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Western University
Biology 1002B
Tom Haffie

Biology 1002B Lecture Outcomes Lecture 1: Chlamydomonas:  single celled, green algae  a sexually active, light-harvesting, carbon-reducing Evolution of Complexity: A eukaryotic cell is much bigger than a bacteria cell, so it should be more complex. Multicellularity is a big thing. Multicellularity is different tissues and different types of cells. Each of these green cells are of different species. The last one is volvox, it has 50, 000 cells. The cells like Chlamy are found on the outside, they are somatic cells. There are reproductive cells on the inside. Chlamy is the first one. Eyespot: This is required for phototaxis (movement towards or away from light). The eyespot just senses photons, it does not produce an image. Eye and Eyespot share a common ancestor, this is homology. For the eye, neurons sit on the retina. We have 6 million cones and 120 million rods. Phototransduction PROCESS:  The photoreceptor gets hit by a photon of light  Then the shape changes  Changes from cis-retinal to trans-retinal  This activates a protein called tranducin  This in turn activates the enzyme phosphoditerase  This enzyme cleaves the 3' bond, the phosphate is only bound to the 5' position  There is a sodium channel on the outer membranes of the photo receptor cell. When you shut off sodium transportation it hyperpolarizes the membrane  This causes an electrical current pulse down the membrane of the photoreceptor cell This is one reaction. Your optical nerve gets 200 billion of these reactions every second, that's how complex it is. Phototaxis is a direct response in response to the presence of light in organism, where a positive effect sees the organism move towards the light and a negative one away from the light. In the mechanism underlying the phototactic behavior of Chlamydomonas, Ca2+ has been thought to control the dominance between the two flagella so as to steer the cell to correct directions. Chlamydomonas would move away from a light source because the presence of light has a negative effect on it. This is to minimize photodamage. Basic structure of Rods and Cones: Lecture 2 Light behaves as a wave. Gamma Rays have shorter wavelengths and Radio waves have longer wavelengths. The energy of the light is inversely related to its wavelength. The shorter the wavelength, the more energy the wave has. There is more energy in blue light (shorter wavelength) then there is in red light. Discrete packages of light is called quanta (photons). If a biological system is going to use energy it has to absorb light The molecules that absorb them are usually pigment, like chlorophyll. What makes pigments absorb photons of light? Conjugated system (double bond-single bond-double bond) This conjugated system indicates a specific kind of electron configuration. These represent an abundance of electrons, they are non bonding, pi orbital. They are readily available to be excited by a photon of light. It is those electrons that are going to interact with the photons of light, because they are not used for bonding. There is some exceptions, like Retinal. Retinal involves bonding electrons. Pigments aren't free, there is a protein that it is non covalently bonded to. This is to produce pigment-protein complexes. Pigments are bound non covalently to the protein. Chlorophyll can absorb blue light. If an electron absorbs a blue photon of light, that is enough for it to go to the higher excited state. You lose energy as heat when the electron goes back down a level. When a red photon of light is absorbed, it only gets it to the lower energy level that the blue goes to after. Weather you absorb blue or red, they end up at the same lower level. The remaining energy, emission of light, is called fluorescence. Since we lost a little energy as heat, the wavelength is longer so it is a different red. You can do work with light. The work is photochemistry, we use energy to change the molecule, the structure of the pigment. You can also transfer the energy to a neighboring pigment. Why is chlorophyll green? Chlorophyll is green because it absorbs every other color of light. When it is hit with green light, it is reflected, so we see it as green. There is no green excited state, nothing between red and blue, if there was it would be able to absorb green light. The photon is either reflected or it goes through that pigment. One photon can excite one electron. When the electron goes to the lower excited state, heat is released. This is the state it would be in if the photon absorbed red light. If you shine blue or red light on chlorophyll, the energy available is always at a lower excited state. 4 fates (competing processes):  HEAT: Energy can be lost as heat, electron returns to ground state  Lose just a little energy as heat (sub-excited state); lose rest of energy as fluorescence Fluorescence is always deep red, slightly longer wavelengths than normal red light (to account for the fact that you have lost some energy as heat)  Photochemistry → light energy used to do work  Energy transfer to another molecule High absorption in blue, less in the red The pigment can only absorb a photon if the energies of the photon and the pigment match. Phototransduction Versus Photosynthesis Photochemistry is essential for both of these. If we look at the rods and the cones, where is the photochemistry taking place? It is occurring in the photoreceptor. (The blue thing in the diagram). The photon is driving (isomerisation) of retinal. Photoreceptors o Located in membrane of discs in rods/cones in the eye o Photochemistry is the isomerization of retinal o If light is absorbed in the eye, it affects retinal. Photosystems o For photosynthesis, the unit of photochemistry is a photosystem (comprised of an antenna and a reaction center) o If light is absorbed during photosynthesis, energy transfer occurs in the antenna of the photosystem (excitation energy can transfer) o Energy can be transferred between pigments (pigments are close together to allow energy transfer). Electrons remain where they are (this is not photochemistry because the chlorophyll molecules do not change). o Chlorophyll is oxidized and an electron is released. This electron is used for electron transport. Chl+ + Chl --> Chl + Chl+ Photon transfer is occurring in the antenna. The excitation moves. Light Absorption and Emission Chlorophyll has two major excited states (higher and lower). Chlorophyll can absorb blue light; because there is more energy in blue light, the electron is excited to the higher excited state. One photon can excite one electron → photochemical equivalence Some energy is lost to heat; electron moves to lower excited state Chlorophyll can also absorb red light; since there is less energy in red light, the electron is excited to the lower excited state. Energy is ALWAYS at lower excited state, regardless of whether chlorophyll absorbs red or blue light Rhodopsin = retinal and opsin  Protein-pigment complex (pigment → retinal, protein → opsin)  When a retinal molecule absorbs a photon, it changes its geometric orientation (from cis to trans). Normally, the double bond prevents rotation. Photoisomerization is when light is absorbed, the retinal is changed from cis to trans molecules. The energy from light breaks the double bond, allowing the single bond to swivel, and when the molecule switches forms, the double bond reforms. This is photochemistry with retinal (which takes place in the eye). Retinal Isomerization Linked to Opsin Change o For the single-transduction pathway, transducin needs to interact with opsin protein o There is a cleft in opsin that enables G-protein (transducin) to interact and bind  No cleft when 11-cis retinal is bound to opsin  When photoisomerization occurs, the opsin cannot accommodate the trans-retinal (detaches from opsin). This does not occur in a photosystem. In a photoreceptor, opsin and retinal are no longer bound (opsin can’t absorb light without pigment – chromophore/pigment loss)  This changes the shape of the protein (opsin), opening up a cleft, allowing the transducin to interact/bind  Pathway is activated Transducin What’s so special about visible light? Everything that uses light in biology uses visible light. Why?  Visible light is the most dominant form of electromagnetic radiation  Energy in visible light is perfect to excite electrons to higher excited state, and to drive isomerization of retinal. Enough energy in visible light for photochemistry Lecture 3: Protein Structure & Function Proteins are cellular macromolecules made up of amino acid polymers (polypeptides). The sequence of amino acids, or primary structure of the protein, is dictated by the nucleotide sequence of the gene coding for that particular protein. Adjacent amino acids in a polypeptide chain will form stable, recurring arrangements based on steric constraints and weak interactions called the secondary structure. The most common of these are the alpha helix and beta sheet conformations. These segments of the protein then form a specific shape (tertiary structure) held by covalent (eg. disulfide) bonds or weaker interactions between amino acids. Why don’t photoreceptors have antennas? Because there is no need to harvest photons; only photosystems use light to create energy Protein Abundance Points of control: o Regulate transcription (creating mRNA)  Transcript abundance (how much mRNA for the protein is there?)  Dependent on rates of transcription (higher rates of transcription = more protein)  Dependent on how long mRNA is there (mRNA can decay)  Therefore, transcription abundance is a balance between the rate of transcription and the mRNA decay o Regulate translation (mRNA to polypeptide) Measuring Transcription Abundance  Large bands → ribosomal RNA  By use of antibody Heat Shock Response  Making a protein takes longer than transcription  For a gene: o Constitutive – expression does not change o Induced – induction of expression o Repressed – expression is reduced Photoreceptor Abundance A defect in what biochemical process could account for the rgIII mutation? o Mutation in opsin gene (therefore small amounts of opsin are available) o Defect in translation o mRNA decay (translation never occurs) o Protein decay (breaks down faster than normal protein) o No/low amounts of retinal Retinal Biosynthesis Biosynthetic pathway: Retinal is NOT coded by a gene Genes code for enzyme which catalyze reactions to convert molecules (but molecules themselves are not coded for by genes) Protein Folding  For a protein to be functional it has to fold correctly (correct three- dimensional shape)  Urea disrupts tertiary bonding  Protein folding: o Spontaneous, in milliseconds o Dependent solely on primary sequence Lecture 4: Energy & Enzymes Entropy and protein synthesis: Proteins are physical biological molecules, and thus break down over time as well. They are constantly breaking down. This explains the requirement for continuous transcription and translation as a means of replacing “broken” proteins. Chloroplasts and mitochondria require many proteins to keep functional. They have their own DNA, but this encodes for a very small number of proteins. The other proteins they require are translated in the cytosol and imported into the organelle. When we isolate chloroplasts or mitochondria, they lose their supply of imported proteins, which is why their function decays over time. This explains the decay in the photosynthesis rate of isolated chloroplasts. Can the reaction occur spontaneously? Spontaneous reactions are those which occur without the requirement of energy. Spontaneity does not reflect the rate of a reaction. There are two conditions required for a reaction to be spontaneous: 1. The products must have a lower potential energy than the reactants, meaning that a negative change in enthalpy describes the exothermic reaction. 2. The products must be less ordered than the reactants, meaning that the entropy of the system has increased due to the reaction. Gibbs Free Energy Gibbs free energy is the amount of energy we actually can use to do work. It is related to the enthalpy (the total energy content of a system) but not equal to it. ΔG = ΔH - TΔS ΔG = ΔGfinal - ΔGinitial Note that the applying the formula is not required, but understanding the relationship between Gibbs free energy change, enthalpy change, and entropy change, is required. Exergonic reactions are those which have a ΔG < 0 and are therefore spontaneous. For these reactions, the reactants have a higher Gibbs free energy than the products. Endergonic reactions are those which have a ΔG > 0 and are non-spontaneous. For these reactions, the products have a higher Gibbs free energy than the reactants. Contributions of enthalpy and entropy *Note that there is an increase in entropy when a system undergoes a phase change from solid to liquid to gas. Using the fermentation of glucose to ethanol as an example, the reaction is exothermic with a negative enthalpy change, and also exergonic with a negative Gibbs free energy change. Using the melting of ice to liquid water as an example, the reaction is exergonic with a negative Gibbs free energy despite being endothermic with a positive enthalpy change. The huge increase in entropy allows for the reaction to be exergonic despite the fact that it is endothermicwith a positive enthalpy change value. These examples show that spontaneous reactions can be endothermic with an increase in entropy or exothermic with a decrease in entropy. The values of ΔH and ΔS can counteract each other to give a ΔG < 0. Free energy, stability, work capacity These three words are interchanged a lot. As Gibbs free energy increases, the system becomes less stable and has more work capacity. There is more Gibbs free energy associated with a higher relative position of an object vs. a lower position, clustered molecules in a solvent vs. uniformly spread molecules in a solvent. Adenosine triphosphate ATP hydrolysis is a spontaneous process with ΔG = -7.3 kcal/mol. In this process, a water molecule severs a bond to produce ADP (adenosine diphosphate) and a free orthophosphate (also known as inorganic phosphate Pi). The hydrolysis of ATP virtually never occurs in cells because the reaction is exothermic and releases heat. If this occurred in aqueous solution in cells, cells would overheat and be damaged. ATP hydrolysis in general doesn’t occur readily in solution. ATP and Energy Coupling Energy coupling refers to driving an endergonic reaction by linking it to an exergonic reaction. For most cases, ATP breakdown serves as the exergonic reaction. For example, the reaction: Glutamic acid --> glutamine, with the use of ammonia as a catalyst, is non-spontaneous with a ΔG = +3.4 kcal/mol. The breakdown of ATP allows for the addition of a phosphate group to glutamic acid (phosphorylation) which makes the amino acid much more unstable and ready to react spontaneously with ammonia. The total Gibbs free energy becomes (3.4 - 7.3) = -3.9 kcal/mol and is now spontaneous. ATP breakdown is occurring, not ATP hydrolysis. The ATP molecule and the glutamic acid are brought in such close proximity that water is unable to enter the active site of ammonia, which is “holding” the substrate and the ATP. There is no ortho
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