Light reactionsConvert light energy into chemical energy stored in NADPH and ATPWater is the initial e- donor to the electron transport chain = water gets oxidized to O2Dark reactionsThe NADPH and ATP from light reaction used to make carbs from CO2Pathway for electron transport in chloroplast thylakoidTravels from photosystem 2 to 1H2O -> PSII -> PQ (lipid) ->ctyb6/f -> PC (peripheral protein) -> PSI -> NADP+ & H+ -> NADPHH2O is split in lumen to generate H+ in the lumenLumen is acidifiedProduct is NADPH and proton gradientStages of e- flow in photosynthesis1. Photolysis – PSII using energy from light to split water -> 3 productsH2O -> H+ (for H+ gradient) & O2 (for mitochondria) & e- (for e- transport)2. Pass e- from PSII to PSI – energy generated -> increases [H+] in the lumen3. Pass e- from PSI to NADP+ - produces NADPHelectron from water is used to start this process, not from the sunsunlight is used to lyse (break) H2OThe “Z-scheme” for e- transport in thylakoidHigh reduction potential -> low reduction potential back and forthCannot go from high to low to pass e-Have to lower reduction potential in order to pass e-Light does thisP680 – part of PSIIP700 is part of PSI1. Light provides the energy to split water and to lower the Eo value of P680(to excited state)2. The excited state of P680 now is low enough to pass e- to PQ but now the Eo of P700 is too high to pass e- furtherlight energy at PSI is needed to lower the Eo of P700 so it can continue to pass e- to NADP+ to make NADPHSummary of Light reactions1. PSII (P680) uses light energy to split water by photolysis: H2O-> H+ & O2 & e-the H+ contributes to the H+ gradient in the lumenthe e- are passed to the e- transport chain and provides energy or more H+ pumping2. Electrons passed to PQ and H+ go to lumen to increase [H+] in lumen3. Cytb6/f complex accepts e- and pumps more H+ into the lumen4. E- passed to PC and PSI (P700)5. E- passed to NADP+ to make NADPH6. Energy from H+ gradient is used to ATP in the stromaplants need water, light, and CO2 to growlight to split water for energyMitochondria and chloroplast e- transportMitochondria – get proton gradientChloroplast – get proton gradient & NADPHSome inhibitors of photosynthesis1. DCMU – blocks e- transport from PSII to PQsteals electronit is an artificial electron acceptor2. Altrazine (herbicide) – blocks e- transport from PSII to PQ3. DCIP – artificial e- acceptor that steals e- from PQ4. Paraquat (herbicide) – steals e- from PSI so that NADP+ doesn’t get reduced to NADPH.The CF1/CF0 can make ATP in vitro (artificially)1. Put thylakoids in pH 4 -> CF1 faces outside = lower H+ insidepmf is inward2. Wait until pH is 4 on inside – load up H+ insidepH = 4 = high H+ inside3. Move thylakoids to pH 7 and add ADP+Pihigh H+ in and low H+ outsidepmf is outward now -> H+ gradient provides energy for ATP synthesise- transport is not needed for ATP synthesis in vitroHow do thylakoid membranes harvest light to make NADPH and ATP5 results of light shining on pigment1. Emitted as heat2. Emitted as fluorescence – absorb as short wavelength and released as longer wavelength (low energy)3. Transmitted – not absorbed, goes straight throughno change in wavelength4. Passed along to another energy carrier5. Reflectednot 100% harvesting of lightchlorophyll are fluorescent but chloroplast are not – pigment in thylakoid will pass the energy to e- transport and not emittedas chloroplast becomes fluorescent = dyingbecause it has no one to pass the energy toAbsorption spectrumLess light transmitted = more light absorbedChlorophyll a – very short wavelength = best absorptionChlorophyll b – short wavelength = best absorptionB-carotene – not orange = best absorptionHigh energy = low wavelengthGreen light on green plant = deathBecause all the light will be reflected = no light absorptionChlorophyllPigment is amphipathic – hydrophobic tail, hydrophilic headProtein analysisElectrophoresis – movement of fully charged (ionic) compounds through a viscous medium by an electric fieldOpposite charges attract each otherAnode = positive pole, Cathode = negative poleAnion -> anode, Cation -> cathodeUsed to separate, purify, and identify RNA and DNA2 main types of gel electrophoresis1. native gel – no detergents addedmost like in vivorate of movement (migration) is affected by the native conformation(shape), size, solubility, and chargepH can affect migration2. Detergent (usually SDS) gelsdetergent gets rid of the shape, everything is soluble -> Assume migration is only affected by sizeHow to look at membrane proteins by SDS-PAGE1. SDS – sodium dodecyl sulfate – anionic detergent at most pH used2. Separates proteins by their sizesmall proteins move through gel fasterassume that all proteins are denatured, uniformly negatively charge, soluble in presence of SDSwith no detergent present – proteins could have a net charge of positive, negative, or zero and be in their native conformation, depending on their pHIdentification and separation of membrane proteins by SDS-PAGE1. SDS is an anionic detergent that solubilizes and coats all proteins with a negative charge2. PAGE – polyacrylamide gel electrophoresis3. In presence of SDS – all proteins migrate to anode (positive pole)4. In SDS-PAGE, protein of same size move together in a bandLarge proteins move slower = top of the gel, farther from the anodeSmall proteins move faster = bottom of the gel, closer to the anode5. Protein bands are stained to be seen and identified by co-migration with known standards(MW)size is expressed as daltons (Da). 1000Da = 1000MWHow to get a membrane protein out of the membrane and solubilized in wateradd ionic detergent like SDSRBC “ghosts” – just plasma membranesMade by hemolysis (Hypotonic lysis) – cells pop and reseal after osmotic shockGeneral features of cytoskeletal components1. Assembled from a pool of protein subunitssubunits -> polymerization -> polymers2. Some polymers (filaments) are dynamic – always changing by assembly and disassembly – easy to change3 main kinds of cytoskeletal filaments1. Microtubules (MT)– biggesttubulin subunits are dimers – joining of 2alpha and beta tubulin – form tubulin heterodimeruse GTPmade in the + end (growing end) – polarityin all eukaryotesfunction – support intracellular transport2. Intermediate filaments (IF) – intermediatemade of polymers of intermediate filamentsmade internal – non polarin all animalsfunction –