LIPIDS. Docent Tuomo Glumoff. Faculty of Biochemistry and Molecular Medicine P Biomolecules for Biochemists (8 op)

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1 Faculty of Biochemistry and Molecular Medicine P Biomolecules for Biochemists (8 op) P Biomolecules for Bioscientists (8 op) P Biomolecules (5 op) LIPIDS Docent Tuomo Glumoff

2 LIPIDS lectures 32 Answers to problems will be given at the lectures and they will also be available in NOPPA after the lectures. - lipids have many functions: fats insulate heat and serve as energy storage form membranes some are vitamins or hormones or other signal molecules enzyme cofactors, electron carriers light-absorbing pigments, protein anchors to membranes emulsifiers - lipids are not polymers, but often associate through noncovalent forces - hydrophilic and hydrophobic parts = amphipathic FATTY ACIDS - simplest lipids are fatty acids (rasvahapot) carboxylic acids with a hydrocarbon side chain straight chain vs. branched chain saturated = contain only single bonds unsaturated = contain double bond(s) configuration about the double bond usually cis mostly even number of carbons COOH COOH COOH COOH - fatty acids are in anionic form at physiological ph: RCOOH RCOO - + H + pka naming and abbreviating fatty acids: number of carbons, double bonds (their position and configuration) see table on page 35

3 33 - fatty acid carbon numbering starts from the carboxy end; however if ω-naming system used, then: - w- (omega) fatty acids: the position of the double bond(s) counted from the methyl end group, e.g. w-3, w-6 - essential: mammals cannot make double bonds between D9 and the methyl end - abundant in some plant seeds and fish oils COOH fatty acids with double bond(s) in this region must be obtained from food fatty acids with double bond(s) in this region can be synthesized in the body - chain length and degree of saturation affects solubility, packing and melting temperature - cis-double bond makes a bend to the fatty acid tail PROBLEM 1. What is one apparent influence to the physical properties of a fatty acid upon introduction of double bond(s)? - in cells not much free fatty acids exist, but most are parts of more complex lipids

4 TRIACYLGLYCEROLS FATS - fatty acyl esters of glycerol - efficient energy stores - may contain different fatty acids - in animals fat stored in adipose cells 34 - fats and oils are mixtures of triacylglycerols; fat is solid, oil is liquid at room temperature - 3 major functions of fat: energy production get ATP to drive metabolic processes heat production (alternative for ATP production) thermal insulation (layers of fat cells under the skin) - 2 advantages of storing energy in the form of fat rather than carbohydrate: fat is less oxidized than carbohydrate, so, fat has more energy-releasing capacity left fat is tightly packed and need no water, which would increases the weight to carry - composition and naming system Molecules in 3D for example from here:

5 classification of lipids 35 Numbering begins from the carboxylic end!

6 PROBLEM 2. Draw the structure or abbreviate the following fatty acids: (NOTE: some may be hypothetical taken here just for the sake of practicing) a) 18:2tD5,8 b) 22:4cD3,9,12,20 c) 36 O OH SOAPS AND DETERGENTS - saponification: fat + alkali (NaOH, KOH) soap - fatty acids are released and form sodium or potassium salts - detergents are synthetic molecules that often have better defined chemical and physical properties than soaps - detergents can be used to denaturate proteins or extract and solubilize membrane proteins (mimic the membrane environment and mask the hydrophobic surface of the membrane protein) O - O S O Na + SDS, sodium dodecyl sulfate O Triton X-100 PROBLEM 3. Chemistry of cleaning: Why do many cleansing agents used to remove stains or spots are basic by ph and what is the practical advantage of this? WAXES - a long-chain fatty acid ester + a long-chain alcohol = wax - the head group is left only weakly hydrophilic waxes are insoluble - used as protective coatings by both animals and plants

7 LIPID CONSTITUENTS OF BIOLOGICAL MEMBRANES - cell membranes contain a variety of different lipids that also affect the structure and function of the membranes 37 i) GLYCEROPHOSPHOLIPIDS (phosphoglycerides) - phosphate-containing head group - derivatives of glycerol-3-phosphate glycerol backbone - major group of lipids in most biological membranes - R1 & R2 derived from fatty acids - R3 possess great variance ii) GLYCOGLYCEROLIPIDS - built on non-phosphorylated glycerol - sugar at R3 - monogalactosyl diglyceride abundant in chloroplast membranes

8 - sphingomyelin is part of membraneous structure that surrounds and insulates nerve axons sphingomyelin contains also protein - sphingolipids at cell surfaces mediate biological recognition: extracellular molecules cell-cell contacts blood group antigens 38 - ganglioside is a soluble lipid due to many carbohydrate units carbohydrate part lipid part iv) CHOLESTEROL - a steroid - rigid structure due to all-chair conformation of the cyclohexane rings - content up to ca. 25% in some membranes - disrupts regularity in the membrane can regulate membrane stiffness and permeability - without cholesterol the flexibility of the membrane could change too rapidly as a response to changes in environment - cholesterol helps membranes to adapt better and so membranes do not break - stiff (jäykkä) breaks more easily - cholesterol adds flexibility to the membrane structure; not as stiff Despite knowing much of the membrane and membrane lipids: why so many different types of lipids in biological membranes - no clear understanding as yet

9 example of lipid function in tissues: lung surfactant (keuhkosurfaktantti) 39 i.e. why do we need to know about lipids... - major lipid component of the surfactant is dipalmitoyl phosphatidylcholine, DPPC - alveoli cells are coated with DPPC - DPPC is tightly packed, since fatty acids in DPPC are saturated - tight packing of DPPC resists collapse of the alveolar space when breathing out - when not collapsed, also less energy is needed in expanding the alveolar space when breathing in - defect in the surfactant causes diseases associated with breathing difficulties knowing about lipid structure-function (biochemistry) makes it possible to understand physiology and disease

10 40 Blood group antigens. ref. Carbohydrate lectures! A case: how will a snake venom kill you? Phosphatidylserine is a glycerophospholipid that contains the amino acid serine as the R3 group, and two myristic acids as the groups R1 and R2. Many snake venoms contain the enzyme phospholipase A2 that can remove the R2 fatty acid in a reaction involving a water molecule. What remains is lysolecithin, which is more amphiphilic and can act as a detergent that disrupts the red blood cell membranes causing hemolysis. a) Draw the molecular formula of phosphatidylserine b) Draw also the molecular formula of lysolecithin. (will be done at the lecture)

11 - membranes of different cells or cell organelles differ in their lipid composition 41 LIPIDS THAT FUNCTION AS SIGNAL MOLECULES - steroid hormones and many vitamins are derived from cholesterol Steroid metabolism: Aineenvaihdunta II course (this topic here just very briefly) EICOSANOIDS - eicosi = 20 - oxygenated derivatives of C20 polyunsaturated fatty acids; synthesized in the body from arachidonic acid - carry messages to nearby cells - various physiological regulation functions, like blood flow to organs, smooth-muscle contraction, body temperature - many cause inflammation and pain aspirin (acetylsalicylic acid) kills pain by inhibiting prostaglandin synthesis PROBLEM 4. Is arachidonic acid an essential lipid? Why?

12 AGGREGATION OF LIPIDS AND STRUCTURE OF BIOLOGICAL MEMBRANES - at the air-water interface lipids cover twice the area of the membrane made of them 42 - membrane monolayers, bilayers and micelles - micelles bilayers liposomes

13 43 - detergents easily form micelles - CMC = critical micelle concentration e.g. SDS 8.2 mm Mw=288; Mw (micelle)= monomers/micelle - liposomes are spherical vesicles containing an aqueous inner compartment surrounded by a lipid bilayer - may form inside cells or can be made experimentally SUMMARY

14 - synthesis of new lipid bilayer is not well understood adding lipids to increase size of membrane different membranes have different composition of lipids and proteins transfer of newly synthesized membrane? 44

15 - fluid mosaic model describes the arrangement of lipid and protein within the membrane 45 what now follows is a trial to combine structural features of both lipids and proteins in order to understand biological membrane structure and function fluid mosaic model (we have to jump a little bit away from lipids every now and then) - there are integral as well as peripheral membrane proteins: either penetrating the membrane or just associated with it

16 - lateral movement of lipids fast - transverse diffusion slow; helped by enzyme flippase 46 fast slow fast fast

17 - biological membrane may exist in an ordered gel phase or as disordered liquid crystal (fluid) or in a transition state having characteristics of both - affected by composition, temperature change in temperature compensated by change of lipid composition to maintain fluidicity 47 - different membrane lipids, including cholesterol, pack well together to form the membrane - lipid side chains affect membrane packing and thus the physical properties of the membrane - head groups add functionality to the membrane

18 48 - notice that the percentage of total saturated fatty acids in cells increases and unsaturated decreases as a function of increased environmental temperature. Proteins as part of membrane structure: - different types of integral membrane proteins, I to VI - transmembrane domain can also be built of b-sheets (e.g. porins) - lipid anchoring (type V) is also considered a type of its own (next page) a porin channel that forms a hole in the membrane 90 o Characteristics of membrane proteins: - topology of an integral membrane protein can be predicted from its sequence (despite shortage of experimentally determined structures of that type of proteins) - continuous sequence of >20 hydrophobic residues indicates a membrane-spanning a-helix - hydropathy plot - aromatic amino acids often found at the lipid-water interface

19 - lipid-anchoring is possible both for integral and peripheral membrane proteins: prenylation fatty acylation GPI-link 49 outside phospholipid sugars protein inside GPI anchor ( ankkuri )

20 lipid and protein composition varies between membranes of different cells/cell organelles 50 - membrane bilayer is also asymmetric: lipid and protein composition varies between different sides of the bilayer

21 lipid and protein composition varies between membranes of different cells/cell organelles - lipids and proteins may also be laterally organized into domains that cover different parts of a cell certain proteins associate to form aggregates specific protein-lipid interactions certain proteins and certain lipids are found together 51 lipid rafts lipid raft ( lipidilautta ) - divalent metal ions ligate certain lipids with negative head groups - glycosphingolipids and cholesterol form rafts that associate with certain proteins lipid raft

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23 - many membrane proteins are glycosylated (=glycoproteins) 53 Basic concept: protein and glycan are synthesized separately and then attached to each other

24 Example: the ebola virus infection mechanism employs specific interaction with components in the lipid rafts i.e. lipid raft is a target for the virus - one of the virus membrane proteins contains a sequence (called fusion peptide ) including a tryptophan and a phenylalanine (1) - the fusion peptide makes a strong interaction with cholesterol molecules in the membrane - virus uses this interaction to integrate its membrane into the endocytotic membrane to release the genetic material into the cytoplasm (2) - cells with less cholesterol in their membrane are less prone to ebola virus infection

25 55 membrane of the same cell may also have a different lipid and protein composition on different sides of the cell, for example apical and basolateral sides of epithelial cells - certain membrane proteins associate with cytoskeleton form distinct localizations - human erythrocyre membrane as an example of studying a membrane structure: peripheral membrane proteins (on the cytoplasmic side) removed by change in ph or ionic strength integral membrane proteins removed by a non-ionic detergent Triton X-100 cytoskeleton-forming proteins are revealed; most notable are spectrin and actin (next page)

26 actin structure 56 electron micrograph of erythrocyte membrane skeleton spectrin structure - examples of plasma membrane composition and functionality (below and next page) Pathogen infections Toxins Leukocyte rolling

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28 FUNCTIONS ASSOCIATED WITH BIOLOGICAL MEMBRANES - barrier against the outside - selective intake and excretion of substances membrane transport - cell-cell and other recognition - fusion and separation events 58 Membrane transport: - passive diffusion through the membrane - permeability depends on the molecule Membrane transport: - facilitated diffusion through pores/channels - facilitated transport by carrier molecules - driving force either ion gradient or coupled to ATP production Energy needed when diffusion goes against the concentration gradient

29 59 - rate of diffusion varies red = open blue = closed substrate - conformational change of proteins is an important issue very often related to protein function, like binding of substrate (enzymes) or other substances (receptors, protein-protein interactions, etc.) - an example of triose phosphate isomerase, where one loop moves ca. 10 Å to give way for substrate to bind

30 Vesicle fusion: - coated vesicles are a transport means for newly synthesized and secreted proteins - both for soluble and integral membrane proteins - endocytosis (transport in), exocytosis (transport out) 60 - caveolae form via association of caveolin proteins, which are asymmetrically positioned in the membrane - make it possible for membrane to curve and finally form a vesicle - so called SNARE proteins insert into both membranes, bind to each other, and anchor the vesicle to the target membrane - in this way mediate the membrane fusion

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32 - physiological phenomena involving membrane fusion 62 IUPAC-IUB Commission on Biochemical Nomenclature (CBN) Nomenclature of Lipids Fahy et al., A comprehensive classification system for lipids Journal of Lipid Research 46, (2005)

33 RECAP QUESTIONS 63 PROBLEM 5. When we classify a certain compound as a lipid, we do so using a different rationale compared to e.g. classifying another compound as a nucleic acid or a protein. What is the difference? PROBLEM 6. Thickness of biological membranes is 5 to 8 nm (50 to 80 Å) with the lipid bilayer part ca. 3 to 5 nm. The carbon-carbon bond length is about 1.5 Å. Estimate the length of a palmitate molecule (consider a fully extended form). If two molecules of palmitate were placed end to end, like in a lipid bilayer, how would their total length compare with the thickness of a lipid bilayer and a biological membrane (i.e. can our understanding of the membrane structure be correct)? PROBLEM 7. Liposomes can be prepared by suspending a suitable lipid in water and sonicating. Any chemicals dissolved in water may then trap inside liposomes. Liposomes may also fuse with cell membranes; each type of liposome with a cell membrane that contains same or similar lipids. What possible practical use could this phenomenon offer? (example) Glycine trapped in phospholipid vesicles Sonication ( sonic disruption )