphingolipids with greater unsaturation form more disorganized bilipids and give a certain “liquidity” to a membrane. Phospho-lipids/sphingolipids at higher temperatures do the same. The conversion of a membrane lipid to a more liquid state ( temp.)occurs at its transition temperature (liquid crystal formation).

PROPERTIES OF TRANSITION IN BILIPID MEMBRANES:

Paracrystalline or gel state

Liquid crystalline state

MOTION TRANSITION Generally, the lipid components of a membrane are under constant motion (vibration). Theamount of vibration depends on temperature and composition.DIFFUSION – As a result the lipids tend to spread out andform a uniform distribution on their half of the bilipid layer. Usually they are confined to that layer. However, in morerecent studies, flippase enzymes have been described thatmove phospholipids from one side of a membrane to another.This seem to be required for limited applications such asinitial membrane formation or maintenance.

OTHER PROPERTIES THAT LIPIDSIMPOSE ON MEMBRANES

ASSYMETRY: THE LIPID COMPOSITION OF THE TRANSVERSESECTIONS (ACROSS THE BILIPID LAYER) CAN VARY BETWEEN THEOUTSIDE AND INSIDE OF A MEMBRANE. AN EXAMPLE IS SHOWN FOR A RED BLOOD CELL (ERYTHROCYTE). SUCH ASSYMETRY CAN IMPOSE SPECIFIC MEMBRANE CURVATURE OR IMMUNE SPECIFICITYTO A CELL.

ASSYMETRY FROM MEMBRANE TO MEMBRANE IN A CELL:

LIPIDS VS.MEMBRANELOCATION

Two extremes in composition of membranes are indicated for mitochondriavs. plasma membranes. In mitochondria, the amount of cholesterol is quitesmall while that of phosphatidyl ethanolamine is substantial (see arrows).On the other hand, cholesterol is 7x higher in plasma membranes while itsphosphatidyl ethanolamine level is only about ½ that of mitochondria. Thesecomponents contribute to the need for a very fluid membrane in mito-chondria (lack of a stiffening molecule [cholesterol] ) and availability of greater amount of unsaturated fatty acids (found in PE).

THE ROLE OF PROTEINS INMEMBRANES?

AT THE TIME THAT DAVSON AND DANIELLI WERE WORKING ON MEMBRANES (1930s – 1940s), THEY REALIZED THAT IT WOULD BE NECESSARY TO EXPLAIN THE ROLE OF PROTEINS IN MEMBRANES TO SHOW HOW –

SUBSTANCES WERE TRANSPORTED THROUGH MEMBRANES

CELL FUNCTION AND SURVIVAL COULD NOT EXIST WITHOUT SUCH TRANSPORT.

FLUID MOSAIC MODEL ACCOUNTS FOR:

1) 2 KINDS OF PROTEINS- EXTRINSIC & INTRINSIC2) INCLUSION OF TRANSPORT3) INTRODUCTION OF CARBOHYDRATES ON PROTEINS

INTRINSIC PROTEINS: ALSOKNOWN AS INTEGRAL PROTEINS, ARE FIRMLY ANCHORED IN BILIPIDLAYERS. THESE PROTEINS PASS THROUGH THE MEMBRANE ANDARE INVOLVED IN MEMBRANE FUNCTIONS SUCH AS TRANSPORTAND HORMONE RECEPTION.

THE EXAMPLE SHOWN IS GLYCO-PHORIN (A RED BLOOD CELL PROTEIN) – NOTE:1) ALPHA HELIX IN BILIPID LAYER (GREEN

ARROW)2) CARBOHYDRATES IN OUTER LAYER (RED ARROW) ABO/ MN BLOOD TYPE

MARKER PROTEINS

Na-K-ATPase IS AN INTRINSIC MEMBRANE TRANSPORT ENZYME

MECHANISM: (shown at right)

(a) It loads up 3 Na+ ions inside the cell(b) ATP causes a decrease in Na+ affinity(c) (d) Na+ release, then K+ uptake occurs(e) dephosphorylation(f) K+ release inside the cell

GENERAL DIAGRAM OF Na-K-ATPase

A FEW INTEGRAL (OR INTRINSIC) MEMBRANE PROTEINS EVEN USEb-STRUCTURES (KNOWN AS STRANDS OR BARRELS) TO CROSS CELLULAR MEMBRANES.

SOME OTHER EXAMPLES OF INTRINSIC MEMBRANE PROTEINS ARE:SODIUM, POTASSIUM STIMULATED-ATPase (transport of Na and K ions)HUMAN LEUKOCYTE ASSOCIATED (HLA) ANTIGENS (antigen presentation)TolC (outer structure membrane protein of E. coli) [uses b-barrell structure]AQUAPORINS (transport of water molecules)

NaK-ATPase

TolC

AQUAPORIN

HLA ANTIGEN

INTEGRAL MEMBRANE PROTEINS PASS THROUGH MEMBRANES WITH ONE OR MORE a-HELICES (sometimes with b-barrels). THOSE PROTEINS THAT ARE INVOLVED WITH TRANSCELLULAR TRANSPORT HAVE BETWEEN 6 AND 12 TRANSCELLULAR HELICES.

A HYRODPATHY PLOT MAY BE MADE OF A MEMBRANE PROTEIN’SAMINO ACID SEQUENCE VS. THE HYDROPATHY SCALE OF ITSAMINO ACIDS (e.g. VAL= 4.2 vs. LYS= -3.9 where more positive numbersare more hydrophobic) TO DETERMINE WHERE THE SEQUENCE CROSSES THE CELL MEMBRANE. AS SHOWN, RHODOPSIN WOULD HAVE SEVEN TRANSMEMBRANE a-HELICES.

EXTRINSIC PROTEINS.ALSO CALLED PERIPHERALPROTEINS. THESE ASSOCIATEWITH THE MEMBRANE BYELECTROSTATIC INTERACTIONSAND HYDROGEN BONDING. THEYARE ISOLATED BY MILD TREAT-MENTS SUCH AS SALTING OUT.(INTEGRAL PROTEIN ISOLATION??)

THE EXAMPLE IS CYTOCHROME C.IT IS ONE OF THE PROTEINS THATIS INVOLVED IN OXIDATION-REDUCTIONS IN THE OX-PHOSPATHWAY. IT IS ATTACHED TO THECRISTAE ON THE INTERMEMBRANE SIDE OF MITO-CHONDRIA.

HOW ARE LIPIDS & PROTEINS “ASSOCIATED” IN MEMBRANES?

IT WAS KNOWN EARLY ON THAT EXTRINSIC MEMBRANE PROTEINSCOULD BE EASILY REMOVED FROM MEMBRANES BY EXPOSING THE MEMBRANE TO SALT SOLUTIONS THAT WERE STRONGER THAN THEIONIC (SALT) SURFACE FORCES ON THE INNER AND OUTER FACES OFTHE LIPIDS THEMSELVES. THESE “FORCES” ONLY SERVE THE ROLE OF KEEPING THE PROTEINS IN PLACE IN A SOMEWHAT LOOSE MANNER.

THE EXTRINSIC PROTEINS ATTACH TO THE MEMBRANES BY: IONICINTERACTIONS WITH THE LIPIDS OR EVEN PARTS OF INTEGRAL MEMBRANE PROTEINS THAT PROJECT OUT OF THE MEMBRANE. EVENSHORT LOOPS OF HYDROPHOBIC AMINO ACIDS THAT STICK INTO THELIPID ARE FOUND, BUT THESE ARE NOT SO PERMANENT THAT THEYCAN’T BE REMOVED BY “SALTING OUT” THE OTHER PARTS OF THE PROTEIN.

THE ATTACHMENTS OF INTEGRAL MEMBRANE PROTEINS WITHIN THELIPID PORTION OF THE MEMBRANE ARE MORE ROBUST (STRONGER)THAN PERIPHERAL PROTEINS AND ARE OFTEN ASSOCIATED WITH SOME MEMBRANE FUNCTION . IT HAS ALREADY BEEN SHOWN THAT a-HELICES AND b-STRUCTURES (i. e., BARRELS AND STRANDS) OF INTEGRAL MEMBRANE PROTEINS ARE USED JUST TO HAVE A PROTEIN CROSS (PENETRATE) THE LIPID MEMBRANE.

IN ADDITION: ANCHORING (= POSITIONING) OF INTEGRAL PROTEINS TO MEMBRANE LIPIDS MAY ALSO REQUIRE OR SUBSTITUTE SEPARATE KINDS OF BONDS FOR PROTEIN FUNCTION TO OCCUR –

AMIDE-LINKED MYRISTOYL ANCHORS; THIOESTER-LINKED FATTY ACYL ANCHORS; THIOETHER-LINKED PRENYL ANCHORS; AND GLYCOSYL PHOSPHATIDYLINOSITOL ANCHORS.

MYRISTOYL = PROTEIN LINKED TO MYRISTIC ACID (C14 FATTY ACID)FATTY ACYL = PROTEIN LINKED TO A FATTY ACID (NON-SPECIFIC)PRENYL = PROTEIN LINKED TO A PRENYL GROUP: C-C=C-C-(CH3)2

HERE ARE TWO EXAMPLES: THE PROTEIN ON THE LEFT IS LINKEDTO MYRISTIC ACID THROUGH AN AMIDE GROUP. THE MYRISTIC ACID,IN TURN IS PART OF THE LIPID BI-LAYER. THE PROTEIN ON THE RIGHTIS LINKED TO PALMITIC ACID THROUGH A THIOESTER. THE PALMITICACID IS PART OF THE LIPID BI-LAYER. HOWEVER, IN ADDITION, THEPROTEIN TRAVERSES THE LIPID BILAYER WITH SEVERAL a-HELICES.

PROTEIN

PROTEIN

PALMITIC ACID

MYRISTIC ACID

A REAL EXAMPLE OF AN INTRINSIC PROTEIN WHOSE POLYPEPTIDECHAINS DO NOT ENTER THE BILIPID LAYER IS THE “REGGIE” PROTEIN.

REGGIE-1 IS HELD TO ITSMEMBRANE BY MYRISTOL-ATION AND PALMITOLATIONNEAR THE N-TERMINALEND OF THE PROTEIN. THESE DOUBLE ANCHORSDIRECT THE REMAINDEROF THE MOLECULE TO CARRY OUT ITS FUNCTIONSON THE INSIDE OF THE CELL.

REGGIE-1 HAS BEENASSOCIATED WITH THEREFORMATION OF THECYTOSKELATIN, CELLADHESION, ENDOCYTOSISAND OTHER FUNCTIONS.

OUTSIDE OF CELL

THE CONCEPT OF A MEMBRANE “RAFT”IT IS CURRENTLY HYPOTHESIZED (=THOUGHT) THAT THERE ARE REGIONS ON A MEMBRANE BILIPID IN WHICH LATERAL DIFFUSION(=MOVEMENT) IS HINDERED. THESE REGIONS ARE DESCRIBED AS BEING RICH IN CHOLESTEROL AND GLYCOSPHINGOLIPIDS (HAVING ACYL GROUPS THAT ARE LONG CHAINED AND SATURATED).

SUCH AREAS ARE ALSO KNOWN AS MICRODOMAINS OR MEMBRANERAFTS. THEY ARE CONSIDERED TO BE SMALL, MOBILE AND WITHSHORT ½ LIVES. ONLY INDIRECT EVIDENCE HAS POINTED TO THEIREXISTENCE. THE ADVANTAGE OF A RAFT IS TO FACILITATE THE FUNCTIONS OF SIGNALING (=RECEPTOR) PROTEINS.

GPI = GLYCOSYL PHOSPHO-INOSITOL ANCHOR.

CAVEOLIN = A PROTEIN INVOLVED IN SIGNALING &ENDOCYTOSIS.

NOTE: ORANGE GLYCO-SPHINGOLIPIDS AND YELLOW CHOLESTEROL.

CARBOHYDRATES IN MEMBRANESCARBOHYDRATES ARE, OF COURSE, NOT COMPATIBLE WITH THEAPOLAR NATURE OF A MEMBRANE. THEY ARE ASSOCIATED WITH MEMBRANE LIPIDS UNDER THE GROUP KNOWN AS SPHINGOLIPIDS(= GLYCOLIPIDS) AND THEY ARE ALSO COVALENTLY BOUND TO MANY MEMBRANE PROTEINS (= GLYCOPROTEINS). THEY ARE POSITIONED ONTHE OUTER FACE OF A MEMBRANE (OFTEN ON THE OUTSIDE). THEY MAY FORM A LOOSE ASSOCIATIONS WITH OTHER CARBOHYDRATES KNOWN COLLECTIVELY AS THE GLYCOCALIX (LITERALLY SUGAR COAT) OF A CELL.

MANY CARBOHYDRATES ON MEMBRANESSERVE THE ROLE OF IMMUNE IDENTIFIERS (“FRIEND OR FOE?”) FOR THE ORGANISM. OTHERS MAKE UP COMPLEX SYSTEMS THATCOAT THE MEMBRANES OF MICROOGANISMS AND SERVE AS PROTECTIVE DEVICES FROM ATTACK OR ACT EVEN AS OSMOTIC STABILIZERS FOR THE ORGANISMS’ MEMBRANE.

SYNDECAN (RIGHT) ACTS AS A MOLECULARGLUE FOR OTHER MOLECULES IN THE EXTRA-CELLULAR MATRIX.

A FEW OTHER FACTS ABOUT CARBOHYDRATESTHAT BELONG TO MEMBRANES:

- The glycocalyx may be involved in a process called “lymphocyte homing” that helps guide white blood cells to a cell requiring immunological intervention – that is they serve as markers.

-Many of the oligosaccharides that make up a cell surface glycocalyx end in sialic acid which has a negative charge. This charge repels substances from approaching a cell surface.

PUTTING IT ALL TOGETHERMEMBRANES ARE COMPOSED OF LIPIDS,PROTEINS AND CARBOHYDRATES FORMEDTO MAXIMIZE THE MEMBRANE’S FUNCTION

Remember that carbohydrates only are associated with membranesby binding to certain lipids and proteins. They do not enter the bilipid layer.

WHAT IS ESSENTIAL TO UNDERSTAND:1. The nature of a bilipid layer in a cell membrane and how phospholipids form the double layer.2. How does one distinguish between a micelle, a bilipid layer and a liposome?3. How do sphingolipids and cholesterol contribute to the characteristics of a membrane?4. What is important about fatty acid chain length and degree of unsaturation in membrane fluidity?5. What is meant by “transition” in a membrane?6. How are membranes asymetric and what does that impose on a membrane?7. What was the contribution of Davson & Danielli to the understanding of membrane structure?8. How does one distinguish between an intrinsic (integral) and extrinsic (peripheral) membrane protein? Give an example.9. How MIGHT a hydropathy plot identify an intrinsic protein?10.How do proteins and lipids associate (bind) together in a membrane?11. How do intrinsic proteins bind to a lipid bilayer if they do not penetrate the the membrane with alpha helices and what are membrane rafts?12. How do carbohydrates associate with membrane bilayers?