Membranes: They function to organize biological systems by formingcompartments within which biological processes take place. Many subcellular organelles are membrane bound, e.g.nuclei, mitochondria, chloroplasts, endoplasmic reticulum…etc. They are organized assemblies of lipids and proteins withsmall amount of carbohydrates. They regulate the composition of the intracellular medium by controlling the flow of nutrients, waste products, ions…. etc. into and out of the cell, through pumps and gates embedded in the membrane, which allow the transport of substancesagainst or with electrochemical gradients, respectively.
Lipids: They are biological substances that are soluble in organic solvents such as chloroform, methanol, acetone….etc. but are almost insoluble in water. They are called neutral fats since they are uncharged molecules. They are classified as Simple, Complex or Derived lipids.3. Simple lipids are esters of fatty acids and alcohols. a. fats; esters of fatty acids with glycerol. b. waxes; esters of fatty acids with higher mol wt. mono- hydric alcohols.4. Complex lipids are esters of fatty acids and glycerol, but they contain groups other than the alcohol and the acid. a. Phospholipids: FA + Alcohol + Phosphoric acid residue. They may also contain a nitrogen-containing base. b. Glycolipids: FA + Alcohol (Sphingosine) + Carbohydrate. c. Lipoproteins: Lipid moiety + Protein part.5. Derived lipids (Precursors): include FA, Alcohols, steroids, fat-soluble vitamins, and hormones.
Fatty Acids are aliphatic carboxylic acids Fatty acids occur mainly as esters (as in fats and oils). They are usually straight chain aliphatic carboxylic acids that contain even number of carbon atoms. They are either saturated (contain no double bonds) or unsaturated (contain one or more double bonds).Saturated fatty acids, are derivatives of acetic acid [CH3-COOH]; CH3-(CH2)n-COOH, where n is an even number, e.g. Palmitic acid: CH3-(CH2)14-COOH and Stearic acid CH3-(CH2)16-COOH.Unsaturated fatty acids are mono-unsaturated or poly-unsaturated, e.g. Oleic, linoleic and linolenic acids are 18 C acids that have 1, 2 and 3 double bonds, respectively; Oleic acid (18:1;9): CH3-(CH2)7-CH=CH-(CH2)7-COOH Linoleic acid (18:2;9,12): CH3-(CH2)4-CH=CH-CH2-CH=CH-(CH2)7-COOH. α-Linolenic acid (18:3;9,12,15): CH3-(CH2)2-CH=CH-CH2-CH=CH-CH2-CH=CH- (CH2)7-COOH. γ-Linolenic acid (18:3;6,9,12): CH3-(CH2)4-CH=CH-CH2-CH=CH-CH2-CH=CH- (CH2)4-COOH. Arachidonic acid (20:4;5,8,11,14):
Functional Roles of Fatty Acids Determine their Structure and Degree of Unsaturation. Melting points of even-numbered-carbon fatty acids increase with chain length and decrease with unsaturation, for example, Triglycerides containing 3 saturated FAs of 12 C or more are solid at body temperature, while if the FAs are 18:2 for example, the triglyceride is liquid at temperatures below 0°C. e.g. Membrane lipids, which must be fluid at all environmental temperatures are more unsaturated than storage lipids, also lipids in tissues that are exposed to cold temperatures such as in extremities or in animals that hibernate are more unsaturated. Fats are solid, and oils are liquids at room temperature (RT). Plants produce triacylglycerols that are rich in unsaturated FAs and thus they are liquid at RT (oils), while Triacyl- glycerols of animal origin are rich in saturated FAs (fats).
Simple lipids are FA esters with Alcohols. Triacylglycerols (Triglycerides; Neutral fats). They constitute most of plant and animal fats. They are non-polar, water-insoluble TRIESTERS of glycerol. They may contain one (simple triacylglycerols), two or three (mixed triacylglycerols) different types of FAs,e.g. Tristearin and Triolein have 3 residues of stearic and oleic acids, respectively.e.g. 1-palmitoyl-2-linoleoyl-3-stearoyl-glycerol, contains palmitic, linoleic and stearic acids esterified with the OH groups of C1, C2 and C3 of the glycerol moiety, respectively.
Simple lipids are FA esters withAlcohols; cont. Monoacylglycerol Diacylglycerol Triacylglycerol / Triglycerides
Triacylglycerols function as energyreservoirs. Triacylglycerols, although are not part of biological membranes, they are a highly efficient form of metabolic energy storage. This is because the main energy producing unit is a 2- carbon atom molecule called acetyl-CoA, which is produced from the oxidation of glucose and FAs. Acety- CoA is also the building units of FAs. In glucose and Fat oxidation for energy production, each molecule of glucose produces 2 acetyl-CoA molecules, while each FA forming an ester in the triacylglycerol produces one acetyl-CoA molecule for each 2-carbon unit of the FA chain.
Triacylglycerols function as energyreservoirs; cont. In animals, the cells that are specialized in synthesis and storage of fats are called adipocytes, which form adipose tissue that is most abundant in subcutaneous (under skin) and abdominal cavity. The subcutaneous adipose tissue serves as a thermal insulation in warm-blooded aquatic and polar animals. Fat content of normal humans (21% for men and 26% for women) enables them to survive starvation for 2-3 months, while glycogen that constitutes short-term energy store, provides less than a day energy supply for body’s metabolic needs.
Complex Lipids: Phospholipids are main lipid constituents of membrane. They are Diacylglycerols (esterified in glycerol’s C1 & C2 with FAs), in which the OH group of glycerol’s C3 is forming an ester with phosphoric acid (simplest form, called phosphatidic acid) or a phosphoric acid derivative (an X-group attached to phosphoric acid via one of its OH groups), e.g. choline, is an amino alcohol that bind with phosphatidic acid through the (OH) group of the phosphate giving rise to phosphatidylcholines (also called lecithins), the most abundant phospholipids of the cell membranes. e.g. inositol is a cyclic alcohol that form phosphatidylinositol, an important constituent of cell membrane phospholipids that act as a 2nd messenger in cell signaling mechanisms.
Complex Lipids; cont. Sphingolipids are also major membrane component. They are derivatives of the C18 amino alcohols (sphingosine and dihydrosphingosine), in which the amino group is forming an ester with FA (i.e. amide derivative, e.g. ceramide). e.g. Sphingommyelins, are sphingolipids that are present in brain and nerve tissues, and consists of choline moiety attached to ceramide phosphate (i.e. sphingosine, FA, phosphoric acid and choline).
Derived Lipids: Steroids play many physiologically important roles. Cholesterol, in addition to its association with atherosclerosis, it is the precursor molecule of large number of biologically important steroids, including Vitamin D, steroid hormones, bile acids….etc. It contains the characteristic steroid nucleus. It is a major constituent of the cell membrane. 18 12 17 19 11 13 1 9 C D 16 2 14 10 8 15 3 A B 7 5 4 6 Steroid nucleus C D A BHO 1, 25-Dihydroxy- Cholesterol cholecalciferol, Vitamin D
Membrane Structure The basic structural characteristic of membranes is due to the physico-chemical properties of phospholipids and sphingolipids that constitute biological membranes. These compounds with their hydrophilic (polar) head and hydrophobic tail, interact in aqueous systems in vitro to form spheres, called “Vesicles” or “Liposomes”, in which the polar heads are directed to the outside (aqueous environment) while the hydrophobic tails interact to exclude water (in the interior side) forming what is called lipid bilayer.
Membrane Permeability Permeability studies of lipid vesicles and electrical-conductance measurements of planar bilayers have shown that lipid bilayer membranes have a very low permeability for ions and most polar molecules. Water is considered an exception of the above rule due to its small size, high concentration and lack of a complete charge. Based on Permeability Coefficient (P) values of different ions and molecules, ions such as Na+ and K+ traverse membranes ~ 109 fold less likely (slower) than does water. Similarly, Tryptophan, which forms a zwitterion at pH 7.0, crosses the membrane 103 times as slowly as does indole, the ring structure of the tryptophan that lacks ionic groups (amino and carboxylic groups).
Membrane Proteins Membrane lipids form a permeability barrier that establish the compartments, which constitute the nature of plasma membranes. Most molecules traverse membranes aided by proteins. Membranes differ in their protein content, where for example “Myelin”, which serves as an electrical insulator around certain nerve fibers contains ~ 18% proteins (pure lipids serve well for insulation) as compared to 50% and 75% protein contents of plasma membrane of other cells and the inner membranes of mitochondria and chloroplasts, respectively. The higher protein contents of other structures (compared to myelin) are mainly because these are metabolically active structures that have proteins embedded in the membrane structure, which serve as channels, receptors, pumps, and enzymes that are required for transport of molecules and signals across the membranes. Membrane proteins are either “Integral Membrane Proteins”, which mainly span the membrane lipid bilayer and can be released only by a detergent or organic solvent (to solubilize the lipid bilayer and thus release the spanning proteins) or “Peripheral Membrane Proteins”, which are bound to membranes primarily by electrostatic and hydrogen bonds interactions with the polar heads of the lipid bilayer or through interaction with integral protein. The association of the peripheral proteins to membranes can be disrupted by increasing the ionic strength (increasing salt concentration) or by changing the pH.
Integral and Peripheral Membrane Proteins Extracellular Peripheral Proteins LipidBilayerIntegral IntracellularProteins
Membrane Proteins,Interaction with membrane structures Proteins may span (traverse) membrane with α-helices. In archaea, there is an integral membrane protein called Bacteriorhodopsin, which acts as a proton pump, where it captures light energy and uses it to move protons across the membrane out of the cell. The resulting proton gradient is subsequently converted into chemical energy. Bacteriorhodopsin is built of 7 closely packed α-helices, arranged almost perpendicularly to the plane of the cell membrane, spanning its entire width (~45 Ao). Most of the amino acid residues in these membrane-spanning α- helices are non polar, where they interact with the hydrophobic hydrocarbon core of the cell membrane or with other hydrophobic residues in adjacent α-helices. The tertiary structure of bacteriorhodopsin is similar to that of rhodopsin, which senses light in the retina of vertebrate animals. Both proteins belong to the 7-transmembrane receptor family. However, their functions are different and there is only slight conservation of the amino acid sequences.
Membrane Proteins,Interaction with membrane structures Proteins may span (traverse) membrane with β-sheets. Gram negative bacteria such as E. coli and some gram positive bacteria contain an outer membrane channel protein called Porin. Porin is formed of β-strands in an anti-parallel arrangement that is forming a single β-sheet that curls up to form a hollow cylinder, which acts a s a pore (hence the name porin), or a channel. Unlike other membrane transport proteins, porins are large enough to allow passive diffusion, i.e. they act as channels that are specific to different types of molecules. Porins are also present in the mitochondrial and chloroplast membranes. Similar to bacteriorhodopsin, porins have hydrophobic outer surface to allow the interaction with the hydrocarbon core of the cell membrane or with other hydrophobic residues in adjacent β-strands. In contrast, the interior of the channel is quite hydrophilic and filled with water to permit the diffusion of the solutes. This characteristic structure of non-polar surface with polar interior of the channel is achieved by a tandem alteration of hydrophobic and hydrophilic amino acid residues along the β-strands.
Porin, Membrane Channel Protein Tertiary Structure Top view of the channel LipidBilayer Hydrophobic Hydrophilic amino acid amino acid residues residues (Yellow) (White) Secondary Structure
Membrane Proteins,Interaction with membrane structures Proteins may be partially embedded in the membrane, i.e. not spanning through the membrane. The membrane-bound enzyme prostaglandin H2 synthase-1 (bound to endoplasmic reticulum), which catalyzes the synthesis of prostaglandin H2 (a pain and inflammation mediator and a modulator of gastric acid secretion) from arachidonic acid (20:4; 5,8,11,14), is a homodimer that firmly attaches to the membrane through the interaction of a set of α-helices with hydrophobic surfaces with the membrane. This association is sufficiently strong to the extent it requires the action of a detergent(s) to be disrupted and thus it’s classified as an integral membrane protein. Since the substrate of this enzyme, the arachidonic acid, is hydrophobic molecule that is generated by the hydrolysis of the membrane lipids, the association of this enzyme to the membrane is crucial to its function, where the substrate reaches the active site of the enzyme from the membrane without entering the aqueous environment of the cytoplasm. This is achieved by travelling of the substrate through a hydrophobic channel in the protein.
Prostaglandin H2 Synthase-1,Catalyzed reaction & Membrane association
Cox-Inhibitors, Blocking substrate channel to active site Drugs that are called COX-inhibitors (cyclooxygenase inhibitors) such as aspirin and ibuprofen act by blocking this channel and prevent prostaglandin synthesis by inhibiting the cyclooxygenase activity of the synthase enzyme. Aspirin, for example (acetyl salicylic acid), acts by acetylating a serine residue in position 530 of the synthase enzyme (Ser530), which lies along the channel that leads the substrate to the active site.
Membrane Proteins,Interaction with membrane structures Soluble proteins may associate with the membranes through enzyme- catalyzed attachment of a hydrophobic group to the protein. Examples of such groups include: – Palmitoyl group attached to a cysteine residue by thioester bond. – Prenyl group (farnesyl or geranylgeranyl) attached to a cysteine residue at a C- terminal end of the protein, e.g. Ras attachment to the membrane. – Glycosylphosphatidylinositol (GPI) anchor attached to the C-terminus.
Prediction of Transmembrane Helices• To identify transmembrane helices is by evaluating the stability of a postulated helical segment and see whether it’s most stable in a hydrocarbon environment or in water, i.e. by estimating the free-energy change when this helical segment is transferred from the interior of a membrane to water.• The sum of the Free-energy changes for the transfer of individual amino acids from a hydrophobic to an aqueous environment, determine whether a segment composed of such amino acids is likely to span the membrane or not.• For example, an α-helix formed of L-Arg (positively charged) from the interior of the membrane to water would be highly favorable (-51.5 kJ mol-1 OR -12.3 kcal mol-1/ residue). In contrast, the transfer of an α-helix formed of L-Phe (hydrophobic) from the interior of the membrane to water would be unfavorable producing an energy change of +15.5 kJ mol-1 OR +3.7 kcal mol-1/ residue.• The hydrocarbon core of a membrane is typically 30Ao, a length of a 20- amino acid residue-α-helix.• Thus transmembrane domain/region of a protein can be identified by estimating the free-energy change that takes place when a hypothetical α- helix formed of any 20-amino acid residues is transferred from the membrane interior to water.• The free-energy change of each set (called window) is plotted to create a “Hydropathy PLOT”. PLOT• A peak of +84 kJ mol-1 (+20 kcal mol-1) or more indicates that the tested poly-peptide segment (20 amino acid residues) could be a membrane- spanning α-helix.