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MCDB 310 1st Edition Lecture 6Outline of Last Lecture I. 4 levels of protein structure (continued from last lecture)a. Secondary Structure: alpha helices and beta sheetsb. Tertiary Structurec. Quarternary Structured. Analyzing protein structure in relation to its functionII. Two major types of protein (fibrous and globular)III. Protein stability and foldingIV. Function of globular proteinsOutline of Current Lecture I. Protein equilibrium constants (Kd and Ka)II. Myoglobin and Hemoglogin: what makes these proteins able to perform their functions?III. EnzymesCurrent LectureI. Protein dissociation constants (continued from the last lecture)a. Remember: the higher the binding affinity —> higher the Ka —> lower the Kd i. Ka=10^6 M^(-1) —> Kd=10^(-6) M (Kd=1/Ka)b. The protein that binds the ligand is called the receptorc. All equilibrium constants are dependent on thermodynamics (Gibbs Free energy),not kinetics (the reaction is given infinite time in order to reach equilibrium)i. Rate constants: represented by lowercase lettersii. Equilibrium constants: represented by uppercase lettersd. At equilibrium: the forward rate is equal to the reverse ratee. Equilibrium constant Ka (affinity) = rate constant (forward reaction)/rate constant (reverse reaction)f. Kd has the units of concentration (usually millimolar or picomolar)II. Ligand binding to a receptor proteina. Theta is binding concentration (it is a unitless value)b. Can plot theta vs concentration of the ligand to determine Kd (the ligand concentration at which 50% of the binding sites are occupied)i. Lower the Kd —> less ligand required to saturate the protein (high affinity)ii. Higher the Kd (shifted to the right) —> more ligand required to saturate the protein (lower affinity)c. Strong binding: Kd < 10nM (strongest=picomolar)d. Weak binding: Kd > 10 micromolarIII. Why Myoglobin is a good storage molecule for OxygenThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.a. Myoglobin accepts the oxygen that hemoglobin releases in the muscles and tissuesb. From the binding curve above:i. Hyperbolic shaped curve (curve governed by equation for theta)ii. Kd = 0.4 kPa (because oxygen is a gas, this value is expressed as a pressure)iii. Pressure of the oxygen in the tissue is about 4 kPa (lower than in lungs - 13 kPa - because it is used for metabolism)iv. Myoglobin has a very high affinity for oxygen, so any oxygen in the tissue will be bound by the myoglobinv. However, myoglobin is NOT a good protein for transporting oxygen because it will not unbind from the oxygen (affinity is too high)IV. Why Hemoglobin is a good transport molecule of Oxygena. The way hemoglobin binds to oxygeni. See the binding curve above (Hb vs Mb)ii. Still hyperbolic, but initially has a sigmoidal (s-shaped) binding curve (starts more sluggishly)iii. 4 binding sites for Oxygen:1. Hb has 4 globin subunits (tetramer)a. Two of these subunits have the same AA sequence (alpha-141 AAs long)b. The other two are the beta subunits (146 AAs long)i. In fetuses: 2 alpha and 2 gamma subunits 2. Hb binds COOPERATIVELY to oxygena. Hb has multiple binding sitesb. Positive cooperativity: the first binding event increases affinity of the second binding site for the ligand c. As shown in above figure, the binding site size increases as more ligands bind to the binging sitesi. In the lungs: the binding sites are the smallest, all oxygen uptake happens here and binding sites growii. In the tissues: the binding sites start large and decrease in size as oxygen is releasedd. In the lungs: Hb has a high affinity for oxygen and is efficiently loaded with oxygene. In the tissues: hemoglobin releases oxygen, and the theta value of myoglobin increases3. Structural Basis for Cooperative Binding: The T and R statesa. T (tense) state: low affinity for oxygen, stable when oxygen levels are lowi. See figure below:ii. Iron is offset from the center of the Heme —> low affinity for oxygen —> deoxyhemoglobinb. R (relaxed) state: high affinity for oxygen, stable when oxygen levels are highi. See figure belowii. Iron molecule is in the center of the hemeiii. Allows binding to one subuint which increases affinity for oxygen and causes more binding -> oxyhemoglobiniv. Smaller hole in the middle of molecule —> higher affinity for oxygen (state that naturally occurs in theR state)4. Hill Equationa. In a simple case (like Mb binding to oxygen), the n is 1 (n represents the number of binding sites)b. n is also the slope of the Hill Ploti. Any slope greater than 1 tells us that positive cooperativity is presentii. Two lagging tails on Hb plot represent the lower affinity and higher affinity states of the moleculec. The hill plot is DIFFERENT from the binding plot5. Spectroscopic Detection of Oxygen Binding to Hemoglobin-Pulse Oximetrya. The oxy and deoxy hemoglobin states have different visible and IR spectrab. Ratio of absorbed light (visible) vs IR can tell us the concentration of oxygen in the blood6. Allostery: Binding to one site of a protein affects binding properties of another site in the same proteina. Requires multiple binding sits (for a ligand and also a modulator-the molecule that binds somewhere else on the protein)b. Allosteric modulators: can either increase (positive) or decrease (negative) the affinity for the ligandi. Homotropic: the modulator and ligand are the same molecule (these are usually positive modulators)1. Example: oxygen on Hbii. Heterotropic: the modulator is a different molecule than the ligand (can be either positive or negative modulator)b. The Bohr effect (effect of pH on hemoglobin binding to oxygen)i. The affinity of Hb for oxygen decreases with decreasing pH (See graph below)ii. Bohr effect: the pH difference between lungs and metabolic tissues increase efficiency of the oxygen transport1. Metabolic tissues accumulate H+ (lactic acid) and CO2 (low pH) —> two negative modulators are in high concentration —> Hb releases oxygen to Mb in the tissues2. In the lungs, H+ and CO2 dissociate from Hb (high pH) —> low concentration of negative modulators —> Hb has a high affinity foroxygen and is loadediii. Mechanism of Bohr Effect:1. Protonation of Histidine-146:a. As the pH drops —> His 146 is proteins —> His 146 forms salt bridges (what keeps the tense state stable) with Asp 94—> decreases affinity for oxygen —> releases oxygen2. Carbamation


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