Steric Factors and Hydrogen Bonding Influence Homopolysaccharide Folding Subunits with a more or less rigid structure dictated by covalent bonds form 3D macromolecular structures 3D structures are stabilized by weak interactions within or between molecules (H-bonds, hydrophobic and van der Waals interactions, and electrostatic interactions in charged subunits) In polysaccharides: many OH groups, so H-bonding is super important Polysaccharide folding: Glycosidic bond: Rigid pyranose rings connected by oxygen atom bridging two carbon atoms Free rotation about both C-O bonds but limited by steric hinderence of substituents Use and (dihedral angles) For starch and glycogen (a14) tightly coiled helix stabilized by interchain hydrogen bonds Amylose: structure is regular enough to allow crystallization and determination of structure 6 residues per turn, 60 angle between each residue core has dimensions that allow complexing with iodine ions Cellulose: 180 between each residue straight, extended chain All OH groups are available for hydrogen bonding with neighboring chains With several chains lying side by side, stabilizing network of interchain and intrachain H-bonds produces straight, stable supramolecular fibers of great tensile strength Low water content: h-bonding between cellulose molecules satisifies capacityBacterial and Algal Cell Walls Contain Structural Heteropolysaccharides Peptidoglycan: heteropolymer Alternating B14 linked N-acetylglucosamine and N-acetylmuramic acid residues Linear polymers Lie side by side in cell wall, cross-linked by short peptides Peptide cross-links: weld polysaccharide chains into strong sheath that envelops entire cell and prevents cellular swelling/lysing due to osmotic entry of water Lysosyme: enzyme that kills bacteria by hydrolyzing B14 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid Agar: Mixture of sulfated heteropolysaccharides made up of D-galactose and L-galactose derivative ether-linked between C-3 and C-6 Complex mixture of polysaccharides: same backbone structures, but substituted to varying degrees with sulfate and pyruvate Agarose: Agar component with fewest charge groups (sulfates, pyruvates) Can form double helix with three residues per turn, trapping water within the central cavity can then form gel from intrahelical interactionsGlycosaminogycans are Heteropolysaccharides of the ECM Groun substance: gel-like material in ECM that holds cells together and provides porous pathway for diffusion Glycosaminoglycans: linear polymers composed of repeating disaccharide units one of monosaccharides is always N-acetylglucosamine or N-acetylgalactosamine Other monosaccharide is normally a uronic acid (like D-glucouronic or L-iduronic acid) Some contain esterified sulate groups that combine with the carboxylate groups of uronic acid to give high density of negative charge extended conformation in solution, forming rodlike helix Negative COOH groups occur on alternate sides of the helix Sulfated glycosaminoglycans are attached to EC proteins to form proteoglycans Unique to animals and bacteria Found in reticular ECM and basement membrane Hyaluronan: glycosaminoglycan: incredibly large Alternating residues of D-glucuronic acid and N-acetylglucosamine Clear, highly viscous solution serves as lubricant in synovial fluid of joints/gives vitreous humor of vertebrate eye its consistency Also a component of ECM of cartilage and tendons contributes tensile strength and elasticity as result of strong interactions with other components of matrix Chondroitin sulfate: Contributes to tensile strength of cartilage, tendons, ligaments and walls of aorta Dermatan sulfate: Contributes to pliability of skin and is present in blood vessels and heart valves Keratan sulfate: No uronic acid and variable sulfate content Present in cornea, cartilage, bone and horns/hair/hoofs/nails/claws Heparan sulfate: Produced by all animal cells an contains variable arrangements of sulfated and nonsulfated sugars Heparin: fractionated form of heparin sulfate derived from leukocytes used to inhibit cogulation by binding antithrombin (protease inhibitor), causing it to bind and inhibit clotting factor thrombin mediated by electrostatic forces: heparin is HIGHLY negative7.3: Glycoconjugates: Proteoglycans, Glycoproteins and Glycolipids glycoconjugate: biologically active molecule that is an informational carbohydrate joined covalently to a protein or lipid proteoglycan: macromolecules of cell surface or ECM with one or more sulfated glycosaminoglycan chains joined covalently to a membrane protein or secreted protein bind through electrostatic interactions with the negatively charged groups on the polysaccharide glycoproteins: one or several oligosaccharides of varying complexity joined covalently to a protein found on outer surface of plasma membrane, in ECM and in blood act as specific sites for recognition by carbohydrate-binding proteins called lectins Glycolipids: membrane sphingolipids in which hydrophilic head groups are oligosaccharides Oligosaccharides serve as specific sites for recognition by lectins Rich in brain and neuronsProteoglycans are glycosasminoglycan-containing macromolecules of the cell surface and ECM Act as tissue organizers and influence various cellular activities Basic unit: core protein covalently attached to glycosaminoglycan via a Serine residue Glycosaminoglycan is joined to serine residue via a tetrasaccharide bridge Ser is generally foundin Ser-Gly-X-Gly sequence Some are in ECM and some are integral membrane proteins Integral membrane proteoglycans: Membrane heparan sulfate proteoglycans: two families Syndecans: single transmembrane domain + extracellular domain bearing 3-5 chains of heparan sulfate (and in some cases, chondroitin sulfate) Glypicans: attached to membrane by lipid anchor (derivative of phosphatidylinositol) Both can be shed into extracellular space: Protease releases syndecan ectodomains Phospholipase releases glypicans Non-random domain structure: Highly sulfated domains (NS domains) alternate with domains having unmodified GlcNAc and GlcA residues (N-acetylated or NA domains) NS domains: bind specifically to extracellular proteins and signaling molecules to alter their activities thru: Conformational change in the protein induced by the binding Ability of adjacent domains to bind two different proteins and bring into closer proximity Binding of extracellular signal molecules (like growth factors) to increase local concentrations and enhance interaction with growth factor receptors in cell surface Eg. Fibroblast growth factor NS domains interact (electrostatically or otherwise) with variety of soluble molecules outside cell, maintaining high local concentrations at the cell surface Proteoglycan aggregates: enormous supramolecular assemblies of many core proteins all bound to single molecule of hyaluronan Aggrecan: Aggrecan core protein, with ser residues joined to multiple chains of chondroitin sulfate and keratin sulfate through trisaccharide linkers Hundreds of these then bind single hyaluronate --> proteoglycan aggregate Interacts strongly with collagen in ECM of cartiliage contributes to the development, tensile strength and resiliency of connective tissue Collagen, elastin and fibronectin: fibrous matrix proteins interwoven with proteoglycans to give ECM strength and resilience Some are have binding sites for several different matrix molecules (fibronectin)Glycoproteins have covalently attached oligosaccharides Carbohydrate-protein conjugates in which glycans are smaller, branched and more structurally diverse than the glycosaminoglycans of proteoglycans Carbohydrate is attached at anomeric carbon through: a glycosidic link to the OH of a serine or threonine residue (O-linked) or sequences for attachment of O-linked chains tend to be rich in Gly, Val or Pro N-glycosyl link to the amide nitrogen of an Asn residue (N-linked) Sequences for attachment of N-linked chains depends on consensus sequence N-[P]- [ST] Some have single oligosaccharide chain, but many have more than one Mucins: secreted or membrane glycoproteins that contain large numbers of O-linked oligosaccharide chains Benefits to oligosaccharide attachment: Very hydrophilic: alter polarity and solubility of protein with which it is conjugated Serve as destination labels and act in protein quality control (tagging for degradation) Charge repulsion between them when multiple are clustered in single region of protein favors formation of extended rod-like structure in that region Bulkiness and negative charge protect proteins from attack by proteolytic enzymesGlycolipids and lipopolysaccharides are membrane components: Gangliosides: membrane lipids of eukaryotic cells in which the polar head group is a complex oligosaccharide containing a sialic acid and other monosaccharide residues Oligosaccharide moeieties (like in glycoproteins) are almost always found on the outer face of the plasma membrane Lipopolysaccharides: dominant surface feature of gram-negative bacterias outer membrane Prime targets of vertebrate immune system Six fatty acids bound to two glucosamine residues (one of which is point of attachment for complex oligosaccharide)

7.4: Carbohydrates as Information Molecules: The Sugar Code:Lectins are proteins that read the sugar code and mediate many biological processes Lectins: proteins found in all organisms Bind carbs with high specificity and moderate to high affinity Serve in variety of cell-cell recognition, signaling and adhesion processes and in intracellular targeting of newly synthesized proteins Plant lectins: found in seeds; probably serve as deterrents to insects and other predators Animal lectins: Some peptide hormones: circulate in blood and have oligosaccharide moieties that strongly influence circulatory half-life For example: N-linked oligosaccharides of LH and throtropin are recognized by lectins on hepatocytes, which mediate uptake and destruction of the hormones Neu5Ac (sialic acid) situated at ends of oligosaccharide chains of plasma glycoproteins Protect proteins from uptake and degradation in liver Responsible for protecting erythrocytes from degradation (when removed by sialidase, the RBCs disappear within hours) Selectins: plasma membrane lectins that mediate cell-cell recognition and adhesion Important for movement of immune cells through capillary walls At infection site: P-selectin on surface of capillary endothelial cells interacts with specific oligosaccharide of glycoproteins of circulating neutrophils Slows neutrophils as they adhere to and roll along endothelial lining of capillaries Second interaction between integrin molecules in neutrophil plasma membrane and adhesion protein on endothelial cell surface stops neutrophil and allows it to move through capillary wall E-selectin (endothelial cell) and L-selectin (neutrophil) bind cognate oligosaccharides on neutrophil and endothelial cell, respectively, to assist in lymphocyte homing Viruses: attach to host cell through interactions with oligosaccharides displayed on host cell surface HA lectin of influenza virus: essential for viral entry and infection Once entering and replicating, newly synthesized viral particles bud out of the cell, wrapped in portion of plasma membrane Viral sialidase trims terminal sialic acid residue from host cells oligosaccharides releases viral particles from their interaction with the cell and prevents aggregation with one another Antivirals: sugar analogs that inhibit sialidase competitivelyLectin-Carbohydrate interactions are highly specific and often polyvalent Essential that oligosaccharide be unique so lectin recognition is highly specific High density of information in oligosaccharides sugar code with essentially unlimited number of unique words small enough to be read by a single protein Lectins: in carb binding sites, have subtle molecular complementarity that allows interaction only with correct carb cognates Affinity between oligosaccharide and lectin may be small BUT lectin is multivalent (multiple carb binding domains) each oligosaccharide on a membrane surface can engage one of the lectins CBDs, strengthening interactions General interactions that contribute to the binding of many carbs to their lectins: Many sugars have a more and a less polar side: more polar side h-bonds with lectin while less polar undergoes hydrophobic interactions with nonpolar amino acid residues Specific interactions: H-bonding of amino acids to oxygen atoms of sugar

Chapter 8: Nucleotides and Nucleic Acids

8.1: Some BasicsNucleotides and Nucleic Acids Have Characteristic Bases and Pentoses Nucleotides have three characteristic components: Nitrogenous base: pyrimidine or purine derivatives Purines: ADENINE and GUANINE Pyrimidines: CYTOSINE and THYMINE or URACIL in RNA Pentose Numbered with prime signs () to distinguish from nitrogenous bases N-1 of pyrimidines and N-9 of purines are bound to 1 carbon of pentose sugar in N-B-glycosyl bond (formed by condensation of OH from pentose and H from base) Two kinds: 2-deoxy-D-ribose D-ribose Phosphate Esterified to 5 carbon Nucleoside: nucleotide without phosphate Nomenclature: Base Nucleoside Nucleotide Adenine Adenosine Adenylate Guanine Guanosine Guanylate Thymin Thymidine Thymidylate Cytosine Cytidine Cytidylate Uracil Uridine UridylatePhosphodiester Bonds Link Successive Nucleotides in Nucleic Acids Phoaphodiester linkage: phosphate group bridges that connect successive nucleotides in DNA and RNA 5 phosphate group of one base with the 3OH group of the next Covalent backbones: alternating phosphate and pentose residues Hydrophilic OH groups of sugar form H-bonds with water Phosphate groups are completely ionized and negatively charged at ph7 Nitrogenous bases: side groups joined to backbone at regular intervals Under alkaline conditions: RNA is hydrolyzed rapidly but DNA is not 2 OH groups in RNA (absent in DNA) directly involved Yields cyclic 2,3-monophosphate nucleotides that then break down into a mixture of 2 and 3 nucleoside monophosphates Sequence is always written in 5 3 directionThe properties of nucleotide bases affect the three-dimensional structure of nucleic acids Free pyrimidines and purines: weakly basic compounds called bases Aromatic when in DNA and RNA Most bonds have partial double bond character pyrimidines are planar and purines are very nearly planar with a slight pucker Free pyrimidine and purine bases may exist in two or more tautomeric forms depending on pH All nucleotide bases absorb UV light: strong absorption at wavelengths near 260nm Purine and pyrimidine bases are hydrophobic and relatively insoluble in water at near neutral pH of cell At non neutral pH: become charged and increase in solubility in water Base stacking: hydrophobic stacking interactions in which two or more bases are positioned with planes of rings parallel involves a combination of van der Waals and dipole-dipole interactions between the bases minimizes water contact H-bonding between complementary strands is what holds them together8.2 Nucleic Acid StructureDNA is a double helix that stores genetic information A=t and G=C for all cellular DNA A+G = T+C Watson and Crick model: 2 helical DNA chains wound right-handedly around the same axis to form a helix hydeophilic backbones of alternating deoxyribose and phosphate groups on outside of double helix, facing surrounding water furanose ring of deoxyribose is in the C2 endo conformation nucleotide bases are stacked within the doube helix, hydrophobic and nearly planar major groove + minor groove 3 hydrogen bonds between G and C and only 2 between A and T strands run antiparallel DNA duplex is held together by two forces: Hydrogen bonding between complementary base pairs Base stacking interactionsDNA can occur in different three dimensional forms Bond rotations can occur in sugar-phosphate backbone Thermal fluctuation bending, stretching, unpairing of strands Three types of structural variation in DNA: Different possible conformations of deoxyribose Rotation about contiguous bonds in phosphodeoxyribose backbone Free rotation about C1-N-glycosyl bond Purines: restricted to syn and anti form with respect to deoxyribose Pyrimidines: restricted to anti form with respect to deoxyribose Watson-Crick structure: B-form of DNA (B-DNA_ Most stable for a random sequence DNA molecule under physiological conditions Standard point of reference Two structural variants: A form: favored in solutions devoid of water Still right handed double helix BUT wider and with 11 bases per turn instead of 10.5 Plane of base pairs tilted about 20 with respect to helix axis Deeper major groove and shallower minor groove Short DNA molecules tend to crystallize in A form Z form: sequences with pyrimidines alternating with purines (especially C and G) Left-handed helical rotation Purines flip to syn conformation, alternating with pyrimidine anti conformation 12 base pairs per turn more slender and elongated zig-zag appearance to DNA backbone barely apparent major groove and narrow/deep minor grooveCertain DNA sequences adopt unusual structures Four or more adenosine residues sequentially in one strand bends in DNA helix Palindrome: inverted repeats with twofold symmetry over two strands Self-complementary within each strand - can form hairpin or cruciform Mirror repeat: inverted repeat within each individual strand of DNA CANNOT form hairpin or cruciform Three to four strand structures: Triplex: Cytidine pairing with G in GC nucleotide pair or thymidine pairing with A in AT pair N-7, O6 and N6, of purines hydrogen bonding of triplex DNA Hoogsteen positions Non-Watson-Crick pairing: Hoogsteen pairing Triplex: most stable at low pH because G=C C triplet requires protonated cytosine Form most readily within long sequences containing only pyrimidines or only purines within strand Either two pyrimidine strands and one purine strand, or two purine strands and one pyrimidine strand Tetraplex: four DNA strands ONLY occurs readily for DNA with high proportion of Guanosine G-tetraplex: quite stable over wide range of conditions Orientation of strands can varyMessenger RNA codes for polypeptide chains mRNA: carries genetic info from DNA to ribosomes (formed via transcription) In Bacteria and Archea: monocistronic: only codes for one polypeptide polycistronic: codes for two or more different polypeptides In eukaryotes: mostly monocistronicMany RNAs have more complex 3D structures Product of transcription: always single stranded RNA Tends to assume right-handed helical conformation dominated by base-stacking interactions (stronger between two purines than between purine + pyrimidine or two pyrimidines) Base pairing between G and U is fairly common No simple, regular secondary structure that serves as a reference point: Weak interactions, especially base-stacking interactions, help stabilize RNA structures Predominant double-stranded structure is an A-form right-handed double helix Z-form helices have been made in laboratory B-form has not been observed Breaks in regular A-form helix caused by mismatched or unmatched bases in one of both strands are common result in bulges or internal loops Hairpin loops: form between nearby self-complementary sequences Hairpins are most common type of secondary structure in RNA Specific short base sequences (like UUCG) are often found at ends of RNA hairpins and form particularly tight and stable loops Other contributions made by H-bonds that are not part of standard Watson-Crick base pairs: E.g. 2 OH group of ribose H-bonding with other groups8.3 Nucleic Acid ChemistryDouble-Helical DNA and RNA Can Be Denatured Extremes of pH or temperature can denature DNA and RNA disruption of H-bonds between paired bases and of base stacking Separates strands Doesnt break covalent bonds Renaturation: quick, one step annealing of strands if strands arent completely disconnection. Otherwise, two steps: first find one another, and then zipper Hypochromic effect: close interaction of stacked bases in nucleic acid decreases UB absorption relative to free nucleotides and furthered when two complementary nucleic acid strands are paired Hyperchromic effect: opposite of hypochromic effect caused by renaturation Thus, can detect transition from double-stranded DNA to single-stranded form by measuring UV absorption at 260nm Each species of DNA has characteristic denaturation temperature (melting point, tm) at which half of DNA present is separated Higher content of G-C higher Tm Bubbles: careful denaturation of weaker AT sections RNA or RNA-DNA duplexes can also be denatured RNA duplex often requires higher temperatures than DNA duplex RNA-DNA is generally intermediateNucleic Acids from Different Species Can Form Hybrids Can be used to detect similar DNA sequences in two different species or within genome of single species Denature both species DNA and then allow to anneal Rate of annealing affected by: Temperature Length and concentration of DNA fragment Concentration of salts in reaction mixture Properties of sequence itself Yields hybrid duplexesNucleotides and Nucleic Acids Undergo Non-enzymatic Transformations Mutations: alterations in DNA structure that produce permanent changes in the genetic information encoded Deamination: spontaneous loss of exocyclic amino groups Under typical cellular conditions: deamination of cytosine uracil (in DNA): 1:107 residues in 24 hours 100 spontaneous events per day product is readily recognized as foreign in DNA strand and is removed by a repair system Deamination of adenine and guanine: 1/100th this rate Depurination: hydrolysis of N-B-glycosyl bond between base and pentose to create DNA lesion (AP site or abasic site) Occurs at higher rate for purines than for pyrimidines Depurination of ribonucleotides and RNA slower than DNA and not considered physiologically significant Can be accelerated by dilute acid Incubation of DNA at pH 3 selective removal of purine bases, resulting in apurinic acid Radiation promoted: UV light: condensation of two adjacent pyrimidine bases to form cycobutane pyrimidine dimers Most frequent between adjacent thymidine residues on the same DNA strand Creation of 6-4 photoproduct Ionizing radiation (X-rays and gamma rays) can cause: ring openings fragmentation of bases Breaks in covalent backbone of nucleic acid Chemical reactions: Deaminating agents (particularly nitrous acid (HNO2) or compounds that can be metabolized to nitrous acid or nitrites) Nitrous acid and Bisulfite: Potent accelerator of deamination of bases Alkylating agents Can alter certain bases of DNA: Dimethylsulfate can methylate a guanine to yield O^6 methylguanine (cannot base pair with cytosine) Oxidative Damage Hydrogen peroxide, hydroxyl radicals, superoxide radicals arise during irradiation or as byproduct of aerobic metabolism Hydroxyl radicals: responsible for most oxidative DNA damage Cells have defense system to destroy ROS (catalase, superoxide dismutase) If not caught, can cause: oxidation of deoxyribose and base moieties, strand breaksSome bases of DNA are methylated A and C are more frequently methylated than G and T Serves as defense mechanism by marking own DNA with methyl groups Or can serve in mismatch repairThe sequences of long DNA strands can be determined Electrophoresis (sanger method)

8.4 Other Functions of NucleotidesNucleotides carry chemical energy in cells: 5 OH of ribonucleotide + 1, 2 or 3 phosphates: nucleoside mono, di, triphosphates three phosphates are labeled alpha, beta, gamma beginning with directly attached to ribose hydrolysis of these provides energy (e.g. ATP) Energy: Bond between ribose and alpha phosphate: ester linkage Hydrolysis: 14kJ/mol Alpha-beta and beta-gamma linkages: phosphoanhydrides Hydrolysis: 30 kJ/molAdenine nucleotides are components of many enzyme cofactors Adenosine: doesnt directly participatein primary function, but removal of adenosine results in drastic reduction of activities Most involve binding enegy between enzyme and cofactor or substrate Nucleotide binding fold: domain that binds adenosineSome nucleotides are regulatory molecules: Primary response from hormones or external chemical signal Leads to production of second messengers inside cell lead to adaptive changes in cell interior Often, second messenger is a nucleotide formed from ATP in a reaction catalyzed by cyclase E.g. cAMP (ATP catalyzed by adenylyl cyclase)

Chapter 10: Lipids10.1: Storage Lipids Fatty acids: hydrocarbon derivatives that are highly reducedFatty acids are hydrocarbon derivatives Carboxylic acids with hydrocarbon chains ranging from 4-36 carbons long In some: unbranched and fully saturated In others: one or more double bonds A few: 3C rings, OH groups, methyl-ground branches Nomenclature: chain length:number of double bonds(^location of double bonds) Numbering begins at carboxyl group Most commonly: even number of carbon atoms in unbranched chain of 12-24 carbons Double bond between C-9 and C-10 in monounsaturated fatty acids, and then 12 and 15 Almost never alternates single and double bonds, but instead separated by methylene group In nearly all: doube bonds are cis (trans are produced by fermentation) Omega-3 fatty acids: polyunsaturated fatty acids (PUFAs) with double bond between third and fourth carbon from the METHYL end of chain Omega carbon: methyl carbon farthest from COOH group Omega-6 fatty acids: double bond between C6 and C7 Humans cant synthesize the required omega-2 PUFA linolenic acid (ALA; 18:3(9,12,15)) must obtain in diet From ALA humans can synthesize two other omega-3 PUFAs: eicosapentaenoic acid (EPA; 20:5(5,8,11,14,17)) and docosahexaenoic acid (DHA; 22:6(4,7,10,13,16,19)) Physical properties of fatty acids: largely determined by length and degree of unsaturation of hydrocarbon chain Nonpolar hydrocarbon chain: poor solubility of fatty acids in water Longer + more saturated --> lower solubility COOH is polar and ionized at neutral pH accounts for slight solubility of short-chain fatty acids in water Melting points: Fully saturated: free rotation around each c-c bond: HC chain has great flexibility most stable conformation = fully extended form that can pack together tightly Unsaturated: cis double bond forces kink, prevents packing (weaker interactions) Less thermal energy needed to disorder them LOWER melting pointsTriacylglycerols are fatty acid esters of glycerol: Composed of three fatty acids in ester linkage with single glycerol If all three fatty acids are same: simple triacylglycerol Named after fatty acid they contain (tripalmitin, tristearin, etc) Mixed triacylgerol: contain 2-3 different FA Name and position of each fatty acid specified in nomenclature Polar OH of glycerol and polar COOH of fatty acids bound in ester Therefore, nonpolar and hydrophobic Insoluble in waterTriacylglyercols provide stored energy and insulation: Typically form separate phase of microscopic, oily droplets in aqueous cytosol Serve as depots of metabolic fuel In vertebrates: adipocytes (specialized fat cells) store large amounts of triacylglycerols as fat droplets Lipases: enzymes that catalyze the hydrolysis of stored triacylglycerols release FA for export to sites where they are required as fuel Two advantages to using as stored fuels (over polysaccharides) C of fatty acids are more reduced oxidation yields twice as much energy Triacylglycerols are hydrophobic and unhydrated Organism that carries fat as fuel does not have to carry weight of water Also serve as insulation sometimesPartial hydrogenation of cooking oils produces trans fatty acids Most natural fats: complex mixtures of simple and mixed triacylglycerols Vegetable oils: composed largely of unsaturated fatty acids liquids at room temperature Animal fats: contain mostly saturated white, greasy solids Rancidity: exposure to oxygen oxidative cleavage of double bonds in unsaturated FA Produces aldehydes and COOH acids of shorter chain length and thus higher volatility Partial hydrogenation of commercial vegetable oils increases shelf life Converts cis double bonds to single bonds Increase Tm to make more nearly solid Converts some cis bonds to trans bonds lead to higher CV risk Raise level of LDL (bad) cholesterol and triacylglycerols in blood Increase body;s inflammatory responseWaxes serve as energy stores and water repellants Esters of long-chain (c14 C36) saturated and unsaturated fatty acids with long chain (C16 C30) alcohols Melting points: generally higher than triacylglycerols Chief storage form of metabolic fuel for plankton Also serve to lubricate and keep hair and skin waterproof (e.g. birds)10.2 Structural Lipids in Membranes Membrane lipids are amphipathic Five general types: Glycerophospholipids (two fatty acids joined to glycerol) Galactolipids and sulfolipids (two fatty acids esterified to glycerol but no phosphate) Archaeal tetraethet lipids (two very long alkyk chains ether linked to glycerol at both ends) Sphingolipids (single fatty acid joined to fatty amine [sphingosine]) Sterols (rigid system of four fused hydrocarbon rings Phospholipids: glycerophopsholipids and some sphingolipids where polar head group is joined to hydrophobic moietry by phosphodiester linkage Glycolipids: sphingolipids lacking phosphate but having simple sugar or complex oligosaccharides at polar endsGlycerophospholipids are derivatives of phosphatidic acid Also called phosphoglycerides Membrane lipids Two fatty acids esterified to first and second carbons of glycerol Highly polar or charged group attached through phosphodiester linkage to the third carbon Phosphate group bears negative charge at neutral pH Named as derivatives of parent compound (phosphatidic acid) according to polar alcohol in head group E.g. phosphatidylcholine and phosphatidylethanolamine Fatty acids are variable: given phospholipid may consist of several molecular species, each with own complement of fatty acidsSome glycerophospholipids have ether-linked fatty acids Ether lipids: one of the two acyl chains is attached to glycerol in ether, rather than ester, linkage Ether-linked chain can be saturated (alkyl ether lipids) or contain a double bond between C1 and 2 (plasmalogens) Platelet-activating factor: ether lipid that is a potent molecular signalChloroplasts contain galactolipids and sulfolipids Galactolipids: one or two galactose residues are connected by glycosidic linkage to C3 of 1,2-diacylglycerol Localized in thylakoid membranes of chloroplasts Sulfolipids: sulfonate on head group (bears negative charge)Archaea contain unique membrane lipids Archaea mostly live in extreme conditions Membrane lipids containing long chain branched hydrocarbons linked at each end to glycerol Linkages are through ether bonds much more stable to hydrolysis at low pH and high temperature than ester Twice the length of phospholipids and sphingolipids Glycerol moiety: central carbon is in R configuration in Archaea and in S configuration in Bacteria and EukaryaSphingolipids are derivatives of sphingosine Also have polar head group and two nonpolar tails Contain NO glycerol one molecule of long-chain amino alcohol sphinosine or one of its derivatives, one molecule of long chain fatty acid, and polar head group (glycosidic or phosphodiester linkage) Ceramide: structural parent of all sphingolipids Fatty acid attached in amide linkage to NH2 on C2 Structurally similar to a diacylglycerol Three subclasses of sphingolipids: all derivatives of ceramide Sphingomyelins: Phosphorcholine or phosphoethanolamine as polar head group Classified along with glycerophospholipids as phospholipids Resemble phosphotidylcholines in general properties and three-dimensional structure + having no net charge on head groups Particularly present in plasma membranes of animal cells and in myelin Glycosphingolipids: Outer face of plasma membranes Head groups with one or more sugars connected directly to the OH at C1 of the ceramide moiety Do not contain phosphate Cerebrosides: Single sugar linked to ceramide (glucose or galactose) Globosides: Glycosphingolipids with two or more sugars (usually D-glucose, D-galactose or N-acetyl-D-galactosamine Gangliosides: Oligosaccharides as polar head groups and one or more residues of N-acetylneuraminic acid (Neu5Ac; a sialic acid) at termini sialic acid gives negative charge at pH 7 distinguishes them from globosidesSphingolipids at cell surfaces are sites of biological recognition humans: at least 60 diff sphingolipids especially prominent in plasma membrane of neurons some are clearly recognition sites on cell surface carb moieties of certain sphingolipids: determine human blood typePhospholipids and sphingolipids are degraded in lysosomes most cells continually degrade and replace membrane lipids for each hydrolysable bond in glycerophospholipid: specific hydrolytic enzyme in lysosome Phosholipase A: remove one of the two fatty acids (yields lysophospholipid) Lysophospholipases: remove other fatty acid Phospholipases C and D: each split one of the phosphodiester bonds in the head group genetic defect in enzymes accumulation medical consequencesSterols have four fused carbon rings: sterol nucleus: four fused rings: three with 6 carbons and one with five almost planar and relatively rigid Cholesterol: major sterol in animal tissues: Amphipathic: polar head group (OH at C3) and nonpolar hydrocarbon body (steroid nucleus and HC side chain at C17) Stigmasterol: plants Ergosterol: fungi BACTERIA CANNOT SYNTHESIZE STEROLS Sterols of all eukaryotes: synthesized from simple five-carbon isoprene subunits (like fat soluble vitamins)10.3: Lipids as Signals, Cofactors and PigmentsPhosphatidylinositols and sphingosine derivatives act as intracellular signalsEicosanoids carry messages to nearby cells: Paracrine hormones (act on cells near point of hormone synthesis) All derviced from arachidonic acid (20:4(5,8,11,14)) Three general classes: Prostaglandins 5C ring originating from chain of arachidonic acid two groups: PGE (ether soluble) - nonpolar PGF (phosfate buffer soluble) Functions: stimulate contraction of smooth muscle of uterus, affect blood flow to specific organs, wake sleep cycle, responsiveness to epinephrine and glucagon Also elevate body temperature and cause inflammation and pain Thrombaxanes: 6 member ring containing an ether produced by platelets and act in blood clotting and reduction of blood flow to site of clot NSAIDS: inhibit prostaglandin H2 synthase catalyzes early step from arachidonate prostaglandins and thomboxanes Leukotrienes First found in leukocytes Contain three conjugated double bonds Contraction of smooth muscle to lung (can cause asthma if overproduced)Steroid hormones carry messages between tissues Steroids: oxidized derivatives of sterols Have sterol nucleus but lack alkyl chain More polar than cholesterol Move through blood on protein carriers to target tissues, where they enter cells and bind to highly specific receptor protein in nucleus Effective at very low concentrations due to high affinity for receptors Major groups: Male and female sec hormones Adrenal cortex hormones (cortisol and aldosterone) Steroid drugs (prednisone and prednisolone) Brassinolide (vascular plants, growth regulator)Vascular plants produce thousands of volatile signals Used to attract pollinators, repel herbivores, attract defenders, communicate with other plantsVitamins A and D are hormone precursors Vitamins: compounds essential to health of animal that must be obtained in diet Two classes: Fat-soluble A, D, E and K A and D hormone precursors D3: UV-driven conversion of 7-dehydrocholesterol in skin Converted into 1,25-dihydroxycholecalciferol (calcitriol) in liver and kidney Hormone that regulates calcium uptake in intestine and calcium levels in kidney and bone Deficiency: defective bone formation and rickets D2: same biological effect, commonly added to milk and butter as dietary supplement A (retinol): functions as hormone and visual pigment of vertebrate eye Vitamin A derivative retinoic acid regulates gene expression in development of epithelial skin Vitamine A derivative retinal: pigment that initiates the response of rod and cone cells of the retina to light producing neuronal signal to brain In vertebrates: B-carotene can be converted into vitamin A E and K and lipid quinones are Redox cofactors E: trocopherols substituted aromatic ring + long isoprenoid side chain Hydrophobic associate with cell membranes, lipid deposits and lipoproteins in blood Biological antioxidants Aromatic ring reacts with and destroys ROS protect unsat FA from ox and prevent ox damage to membrane lipids K: aromatic ring which undergoes cycle of oxidation and reduction in formation of active prothrombin (blood clotting factor) Ubiquinone (coenzyme Q) and plastoquinone: isoprenoids Function as lipophilic electron carriers in ETC Both can accept one or two electrons and either one or two protons Water-solubleMany natural pigments are lipid dienes: Conjugated dienes: carbon chains with alternating single and double bonds Allows for delocalization of electrons compounds can be excited by low-energy EMR gives visible colorsPolyketides Secondary metabolites that are not central to organisms metabolism but offer producers an advantage in some ecological niche

Chapter 11: Biological Membranes and TransportComposition and Architecture of MembranesEach type of membrane has characteristic lipids and proteins Relative proportions of protein and lipid vary with type of membrane E.g. nerves have myelin more lipid E.g. bacteria and mitochondria more protein Type of lipids and proteins also varies from membrane to membrane Some membrane proteins are covalently attached to one or more lipids serve as hydrophobic anchors that hold proteins to membraneAll biological membranes share some fundamental properties Impermeable to most polar or charge solutes but permeable to nonpolar compounds 5-8nm (50-80A) thick appear trilaminar in cross section fluid mosaic model phospholipid bilayer embedded with proteins (held by hydrophobic interactions between membrane lipids and hydrophobic domains on proteins) protein orientation is asymmetric: sidedness of membrane molecules are free to move laterally in plane of membraneA lipid bilayer is the basic structural element of membranes glycerophospholipids, sphingolipids, sterols: insoluble in water in water, will spontaneously form lipid aggregates to minimize surface area exposed to water driving force: increase entropy at lipid-water interface three types of aggregates can form: micelles: spherical structures that contain anywhere from a few dozen to a few thousand amphipathic molecules favored when cross sectional area of head is greater than that of acyl side chain (like free fatty acids, lysophospholipids, detergents) bilayer: two lipid monolayers form two-dimensional sheet favored if cross section area of head and tails are similar (like glycerophospholipids and sphingolipids) relatively unstable and spontaneously folds back on self to form vesicles 3 nm (30A) thick vesicle: formed as result of interaction of hydrophobic edges of bilayer with water asymmetry of plasma membrane: erythrocyte: choline-containing lipids (phosphatidylcholine and sphingomyelin) typically found on outer leaflet while phosphatidylserine, phosphatidylethanolamine and phosphoatidyinositols are more common on inner leafletThree types of membrane proteins differ in their association with the membrane Integral membrane proteins: firmly associated with lipid bilayer Only removable by agents that interfere with hydrophobic interactions Peripheral membrane proteins: associate with membrane through electrostatic interactions and hydrogen bonding with hydrophilic domains of integral proteins and polar head groups of membrane lipids Removable by relatively mild treatments that interfere with electrostatic interactions or h-bonds E.g. carbonate at high pH Amphitropic proteins: found both in cytosol and in association with membranes Noncovalent interaction with a membrane protein or lipid OR presence of one or more lipids covalently attached to amphitropic protein Reversible and regulated association: e.g. by phosphorylationMany membrane proteins span the lipid bilayer Localization of protein domans relative to lipid bilayer: membrane protein topology Determined with reagents that react with protein side chains but cannot cross plasma membranes Glycophorin: erythrocyte glycoprotein that spans plasma membrane Amino terminal: on outer surface (bears carb chains) Carboxyl terminus insude of cell Both terminals are charged or polar and are both hydrophilic Transmembrane segment is hydrophobic Each protein has specific orientation in bilayer, giving membrane distinct sidedness Glycoproteins: glycosylated domains are invariably found on outer faceIntegral Proteins are held in the membrane by hydrophobic interactions with lipids Attachment of integral proteins to membrane: result of hydrophobic interactions between membrane lipids and hydrophobic domains of protein 6 types: Types I and II: single transmembrane helix Amino terminal outside in Type I Amino terminal inside in Type II Type III: multiple transmembrane helices in one single polypeptide Type IV: transmembrane domains of several polypeptides form channel through membrane Type V: held to bilayer primarily by covalently linked lipids Type VI: have both transmembrane helices and lipid anchors Annular lipids: phospholipid molecules that lie on protein surface, head groups interacting with polar amino acid residues at inner and outer membrane water interfaces and side chains associated with nonpolar residues Form bilayer shell around protein Found at interfaces between monomers of multisubunit membrane proteins form grease shell Embedded deep within membrane protein with head group well below plane of bilayerThe topology of an integral membrane protein can sometimes be predicted from its structure Presence of unbroken sequences of more than 20 hydropobic residuces: evidence of transmembrane domain A-helical sequence of 20-25 residues: just long enough to span thickness of bilayer Polypeptide chain surrounded by lipids with no water molecules to bond to will tend to form a helices or B sheets to maximize intrachain h-bonding If side chains of helix are all nonpolar, hydrophobic interactions with surrounding lipids further stabilize helix Hydropathy index: used to find regions of 20 or more hydrophobic residues Overall hydrophobicity of sequence of amino acids estimated by summing hydropathy indices calculated for successive segments of given size (e.g. 1-7, 2-8, 3-9) Presence of Tyr and Trp residues at interface between water and lipid Side chains serve as membrane interface anchors: able to interact simultaneously with central lipid phase and peripheral aqueous phase Positive-inside rule: positively charged Lys, His and Arg residues: occur more commonly on cytoplasmic face of membranes B-barrel: common in bacterial membrane proteins 20 or more transmembrane segments form B sheets that line cylinder stabilized by same factors as alpha-helix porins: proteins that allow certain polar solutes to cross outer membrane of gram (-) bacteria have many-stranded B-barrels lining polar transmembrane passage more extended than a-helix: only 7-9 residues needed to span membrane every other residue is hydrophobic and interacts with lipid bilayer other residues may or may not be hydrophobic at lipid-protein interface: aromatic side chainsCovalently attached lipids anchor some membrane proteins some membrane proteins contain one or more covalently linked lipids of one of several types: long-chain fatty acids isoprenoids sterols glycosylated derivatives of phopsphatidylinositol (GPIs) provides hydrophobic anchor that inserts into lipid bilayer and holds protein at membrane surface strength of hydrophobic interaction between bilayer and single hydrocarbon chain barely enough to anchor protein securely most proteins have more than one lipid moiety other interactions (ionic attractions between positive Lys and negative lipid head groups) contribute to stabilitiy integral proteins: treatment with alkaline carbonate does not release them however, weaker than that for other integral membrane proteins and reversible11.2 Membrane Dynamics: noncovalent interactions among lipids in bilayer ability to change shape without becoming leakyAcyl groups in the bilayer interior are ordered to varying degrees structure and flexibility of lipid bilayer: depend on kinds of lipids present at physiological range: long chain saturated fatty acids pack together liquid ordered state long chain unsaturated fatty acids have kinks liquid disordered state shorter chain fatty acyl groups: same effect changes in temperature below normal physiological temp: lipids form semisolid gel phase all types of motion of individual lipid molecules are strongly constrained bilayer is paracrystalline above: fluid, liquid-disordered state individual hydrocarbon chains of fatty acids are in constant motion (rotation about carbon-carbon bonds of long acyl side chains interior of bilayer is more fluid than solid intermediate: liquid ordered state less thermal motion in acyl chains but lateral movement still takes place sterol content: rigid planar structure of steroid nucleus reduces freedom of neighbouring acyl chains to move by rotation forces chains into fully extended conformation reduces fluidity in core of bilayer liquid ordered phase increases thickness of lipid leafletTransbilayer movement of lipids requires catalysis transbilayer (flip-flop) diffusion of a lipid molecules from one leaflet of bilayer to other occurs slowly if at all lateral diffusion, on other hand, is very rapid transbilyer movement: requires that polar or charged head group leave aqueous environment and move into hydrophobic interior of bilayer large, positive G however, necessary: in ER, glycerophospholipids are synthesized on cytosolic surface, sphingolipids synthesized on luminal surface flip flop diffusion Flippases, Floppases, Scramblases: facilitate the transbilayer movement of lipids Flippases: catalyze translocation of aminophospholipids phosphotidylethanolamine and phosphatidylserine from extracellular cytosolic Phosphatidylethanolamine and phosphatidylserine: primarily in cytosolic leaflet Phosphatidylserine Sphingolipids and phosphatidylcholine in outer leaflet Also act in ER to move newly synthesized phospholipids from site of synthesis in cytosolic leaflet to luminal leaflet One ATP per molecule of phospholipid transported structurally and functionally related to P-type ATP-ases Floppases: phospholipids FROM cytosolic TO extracellular leaflet ATP-dependent Members of ABC transporter family Scramblases: move any membrane phospholipid across bilayer down its [] gradient NOT ATP-dependent Controlled randomization of head-group composition on two faces of bilayer Activity rises sharply with increase in cytosolic Ca2+ concentration Results from cell activation, injury or apoptosis could trigger apoptosis by bringing phosphatidylserine to outer face Phosphatidylinositol lipids: move phosphatidylinositol lipids across lipid bilayers for lipid signaling and membrane traffickingLipids and proteins diffuse laterally in the bilayer Brownian motion within bilayer lipid molecules moving laterally by changing places with neighboring lipid molecules Rapid Tends to randomize the positions of individual molecules in a few seconds FRAP: rate of fluorescence recovery after photobleaching Measure of rate of lateral diffusion of lipids Single particle tracking: allows one to follow the movement of a single lipid molecule on shorter time scale Movement inhibited to small discrete regions, as though corralled by fences that can occasionally be hopped (hop diffusion) Membrane proteins: free to diffuse laterally in plane of bilayer and are in constant motion (also shown by FRAP) Some membrane proteins associate to form large aggregates (patches) on surface of cell or organelle in which individual protein molecules do not move relative to one another Some are anchored to internal structures that prevent free diffusion Immobilization may form the fences that corral lipidsSphingolipids and Cholesterol cluster together in membrane rafts Glycosphingolipids (cerebrosides and gangliosides) typically contain long-chain saturated fatty acids Form transient clusters in outer leaflet that large exclude glycerophospholipids (one unsat fatty acyl + shorter saturated acyl group) Sphingolipids: long saturated acyl groups can form more compact, more stable associations with long ring system of cholesterol than shorter, often unsat chains of phospholipids Cholesterol-sphingolipid microdomains in outer monolayer of plasma membrane are slightly thicker and more ordered (less fluid) than neighboring microdomains rich in phospholipids More difficult to dissolve with nonionic detergents Behave like liquid-ordered sphingolipid rafts adrift on ocean of lipid disordered phospholipids Enriched in two classes of integral membrane proteins: those anchored to membrane by two covalently attached long-chain saturated fatty acids GPI anchored protins Lipid anchors form more stable associations with cholesterol and long acyl groups in rafts than with surrounding phospholipids Membrane proteins can move into and out of rafts rapidly Likely that segregation is functionally significant: for example, getting two proteins that need to interact to be more likely to do so Calveolin: integral membrane protein two globular domains connected by hairpin shaped hydrophobic domain, which binds protein to cytoplasmic leaflet of plasma membrane three palmitoyl groups attached to carboxyl terminal globular domain further anchor it to the membrane binds cholesterol in the membrane forces lipid bilayer to curve inward, forming caveolae unusual rats: involve both leaflets of the bilayer (cytoplasmic and extracellular) implicated in variety of cellular functions (membrane trafficking, transduction of external signals into cellular responsesMembrane curvature and fusion are central to many biological processes three mechanisms for inducing membrane curvature: protein that is intrinsically curved forces bilaryer to curve by binding to it binding energy provides the driving force for the increase in bilayer curvature many subunits of scaffold protein assemble into curved supramolecular complexes and stabilize curves that spontaneously form in the bilayer protein inserts one or more hydrophobic helices into one face of the bilayer, expanding area relative to the other face and forcing curvature specific fusion of two membranes: requires: recognize one another surfaces become closely apposed (involves removal of water normally associated with polar head groups of lipids) bilayer structures become locally disrupted results in fusion of outer leaflet of each membrane in receptor mediated endocytosis: process must be triggered at appropriate time or in response to a specific signal fusion proteins: integral proteins that mediate fusion events example: intracellular vesicles at synapses involves family of SNAREs v-SNAREs: cytoplasmic face of intracellular vesicle t-SNAREs: target membrane with which vesicle fuses SNAP25 and NSF also involved During fusion: vSNARE and tSNARE bind and undergo structural change produces bundle of long ting rods made up of helices from both SNARES and two helices from SNAP25 Two SNARES initially interact at their ends and then zip up into bundle of helices, pulling membranes into contact and initiation bilayer fusion Clostridium botulinum: protease that cleaves specific bonds in SNARES and SNAP2511.3 Solute Transport Across MembranesPassive Transport is Facilitated by Membrane Proteins Simple diffusion you know what this is Membrane potential = Vm force that drives simpl diffusion Electrochemical gradient Polar or charged solute must first give up interations with water molecules in hydration shell, then diffuse through lipids (n which it is insoluble) energy lost is regained on other side Activation barrier exists: can be large enough to prevent simple diffusion from occurring Facilitated diffusion: activation energy lowered by membrane proteins (transporters or permeases) Bind substrates with stereochemical specificity through multiple weak, noncovalent interactions Negative Gbinding of these weak interactions counterbalances positive Gdehydration, thereby lower G for transmembrane passage Transporters: span lipid bilayer sevel times, forming transmembrane channel lined with hydrophilic amino acid side chains Transporters can be grouped into superfamilies based on structures Carriers: bind substrates with high stereospecificity Catalyze transport at rates well below limits of free diffusion Saturable in the same sense as enzyme Monomeric proteins Two families: Passive transporter simply facilitate diffusion down a concentration gradient Active transporter can drive substrates across membrane against concentration gradient Use energy provided directly from chemical reaction (primary active transporters) or couple with something (secondary) Channels: generally allow transmembrane movement at rate orders of magnitude greater than carrier (approaching unhindered diffusion) Show less stereospecificity than carriers Are not saturable Most are oligomeric complexes of several identical subunitsThe glucose transporter of erythrocytes mediates passive transport Type III integral protein with 12 helices Analogous to enzyme, where glucose outside = substrate, and glucose inside = product Glucose binds outside cell, conformational switch, glucose exits inside cell PASSIVE: down concentration gradient Can use michaelis-menten energetics to solve for Vmax UNIPORT: only one substrateThe chloride-bicarbonate exchanger catalyzes electroneutral cotransport of anions across the plasma membrane COTRANSPORT SYSTEM Anion exchange protein (AE protein) increases rate of HCO3 transport across erythrocyte membrane Integral protein that spans membrane at least 12 times Mediates simultaneous movement of two anions: for each HCO3- ion that moves in one direction, one Cl- ion moves in the opposite direction ELECTRONEUTRAL ANTIPORT: moving in opposite directions Symport is moving in same directionsActive transport results in solute movement against a concentration or electrochemical gradient Thermodynamically unfavorable Takes place only when couple to exergonic process Primary active transport: solute accumulation coupled directly to exergonic chemical reaction (like ATP hydrolysis) Secondary active transport: endergonic transport of one solute is coupled to exergonic flow of different solute that was originally pumped uphill by primary active transport Amount of energy needed: calculated by the initial concentration gradient Free energy of transport of uncharged molecule from area of []C1 to []C2G = G + RTln(C2/C1) When solute is an ion, movement without accompanying counterion endergonic separation of positive and negative charges produces electrical potential ElectrogenicGt = RTln(C2/C1) + ZFpsi (where Z = charge on ion, F = Faradays, psi = transmembrane electrical potential in volts)P-Type ATPases undergo phosphorylation during catalytic cycles Cation transporters that are reversibly phosphorylated by ATP Phosphorylation forces conformational change that is central to movement of the cation across the membrane Ca2+ ATPase (uniporter for Ca2+ ions) and Na+K+ ATPase are P-type Plasma membrane Ca2+ pump: maintains low concentration of Ca2+ in cytosol of virtually all cells Moves calcium ions out of cell SERCA pumps: sarcoplasmic and endoplasmic reticulum calcium pumps: P-type ATPases closely related in structure and mechanism Single polypeptide that spans membrane 10 times Three cytosolic domains formed by long loops that connect transmembrane helices N domain: nucleotide ATP and Mg2+ bind P domain: phosphorylated Asp residue characteristic of P-type pumps Does not directly affect Ca2+ binding A domain: actuator domain (communicates movement of N and P domains to the two Ca2+ binding sites) M domain: contains transmembrane helices and Ca2+ binding sites Mechanism: E1 conformation: two Ca2+ binding sites are exposed on cytosolic side of ER or SR and bind Ca2+ with high affinity ATP binding and Asp phosphorylation conformational change from E1 E2 E2: Ca2+ binding sites are now exposed on luminal side of membrane and affinity for Ca2+ is greatly reduced Ca2+ release into lumen Dephosphorylation returns to E1F-Type ATPases: reversible, ATP-driven proton pumps Energy coupling factors Fo integral membrane protein complex transmembrane pathway for protons Fi uses energy of ATP to drive protons uphill into higher H+ region Reversible reaction Proton gradient can supply the energy for ATP synthesis (reverse reaction) ATP synthaseV-type ATPases Class of proton-transporting ATPases structurally and possible mechanistically related to F-type Responsible for acidifying intracellular compartments in many organisms Similar complex structure: integral domain (Vo) that serves as proton channel and peripheral domain (Vi) that contains ATP binding site and ATPase activityABC transporters use ATP to drive the active transport of a wide variety of substrates Pump amino acids, peptides, protons, metal ions, etc etc out of cell against [] gradient MDR1: multi-drug transporter: responsible for tumor resistance to drugs Broad substrate specificity for hydrophobic compounds Pumps drugs out of cells and prevents therapeutic effects Integral membrane protein with 12 transmembrane segments and 2 ATP binding domains All ABC transporters: two nucleotide binding domains (NBD) and two transmembrane domains Sometimes: all are in single long polypeptide Others: two subunit, each with 1 NBD and 6 helices Most act as pumps, but at least some are ion channels ATP motors When coupled with a pump, ATP driven motor moves solutes against [] gradient When coupled with ion channel, motor open and closes channelIon Gradients provide energy for secondary active transport Ion gradients formed by primary transport of Na+ or H+ can provide driving force for cotransport of other solutes Lactose transporter (lactose permease): proton driven cotransporter Single polypeptide chain that functions as monomer to transport one proton and one lactose molecule into cell with net accumulation of lactose Lactose transporter provides route for proton reentry, simultaneously carrying lactose into cell by symport 12 transmembrane helices and connecting loops protrude into cytoplasm or periplasmic space large cavity on cytoplasmic side of membrane, where substrates bind side facing outward: closed tightly, no channel big enough for lactose to enter proposed mechanism: rocking motion between two domains, driven by substrate binding and proton movement, alternatively exposing substrating binding domain to cytoplasm and periplasm