Bio285 F24 Topic Notes on Protein Misfolding, Enzymes, and Regulation PDF

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SophisticatedSnowflakeObsidian1894

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Prof. Francis

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protein regulation enzymes biology protein folding

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This document provides notes and resources for a biology course, covering protein misfolding, enzyme function, and protein regulation. It includes learning objectives and external resources.

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Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Introduction to topics covered this week: This week we will discuss how protein folding can be perturbed and h...

Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Introduction to topics covered this week: This week we will discuss how protein folding can be perturbed and how the cell deals with that. We will also discuss overarching aspects of protein function such as how proteins interact with other molecules, the mechanism of enzyme function, and how an overview of how protein activity is regulated in the cell. Unit Objectives: After covering the content in this section, students should be able to: ✓ Describe the effects of environmental factors on protein folding and how cells have a stress response when proteins unfold or misfold within the cell ✓ Discuss what determines how strong the interaction is between a protein and a ligand and how the dissociation constant Kd is measured and how that relates to the strength of the interaction ✓ Describe why cells need enzymatic function and how enzymes speed up chemical reactions ✓ Discuss general mechanisms by which enzymes catalyze chemical reactions ✓ Identify and describe general mechanisms by which protein function is regulated in the cell *As you read through the topic notes, have the pre-recorded lecture slides open to view the figures and diagrams. The notes should approximately follow the concepts discussed in the pre-recorded lectures. Reading and Videos Resources: Use these resources, and any other useful resources you can find online, to help fill in any gaps in your knowledge, help solidify your understanding of a concepts, get alternate explanations of concepts, put concepts into context, etc. Protein-Ligand Interactions Introduction to protein-protein interactions: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein- biology-learning-center/protein-biology-resource-library/pierce-protein- methods/overview-protein-protein-interaction-analysis.html Protein-ligand interactions – see from 1:30-3:10 in this video Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis https://www.youtube.com/watch?v=5-hLq8DmtZs See the section on protein-ligand interactions: https://en.wikipedia.org/wiki/Dissociation_constant How is protein activity regulated? https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/Book%3A_Biofundam entals_(Klymkowsky_and_Cooper)/08%3A_Peptide_bonds_polypeptides_and_proteins/8. 12%3A_Regulating_protein_activity https://www.ncbi.nlm.nih.gov/books/NBK9923/ Enzymes Introduction to enzymes (reading): https://courses.lumenlearning.com/wm-biology1/chapter/reading-enzymes/ Introduction to enzymes pdb-101 (video) https://pdb101.rcsb.org/learn/videos/how-enzymes-work Also watch movie 4.8 in the movies folder (mechanism of lysozyme enzyme function) Read some select sections from MBOC: https://www.ncbi.nlm.nih.gov/books/NBK26838/ About 1/3 of the way down the page read the following sections: Enzymes Lower the Barriers That Block Chemical Reactions How Enzymes Find Their Substrates: The Importance of Rapid Diffusion The Free-Energy Change for a Reaction Determines Whether It Can Occur Topic Notes: What Affects Protein Folding, Protein Function, and Regulation? What affects protein folding? o Impacts of environment on protein folding ▪ The environment that a protein is in is very important for its function Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis Environmental factors such as heat, salt (type or concentration) conditions, pH conditions, exposure to high- intensity light (e.g. X rays) can all affect the stability of the non-covalent bonds that hold proteins together ▪ If the bonds that hold a protein’s structure together become destabilized the protein can unfold and/or misfold and lose function ▪ If the bonds that hold a protein’s structure together become more stable than they should be, that can affect the function of the protein as well (protein’s do require a certain amount of flexibility) ▪ Thermal energy (heat) can also affect diffusion rates, which can also affect the ability of molecules to bump into each other and interact with one another ▪ Cells carefully control environmental factors like temperature (thermal energy), pH, and salt concentration because they can have an impact on charge interactions, which make up the large majority of non-covalent bonds in the cell ▪ When a protein unfolds, this is called denaturation ▪ In the cell, there are proteins that help other proteins fold, or stabilize them when they’re unfolding or misfolding Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis These proteins are called chaperones Some chaperones help proteins when they’re made and some chaperones are more involved in trying to stabilize protein folding during times of cellular stress Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis Temperature: thermal energy affects the stability of non-covalent ▪ bonds (it affects the stability of covalent bonds as well, but not within our normal environmental parameters – even temps around water’s boiling point don’t affect covalent bonds, but can certainly disrupt non-covalent bonds) Less thermal energy means more stable bonds More thermal energy means less stable bonds Don’t fall into the trap of thinking more stable is always better – here is where I introduce the Goldilocks Hypothesis of Cellular Function (yes, this is something I made up J) o If a protein is too stable, that’s not necessarily a good thing – sometimes proteins need to be flexible or shift conformations o If there is too little thermal energy in a system, random diffusion/Brownian motion of molecules may be too slow and that’s not good for cellular function (which largely depends on diffusion) o This means that cells have evolved the mechanisms that work best for them – for example, our cells function best at 37 degrees Celsius – our proteins and cellular pathways have evolved to work the way they need to work at that temp ▪ pH and salts can affect charge interactions – cells have a particular pH and control the amount or type of salt that is around ▪ Light energy can affect non-covalent bonds by introducing energy into a system – higher energy wavelengths of light can disrupt noncovalent bonds holding proteins together (e.g. UV, lasers) o How do cells respond to a protein unfolding/misfolding stress response? ▪ In the cytosol or nucleus the proteins are sent to the proteasome in a stress response pathway called the Heat Shock Response ▪ For proteins going into the endomembrane system, there is an ER- specific stress response called the Unfolded Protein Response ▪ Upregulation of production of chaperones is common in stress responses such as heat shock, and in fact is how chaperones were discovered Protein Function and Regulation: o Protein-ligand interactions (note that a ligand can be any molecule, including another protein with which that the protein interacts) Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis ▪ What determines the strength of interaction (affinity) between a protein and the molecule it’s binding to? The number and strength of the chemical bonds formed between the protein and its ligand determines the affinity of the protein for that ligand ▪ How we measure and describe protein-ligand interactions: the dissociation constant Kd The dissociation constant is an average rate measurement of how often the protein dissociates from its ligand Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis The dissociation constant is measured by setting a constant amount of protein and titrating in increasing amounts ligand o For each concentration of ligand, the mixture is allowed to go to equilibrium and the amount of free protein, and protein in complex with ligand, is measured at that binding equilibrium; Kd = [P][L]/[C] o The ligand concentration at which (at equilibrium) the amount of free protein [P] is equal to the amount of protein in complex with ligand (this is called the “half bound state”) [C] is expressed as the Kd of a protein- ligand interaction – this is the point at which [P] = [C] so [P]/[C] = 1 and so Kd = [L] the ligand concentration at which [P] = [C] Because Kd is a measure of rate of dissociation, there is an inverse relationship between affinity (strength of interaction) and Kd o The lower the Kd, the higher the affinity o The higher the Kd, the lower the affinity o Enzyme function and mechanism ▪ The typical reaction that enzymes catalyze in the cell are to make or break covalent bonds – so they change the chemical make-up and structure of molecules ▪ Why are enzymes needed by the cell? There are several reasons: o Enzymes speed up reactions that might normally happen on their own (spontaneously), but too slow for the functions of the cell o Enzymes are regulatable – so cellular processes can be regulated, which is very helpful for cell function ▪ What are cellular enzymes made of? Most enzymes are proteins, but there are a few made of RNA Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis RNA enzymes are called “ribozymes” o Ribosomes and the spliceosome falls under the category of ribozymes because it is the RNA component of those molecules that catalyze the relevant chemical reactions ▪ Reaction progress diagram A reaction diagram tracks the progress of a reaction over time and shows the free energy associated with the molecules involved in the reaction On the x axis is progression in time, on the y axis is the free chemical energy associated with the molecules taking part in the chemical reaction When the free energy of the reactants is higher than products, the reaction is considered spontaneous or exergonic because energy is given off in the reaction When the free energy of the reactants is lower than the products, the reaction is not spontaneous and is called endergonic because an input of energy is required to create the products Note that there is always a “transition state” that the reactants must go through to become the product of the reaction o This transition state is often quite unstable and so is associated with a higher free energy state Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis o In spontaneous reactions, the height of the energy of the transition state determines the rate of the reaction o The difference between transition state energy and energy of reactants is called the “activation energy” How do enzymes speed up chemical reactions? o Enzymes typically stabilize the transition state and so they lower the “activation energy” required to get the reaction to happen ▪ This is what speeds up the rate of the reaction o Mechanistically, the general idea is that the enzyme “active site” creates a little microchemical niche that positively affects the ability of the reactants to reach the transition state o What are some general ways that enzymes might catalyze chemical reactions? ▪ Enzymes can bring two reactants closer together to encourage the reaction to occur ▪ Enzymes can change charges – for example, if the transition state requires two negative charges to be near each other, the enzyme could facilitate that by placing a positively charged functional group in between ▪ Enzymes can force and stabilize reactants into conformations that more closely resemble the transition state o Regulation of protein function ▪ Allostery Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis I don’t use this term, but some do, so you should know it The old biochemical definition of this term basically says that an enzyme can have a regulator that binds somewhere other than the active site, causes a conformational shift in the enzyme, which disrupts or helps its ability to perform its activity The definition used nowadays is more general and says that allostery is the process by which molecules transmit the effect of binding at one site, to another site, by a change in structure, allowing for regulation of activity Other than understanding what the word means, the general concept that I want you to keep in mind is that protein function is typically regulated by changing the protein’s structure o Structure = Function ▪ Post-translation modification Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis Yellow Green – ubiquitin phosphorylation Blue – –acetylation Proteins are often regulated by chemically modifying (covalently) the protein after the protein has been made (hence “post-translational) These modifications are typically reversible (although in some cases the modification is never removed) Post-translational modification is often used as a switch mechanism to control a protein’s function – change the protein’s surface chemistry, change its structure, change its function – and be able to reverse that by removing the modification Proteins like p53 have been shown to be post-translationally modified in a number of different ways o This creates the power of combinatorics – different combinations of the different modifications allows p53 to act as a central coordinator of cell stress response in many different pathways ▪ Nucleotide binding and hydrolysis We will talk about many examples of ATPases and GTPases during this course Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis o These are proteins that are categorized as enzymes because they have the ability to bind ATP or GTP (never both – they do one or the other) and can hydrolyze the tri-phosphate to a di-phosphate so that they are then bound to ADP or GDP respectively o These ATPases and GTPases may use this activity to control other protein’s functions (as regulators) – they are common in signal transduction pathways ▪ We’ll discuss a few examples such as Ran (involved in nuclear import and export), and Ras (involved in cell division signaling) – these are both GTPases o Or they may be used a molecular “machines” using the energy and structural changes to power their activities ▪ Some examples of this: Helicases are ATPases – they are 6- subunit hexamers where each subunit has ATPase activity – they use the coordinated processes of ATP binding, ATP hydrolysis, ADP binding, ADP release to drive the coordinated movement needed to unwind DNA Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis Motor protein movement is regulated by ATP binding, hydrolysis, ADP binding and release: Motor proteins that walk along microtubules or actin are ATPases – similarly to helicases, they use the stepwise nature of ATP binding, hydrolysis, etc to move “feet” in a coordinate fashion along a microtubule or actin Bio285 F24 Topic Notes and Resources for Topic 2: Protein Misfolding, Enzymes, and Protein Regulation Written by Prof. Francis

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