BCH 369 Week 5 Study Guide - Protein Study Techniques and Enzyme Behavior PDF

BCH 369 Week 5 Study Guide Protein Study Techniques and Enzyme Behavior Chapter 5 Extracting Cellular Protein: Rupturing Cells (Section 5-1 part 1) Learning Objectives: Know the general steps of extracting cellular proteins o Cell rupture  Homogen...

BCH 369 Week 5 Study Guide Protein Study Techniques and Enzyme Behavior Chapter 5 Extracting Cellular Protein: Rupturing Cells (Section 5-1 part 1) Learning Objectives: Know the general steps of extracting cellular proteins o Cell rupture  Homogenization – breaking open the cells  Know purpose of this step o Differential centrifugation  Know the general steps of differential centrifugation  Different speeds (low to high)  As you increase the force of gravity, smaller and smaller components are in the pellet Know the general order of precipitation o Purify soluble proteins “Differential” means we are doing a series of steps to get to the components we want. Consider three different centrifuge speeds: 600 x g (600 times the force of gravity) – only the biggest pieces will pellet out (be in the precipitate at the bottom of the test tube). This would include unbroken cells and cell nuclei 15000 x g – now smaller pieces will pellet out, such as mitochondria 100000 x g – this will pellet out small items such as ribosomes and membrane fragments Often what we want is not in the pellet but is in the supernatant (still dissolved in the liquid). We can spin to the speed needed to pellet out the things we don’t want and then work with the soluble components that are still in the supernatant. Review Exercises Recommended: 2 Extracting Cellular Protein: Protein Purification (Section 5-1 part 2) Learning Objectives: Know the general principles of salt fractionation (salting out) o What presence of salt accomplishes o Protein precipitates in native conformation o Purpose of this method  Know the general order of protein purification steps Know the difference between total activity and specific activity o Know how these change with each step in the purification As you increase the salt concentration, which proteins would precipitate first? Ones with more hydrophobic areas or ones with less hydrophobic areas? Total activity: total amount of target protein in sample Specific activity: total activity / total amount of protein A measure of protein purity Review Exercises Recommended: 3, 4 Column Chromatography: Size Exclusion (Section 5-2 part 1) Learning Objectives: Know the two phases of chromatography and their purpose o Stationary phase o Mobile phase Know the method of column chromatography o Stationary phase packed in column o Mobile phase is eluent Know the method of size exclusion (gel filtration) chromatography o Stationary phase is cross-linked gel particles o How particles separate based on size Review Exercises Recommended: 12, 19, 20 Column Chromatography: Affinity and Ion Exchange (Section 5-2 part 2) Learning Objectives: Know the general principles of affinity chromatography o Based on specificity of binding to ligand o Very pure sample in one or few steps Know the general principles of ion exchange chromatography o Know the difference between anion and cation exchangers o Know how pH ensures protein carries set charge and why o Know which molecules flow through o Know how protein eluted from column o Know how to determine order of elution of components, based on charge Know general purpose of HPLC  o Fast and clean purifications Review Exercises Recommended: 11, 14*, 15, 23**, 27, 28, 29 Note the terminology for ion exchange chromatography: A cation exchanger binds cations. Hence the resin in the column must have a negative charge. Also note the importance of pH for ion exchange chromatography. We need to find a pH where our target protein has a positive charge (if we are using a cation exchange column) but our non-target proteins have neutral or negative charges so that we achieve separation. *For #14, the video doesn’t talk about changing the pH, but that is a second option for getting the protein to elute. We originally set the pH such that the protein has a net positive charge, so it will bound to the negatively charged particles in the column. When we are ready to release the proteins, we could push liquid through the column with a different pH, causing the charge on the protein to change. A challenge is that extremes of pH can denature the proteins, so raising the salt concentration is often the way to go. **The answer to #23 is correct, but consider: Would this necessarily entirely separate protein X from all other proteins? No, just from ones that do not have a negative charge at pH = 8.5. Electrophoresis (Section 5-3) Learning Objectives: Know general principles of separation by electrophoresis o Differences in charge, shape, size Know the methods of SDS-PAGE o Purpose of SDS o How size relates to mobility (smaller is faster) Know method of isoelectric focusing o Gradient used and how separation accomplished General method of 2D electrophoresis o What are the two dimensions? o What is the purpose? Review Exercises Recommended: 31, 35, 37, 38, 39* A key point made right at the beginning of the video: This technique is often used for sample analysis (what is in the sample?) rather than sample purification. *Q39: The solution to this problem is wrong – though I like the question. If you look at the image – you can plainly see that the protein size cannot be more than 36,000. If you use Excel to plot distance traveled by the log of the MW – you should get something on the order of 32,400 Daltons. An estimation of a MW between 24,000 and 36,000, closer to the 36000 side would be a sufficient answer to this question. Primary Structure Determination: Cleavage into Peptides (Section 5-4 part 1) Learning Objectives: Know general method of determining amino acid composition o Content, N- and C-terminal o Peptide fragments sequenced and pieced together o Use of HPLC  Quantitative and qualitative analysis Know methods of determining primary structure o Use of proteolytic enzymes  Trypsin Cleaves at C-terminal side of basic AA  Chymotrypsin Cleaves at C-terminal of aromatic residues  Cyanogen bromide Chemically reacts and cleaves at C-terminal of methionine residues  Know the specificities of these enzymes o Know how to determine protein sequence based on cleavage products Review Exercises Recommended: 45, 46 Primary Structure Determination: Edman Sequencing (Section 5-4 part 2) Learning Objectives: Know purpose for producing various mixtures of peptides Know the benefits and limitations of Edman sequencing Know the general steps o Reaction with PITC o Formation of cyclic structure o Hydrolysis of peptide bond by anhydrous TFA o How identity of derivative determined Note: I did not find the extra tutorial video for this technique as useful as the previous two, but I left it in because I did find it useful to see the reactions laid out differently and to run through the pros and cons of the technique. The key idea here is that each N-terminal AA is separated sequentially. The first one is pulled off and then the next one and then the next one…. Proteomics (Section 5-6) Learning Objectives: Know what constitutes a proteome Know the purpose and types of studies Know the general steps of using protein bait to analyze protein interactions Review Exercises Recommended: 64 Protein Detection Techniques (Section 5-5) Learning Objectives: Know general method and purpose of mass spectrometry Know general steps and purpose of ELISA o Purpose of various components o How protein detected Know the general methods associated with protein chips o What they are o How a protein detected Know the general principles of the Western blot o Why protein transferred to nitrocellulose o How detection accomplished Note the two steps in the Western Blot: SDS-Page electrophoresis separates proteins based on MW, but does not identify specific proteins Western blot moves those separated proteins onto a protein binding membrane and allows them to be exposed to specific antibodies. Knowing which specific antibodies bind to the protein allows us to identify unknown proteins. Review Exercises Recommended: 56 Chapter 6 Thermodynamic Principles of Enzymes (Section 6-1) Learning Objectives: Know the general characteristics of enzymes as biological catalysts o Influence on reaction rate o Highly specific o Regulated processes o Don't alter equilibrium Know the standard state conditions Understand the concept of activation energy and the transition state o Energy needed to reach transition state o Intermediate state between reactants and products o Enzymes lower this value but do not change ∆G° Know the relationship between temperature and catalysis o Rate increases with temperature o Raising temp too much denatures enzyme Review Exercises Recommended: 2, 4*, 5, 7, 10, 11, 12 *Neither the video nor the text really cover the answer to #4 at this point, but this is a key idea, so make sure to look at the question and provided answer. I created new videos for sections 6-2 through 6-4. Enzymes: Free E and Kinetics Enzymes: Active Sites Enzyme Kinetics, part 1 Enzyme Kinetics, part 2 Enzyme Kinetics, part 3 Enzyme Kinetics, part 4 Important points: Rather than sticking exactly by each text section, I covered topics in an order I thought would make sense. I am leaving the learning objectives separated by textbook section below rather than by video so that those of you who prefer to read the text can follow along. Plus I put all my time and effort into making the videos; no energy left for reorganizing the learning objectives.  The sections of learning objectives below (with just section numbers and not titles) are still the learning objectives for the new enzyme videos. They are just not grouped by video. After you study all the videos, you should be able to meet all the learning objectives though. (Section 6-2) Learning Objectives: Know how to express enzyme reaction rate as loss of substrate or increase in product concentration over time Understand the concept of the rate or proportionality constant Know how to determine the order of a reaction o With respect to each substrate o Overall order of reaction Know what constitutes a zero-order reaction o How enzymes achieve this state ([S] >>>[E]) Review Exercises Recommended: 16 (Section 6-3) Learning Objectives: Know the general principles of binding of substrates to enzyme o Where it binds (active site) o Types of intermolecular forces involved o Know the two models for binding  Lock and key  Induced fit o Energy changes associated with formation of ES complex o Importance of proximity and orientation to speed of reaction Know the changes in structure and energy associated with product formation and release Review Exercises Recommended: 19, 20, 21 (Section 6-4a) Learning Objectives: Know the simple mechanism for a one-substrate/one-product reaction o Know the three rate constants and to what they relate  o Know the basic assumptions in this model Know the relationship between velocity and substrate concentration o Hyperbolic plot o Initial velocity is first order (why?) o Reaches Vmax and zero order at high [S] Know the significance of KM o Equilibrium constant that relates to substrate affinity Be able to write expressions for rate of formation and breakdown of ES Know the definition of steady state kinetics o Rate of formation of ES = rate of breakdown of ES o No change in [ES] over time o [S]>>[E]T Know the relationship between enzyme concentration and velocity o More enzymes  higher reaction velocity Review Exercises Recommended: 22*, 23 *The solution to this question in your book is stated INCORRECTLY. The amount of enzyme present certainly impacts the amount of product formed per unit time. If you have twice the machines running – you will get twice the output you would otherwise get. Perhaps the intent was to specify the catalytic rate, which is NOT influenced by enzyme concentration. In other words, Vmax may vary with [E], but kcat will not. (Section 6-4b) Learning Objectives: Michaelis-Menten Model o Know that KM is a collection of rate constants o Know that when [S] >>> [E], [ES] = [E]t and Vmax = k2[E]t Know the significance of the Michaelis-Menten equation and how to use it o You do NOT have to know how to derive it o Know that when V0 = ½ Vmax, [S] = KM o Know how to estimate Vmax and KM from a substrate saturation curve Review Exercises Recommended: 25 (Section 6-4d) Learning Objectives: Know the significance of KM  o Substrate concentration where reaction half-maximal o Good estimate of [S] in vivo o Dissociation constant and inverse measure of substrate affinity Know the significance of kcat and its relationship to Vmax o This is the turnover number – amount of product formed per enzyme per unit time Review Exercises Recommended: 24, 31 Enzyme Mechanisms and Lineweaver-Burk (Section 6-4c) Learning Objectives: Know the definition and types of Bi-Bi reactions o Ordered o Random o Ping-pong Know the significance of the Lineweaver-Burk equation and be able to use it o Know what an L-B plot looks like and how to determine Vmax and KM from the graph o Know the kinetic values associated with the slope and x- and y-intercepts Review Exercises Recommended: 26, 27*, 33, 39, 40, 41 *The textbook solution to this problem gives you the correct value for Vmax but not for KM. The latter should be 0.010 M. Examples of Enzyme-Catalyzed Reactions (Section 6-5) Learning Objectives: Know the example of chymotrypsin o Hyperbolic plot of Velocity vs. [S] Know the example of ATCase o Sigmoidal plot of Velocity vs. [S] Review Exercises Recommended: 44, 46 Big picture: These two enzymes are examples of the two general classes of enzymes: Michaelis-Menten enzymes and allosteric enzymes. The difference in the shape of the curves of [S] vs velocity tells us which each is. Allosteric here means the same thing as it did when we were talking about hemoglobin – that it undergoes cooperative effects. In allosteric proteins (including allosteric enzymes), subtle changes at one site of the protein chain affect the structure and function at other site(s). Competitive Inhibition (Section 6-6a) Learning Objectives: Know the definition of inhibitor and reversible inhibitor Know the characteristics of competitive inhibitors o To what form of the enzyme they bind and where o Often resemble either substrate or transition state o Can be overcome by adding more substrate Know the significance of KI o Inverse measure of enzyme affinity for inhibitor Know which kinetic parameters change and how (and why) o Know the significance of a and how it relates to inhibition Know how to recognize a competitive inhibitor, graphically Noncompetitive Inhibition (Section 6-6b) Learning Objectives: Know the characteristics of noncompetitive inhibitors o To what form of the enzyme they bind and where o Cannot be overcome by adding more substrate Know which kinetic parameters change and how (and why) Know how to recognize a noncompetitive inhibitor, graphically