MBS 320 Final Exam Lecture Slides FA24 PDF

MBS 320: Cell Biology Final Exam Lecture Slides  General Information on Final Exam Exam will consist of 24 multiple choice (48pts) and 8 free-response questions (52pts); this exam will cover All Lectures. Content from papers 1-3 will not be covered. Questions will...

MBS 320: Cell Biology Final Exam Lecture Slides  General Information on Final Exam Exam will consist of 24 multiple choice (48pts) and 8 free-response questions (52pts); this exam will cover All Lectures. Content from papers 1-3 will not be covered. Questions will be based on previous exams, practice exams, and study guides. About 25% of the questions on the final will be from previous exams and practice exams. Short answer questions will not be multi-part questions. This will be a paper exam, and you need to be on campus to take the exam. Taking the final as an online exam is not an option for this course. You can use pen or #2 pencil on exam. If you are using pencil, make sure you write dark enough for scanning. Exams will be scanned, uploaded to Gradescope and graded by instructor and TAs. Graded exams will not be published. To view graded final exam, you need to schedule a one-on-one meeting with the instructor. To prepare for the exam, you should look over “Final Exam Lecture Slides”, the final exam study guide, and previous midterm exams (passive learning). You should also try to identify concepts that you don’t fully understand. Print out blank practice exams, homework assignments, or discussion activities and try to fill them out without your notes (active learning). Macromolecules in Cells Building blocks = small organic molecules = subunits Building blocks of cells fall into four categories: sugars, amino acids, fatty acids, and nucleotides Building blocks are used to form macromolecules (large organic molecules) Macromolecules are typically formed by generating strong covalent bonds between small organic molecules Macromolecules in Cells Building blocks = small organic molecules Building blocks of cells fall into four categories: sugars, amino acids, fatty acids, and nucleotides Building blocks are used to form macromolecules (large organic molecules) Macromolecules are typically formed by generating strong covalent bonds between small organic molecules Covalent bonds between subunits are created by a condensation reaction: two molecules combine to form single molecule, often by removal of water Problem: condensation reactions are energetically unfavorable; will not happen spontaneously at high rate Macromolecules in Cells Solution: cells use both condensation and hydrolysis reactions to make macromolecules from subunits Condensation reaction: molecules combined by removing water (energetically unfavorable) Hydrolysis reaction: molecules broken by adding water (energetically favorable)  Macromolecules in Cells Solution: cells use both condensation and hydrolysis reactions to make macromolecules from subunits Condensation reaction: molecules combined by removing water (energetically unfavorable) energetically Hydrolysis reaction: molecules broken by adding water favorable (energetically favorable) energetically unfavorable Cells create macromolecules by coupling energetically favorable reactions (C to D) with energetically unfavorable reactions (X to Y)  Macromolecules in Cells Solution: cells use both condensation and hydrolysis reactions to make macromolecules from subunits C Condensation reaction: molecules combined by removing water (energetically unfavorable) Y Hydrolysis reaction: molecules broken by adding water (energetically favorable) Cells create macromolecules by coupling energetically favorable reactions (C to D) with energetically unfavorable reactions (X to Y) X D Amino Acids All amino acids have an amino group and carboxyl group connected by ⍺-carbon In water (pH 7), free amino acids exist in ionized form; amino group accepts a proton and carboxyl group donates a proton Amino Acids All amino acids have an amino group and carboxyl group connected by ⍺-carbon In water (pH 7), free amino acids exist in ionized form; amino group accepts a proton and carboxyl group donates a proton Peptide bonds link amino acids with condensation reaction; charges on amino and carboxyl groups disappear N-terminus: first amino acid in polypeptide backbone C-terminus: last amino acid in polypeptide backbone Amino Acids Each amino acid has a side chain (aka R group) attached to the ⍺-carbon Structure of side chain is what distinguishes one amino acid from another Side chains have different chemical properties that are critical for protein structure and function Polar amino acids: hydrophilic amino acids that form hydrogen bonds and prefer to interact with water Nonpolar amino acids: hydrophobic amino acids that cannot form hydrogen bonds and repel water  Protein Structure Noncovalent Bonds in Protein Structure Hydrogen bonds Electrostatic interactions Van der Waals attractions Noncovalent bonds between amino acids influence three dimensional shape of proteins  Protein Structure Noncovalent bonds between amino acids influence three dimensional shape of proteins Hydrophobic interactions play a critical role in determining shape of protein; nonpolar amino acids cluster in core Protein Structure Proteins have different levels of protein structure Primary: amino acid sequence (polypeptide chain) Secondary: alpha-helices and beta sheets Alpha helices and beta sheets are observed in most proteins. These structures do not require a specific amino acid sequence. How is this possible? Protein Structure Proteins have different levels of protein structure Primary: amino acid sequence (polypeptide chain) Secondary: alpha-helices and beta sheets Tertiary: collection of secondary structures in protein All proteins have primary, secondary, and tertiary structures  Protein Structure Proteins have different levels of protein structure Primary: amino acid sequence (polypeptide chain) Secondary: alpha-helices and beta sheets Tertiary: collection of secondary structures in protein All proteins have primary, secondary, and tertiary structures Proteins are often organized into protein domains: segment of amino acids that can perform specific function Protein Structure Proteins have different levels of protein structure Primary: amino acid sequence (polypeptide chain) Secondary: alpha-helices and beta sheets Tertiary: collection of secondary structures in protein Quaternary: binding of different polypeptide chains All proteins have primary, secondary, and tertiary structures; some proteins have quaternary structure Protein Structure Proteins have different levels of protein structure Primary: amino acid sequence (polypeptide chain) Secondary: alpha-helices and beta sheets Tertiary: collection of secondary structures in protein Quaternary: binding of different polypeptide chains Protein subunit: one polypeptide chain in protein with quaternary structure  Protein Structure Figure 3-25 Noncovalent bonds allow different polypeptide chains to combine to make one large protein (quaternary structure) Disulfide bonds: covalent bonds between cysteines that stabilize protein structure or combine different polypeptide chains Disulfide bonds are often observed in proteins that are secreted by cell or are attached to outer surface of plasma membrane Protein Function Protein shape dictates protein function Protein function is typically dependent on ability of protein to physically interact with other molecules Ligand: any substance that is bound by a protein Proteins use many weak, noncovalent bonds to bind specific ligands and to other proteins Protein Function Protein shape dictates protein function Protein function is typically dependent on ability of protein to physically interact with other molecules Ligand: any substance that is bound by a protein Proteins use many weak, noncovalent bonds to bind specific ligands and to other proteins Binding site: cavity with amino acid side chains that bind to ligand using noncovalent bonds Binding sites often use electrostatic interactions and hydrogen bonds to selectively bind to one specific ligand  Antibodies Antibodies are proteins composed of four polypeptide chains; two identical light and two identical heavy chains Antibodies have variable regions (VH and VL) that can bind to an antigen (substance that stimulates immune response) Antibodies Producing antibodies in the lab Antibodies that bind to specific proteins can be produced for new drug treatments and scientific research Protein/antigen is injected into animal; antibodies that bind to antigen are secreted into blood by B cells Blood is collected and antibodies are purified Antibodies Producing antibodies in the lab Antibodies that bind to specific proteins can be produced for new drug treatments and scientific research Protein/antigen is injected into animal; antibodies that bind to antigen are secreted into blood by B cells Blood is collected and antibodies are purified Purified antibodies are polyclonal: mixture of antibodies that bind to different places on same antigen (different antibodies made by different B cells) Antibodies Producing monoclonal antibodies Step one: inject mouse with antigen to stimulate immune response Step two: isolate B cells from spleen from mouse Step three: fuse B cells with myeloma cells; hybrid cells produce antibodies but do not have cell senescence (immortal) Step four: identify which hybrid cells produce effective antibody Monoclonal vs Polyclonal Antibodies Monoclonal antibodies are purified from hybrid cells; expensive Hybrid cells (hybridomas) provide infinite supply of antibodies Monoclonal antibodies have low variability between batches (ideal for research and drug treatment)  Protein Denaturation Most proteins begin to fold into correct shape/conformation as they are synthesized Protein denaturation: change in protein shape that reduces protein activity Protein denaturation occurs because protein shape is dependent on weak, noncovalent interactions Heat Protein denaturation is caused by heat, extreme pH, and chemicals Chaperones Most proteins cannot spontaneously refold once they have been completely denatured Cell use chaperones to refold proteins that have become denatured or are not properly folded Chaperones can refold proteins using different strategies Chaperones Heat shock proteins (Hsp): chaperone proteins that refold proteins that have been denatured (often due to an increase in temperature) Hsp70 and Hsp60 are two different heat shock proteins that are made by the cell in response to stress Protein folding by Hsp70: Hsp70 binds to segment of 4-5 nonpolar/hydrophobic amino acids Hydrolysis of ATP allows Hsp70 to bind to hydrophobic amino acids Hsp70 can bind to protein as soon as it is translated by ribosome Binding of Hsp70 to protein causes hydrophobic segment to extend Rebinding of ATP releases segment in extended form Repeated cycles of bind, extend, release helps protein fold properly Hsp70 is constantly extending polypeptide chain until enough protein is made for correct folding Hsp70 can also refold a misfolded protein that has already been translated Chaperones Heat shock proteins (Hsp): chaperone proteins that refold proteins that have been denatured Hsp70 and Hsp60 are two different heat shock proteins that are made by the cell in response to stress Protein folding by Hsp70 Protein folding by Hsp60: Hsp60 is a multi-subunit complex (Hsp60 x 14 + accessory proteins) Hsp60 uses isolation chamber to help protein fold Protein in chamber folds without risk of aggregation Hsp60 works in partnership with Hsp70 (delivers unfolded protein to Hsp60) Protein Degradation Proteins that cannot fold properly need to be degraded to prevent protein aggregation Protein Degradation Three steps for ubiquitylation: 1. Ubiquitin added to ubiquitin-activating enzyme (E1) 2. Ubiquitin on E1 transferred to ubiquitin-conjugating enzyme (E2) 3. E2 binds to ubiquitin ligase (E3); ubiquitin ligase adds ubiquitin to target protein and process is repeated to produce polyubiquitin chain Protein Degradation Protein is denatured by cap, degraded in cylinder; ubiquitin and amino acids are recycled Each cell has about ~16,000 different proteins E1, E2, E3, proteins work together to target specific proteins Cells use combinations of E1, E2, and E3 to target proteins Cells activate a specific set of E1, E2, and E3 enzymes to target a specific protein (substrate) Cell Fractionation Step two: lyse cells After desired cells are collected they are exposed to a lysis buffer: solution with detergent that breaks plasma membranes Lysis buffer produces cell lysate or cell homogenate: plasma membranes disrupted, sample has all cellular components (nuclei, organelles, membranes, etc.) Step three: cell fractionation Cell fractionation: separate cellular components from one another and isolate organelles; often done with centrifuge  SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Cell fractionation can separate cellular components but scientists also need to be analyze specific proteins SDS-PAGE separates proteins in a mixture by size SDS is a detergent that denatures proteins and coats proteins with negative charge Beta-mercaptoethanol (BME) is a reducing agent that eliminates disulfide bridges Denatured and reduced proteins are loaded onto polyacrylamide gel and electric field is applied Proteins travel towards positive electrode Large proteins migrate slow, and small proteins migrate fast  SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) After separating proteins with SDS-PAGE, gel is stained to visualize protein Coomassie blue: stain commonly used to stain protein in gel after SDS-PAGE; stain binds to basic amino acids Coomassie blue is a non-specific stain; all proteins in sample are visible in gel Western Blot Western blot measures amount of specific protein Step one: use SDS-PAGE gel to separate protein mixture in sample with a gel Step two: transfer separated proteins from gel to membrane Step three: primary antibody recognizes specific protein on membrane Step four: secondary antibody binds to primary antibody and is used for visualization; more signal on membrane indicates more protein in cells  Immunoprecipitation Immunoprecipitation is used to purify proteins and assess protein-protein interactions Challenge: proteins interact using weak, noncovalent interaction; need to stabilize interaction  Immunoprecipitation Immunoprecipitation is used to purify proteins and assess protein-protein interactions Step one: generate cell lysate; cross-link proteins with formaldehyde to stabilize protein-protein interactions Step two: add antibody conjugated to bead; antibody binds to protein of interest Step three: use centrifuge to pull down bead; proteins not bound by antibody are discarded and proteins bound by antibody are purified Step four: reverse cross-linking; use Coomassie blue or western blot to determine which proteins are interacting with protein of interest Microscopy Different Microscopes in Research Light microscopes use light source to view single cells and internal structure of a cell Fluorescence microscopes also use a light source but specific structures within cells can be seen in greater detail because of fluorescent labels Electron microscopes use a beam of electrons instead of light to achieve highest magnification Fluorescence Microscopy How do scientists attach fluorescent dyes to specific proteins for fluorescence microscopy? Fluorescent dyes can be added to DNA sequence to create fluorescent protein (aka fusion protein) Green fluorescent protein (GFP) is a common fluorescent dye used in fluorescence microscopy Cells are often “fixed” to glass slides. Cell fixation: cells are locked in place on glass slide and have permeable membranes that allow large molecules to pass through Fluorescence Microscopy How do scientists attach fluorescent dyes to specific proteins for fluorescence microscopy? Fluorescent dyes can be added to DNA sequence to create fluorescent protein (aka fusion protein) Green fluorescent protein (GFP) is a common fluorescent dye used in fluorescence microscopy Cells are often “fixed” to glass slides. Cell fixation: cells are locked in place on glass slide and have permeable membranes that allow large molecules to pass through Immunofluorescence: antibody with fluorescent dye to detect specific protein using fluorescence microscopy  Fluorescence Microscopy How do scientists attach fluorescent dyes to specific proteins for fluorescence microscopy? Two different proteins are analyzed with two different antibodies Fluorescent dyes can be added to DNA sequence to Same cells are analyzed in one column; rows represent different antibodies create fluorescent protein (aka fusion protein) Green fluorescent protein (GFP) is a common Protein of fluorescent dye used in fluorescence microscopy interest Cells are often “fixed” to glass slides. Cell fixation: cells are locked in place on glass slide and have permeable membranes that allow large molecules to pass through Nuclear protein Immunofluorescence: antibody with fluorescent dye to detect specific protein using fluorescence microscopy A B Immunofluorescence provides information about Protein of interest (column A): mostly in nucleus location of protein and amount of protein in cell Protein of interest (column B): mostly in cytoplasm Plasma Membrane All living cells must have a plasma membrane Plasma membrane defines boundary of cell and separates cytosol from extracellular environment Internal membranes from organelles provide cell with intracellular compartments to perform specific reactions Membranes allow selective passage of molecules and ions into and out of the cell Small, non-polar molecules diffuse rapidly across membrane Molecules that are large, polar/charged diffuse slowly across membrane Plasma Membrane Fatty acids are the building blocks of the plasma membrane Fatty acids are amphipathic; have hydrophilic, polar head and hydrophobic, non-polar tail Fatty acids can be saturated (no double bonds) Fatty acids can be unsaturated (one or more double bonds) Membrane lipid: modified fatty acid in plasma or internal membrane; two fatty acid tails, one polar head Lipid bilayer formed by hydrophobic tails clustering together and polar/hydrophilic heads contacting water Plasma Membrane Membrane fluidity determined by packing of hydrocarbon tails in lipid bilayer Close packing of tails = less fluid, little movement Loose packing of tails = more fluid, more movement Length of hydrocarbon tail affects membrane fluidity Long tail = more interaction between tails, less fluid Short tail = less interaction between tails, more fluid Number of double bonds in tail affects membrane fluidity No double bonds (saturated) = more interaction, less fluid One or more double bonds (unsaturated) = less interaction, more fluid Animal cells regulate membrane fluidity using cholesterol More cholesterol = less fluid, little movement Less cholesterol = more fluid, more movement Membrane Proteins Membrane proteins often use hydrophobic amino acids in alpha helix to pass through lipid bilayer Hydrophobic amino acids exposed to hydrophobic lipids; peptide bonds form hydrogen bonds with one another Single-pass transmembrane proteins only require one alpha helix to pass through lipid bilayer Membrane Proteins Membrane proteins often use hydrophobic amino acids in alpha helix to pass through lipid bilayer Hydrophobic amino acids exposed to hydrophobic lipids; peptide bonds form hydrogen bonds with one another Single-pass transmembrane proteins only require one alpha helix to pass through lipid bilayer Multi-pass transmembrane proteins require multiple alpha helices to pass through lipid bilayer to form aqueous pore Polar amino acids in alpha helices aggregate in lipid bilayer to form hydrophilic environment for aqueous pore Membrane Proteins Hydrophobicity Plot Scientists can use various tools to determine whether proteins have transmembrane segments Transmembrane segment: stretch of 20 amino acids in protein used to pass through lipid bilayer Proteins can have zero, one, or several transmembrane segments; number provides information about function Membrane Proteins Hydrophobicity Plot Scientists can use various tools to determine whether proteins have transmembrane segments Transmembrane segment: stretch of 20 amino acids in protein used to pass through lipid bilayer Proteins can have zero, one, or several transmembrane segments; number provides information about function Hydrophobicity plot (aka hydropathy plot): visualization of hydrophobicity in amino acid sequence of protein High positive value in hydrophobicity plot for ~20 amino acids indicates a transmembrane segment Membrane Proteins Hydrophobicity Plot Scientists can use various tools to determine whether proteins have transmembrane segments Transmembrane segment: stretch of 20 amino acids in protein used to pass through lipid bilayer Proteins can have zero, one, or several transmembrane segments; number provides information about function Hydrophobicity plot (aka hydropathy plot): visualization of hydrophobicity in amino acid sequence of protein High positive value in hydrophobicity plot for ~20 amino acids indicates a transmembrane segment Figure 10-20 Membrane Transport Cells maintain concentrations of ions by using membrane transport proteins Transporters: transmembrane proteins that change shapes to transport specific molecules/ions Channels: transmembrane proteins that form pores which can open and close to transport ions Membrane Transport Direction of transport for solutes often depends on concentration between inside and outside of cell Passive transport: solutes spontaneously (no energy required) travel from high to low concentration All channels and some transporters use passive transport Active transport: solute travels from low to high conc.; energy is required and is carried out by transporters Electrochemical gradient: charged molecules (ions) have two gradients: electrical + concentration (chemical) Electrochemical gradient determines if passive or active transport will occur and at what rate for an ion  Membrane Transport Cytosolic side of plasma membrane usually has negative charge so positive ions tend to be pulled into the cell Sodium (Na+): low conc inside cell, high conc outside cell Sodium has high electrochemical gradient because concentration and voltage work in same direction Potassium (K+): high conc inside cell, low conc outside cell Sodium (Na+) Potassium (K+) Potassium has low electrochemical gradient because concentration and voltage work in opposite directions Membrane Transporters In active transport, membrane transporter linked to source of energy to move against gradient Gradient-driven pump: transport solutes against gradient by linking one solute that goes down the gradient to another going against the gradient ATP-driven pumps: use energy from ATP to transport solutes against gradient Light-driven pumps: use energy from sunlight to transport solutes against the gradient (bacteriorhodopsin); not covered in this course Cells use transporters and pumps to actively maintain various ion concentrations in cell Membrane Transport Na+- K+ pump: ATP - driven pump that transports Na+ outside of cell, and brings K+ inside cell (against gradient) Phosphorylation by ATP causes Na+- K+ pump to undergo several conformational changes for active transport Effects of Na+- K+ pump: Three sodium ions out, two potassium ions in Ion concentrations for sodium and potassium maintained Negative charge inside cell maintained High electrochemical gradient of Na+ is used to transport other solutes across membrane Membrane Transport Coupled transport: solute that travels down its gradient provides energy for different solute that travels against its gradient Glucose-Na+ symport pump transports glucose into intestinal cells by using high electrochemical gradient of Na+ Pump restricted to apical domain by tight junctions Glucose transported from the gut into the cell (against concentration gradient, active transport) Glucose transporters (uniports) in lateral and basal domains release glucose into bloodstream (with concentration gradient, passive transport) Neuronal Signaling Three Critical Steps for Neuronal Signaling Step one: neuron (with a negative resting membrane potential) receives a depolarizing stimulus Neuronal Signaling Three Critical Steps for Neuronal Signaling Step one: neuron (with a negative resting membrane potential) receives a depolarizing stimulus Step two: depolarizing stimulus exceeds threshold potential and activates voltage-gated Na+ channels (action potential) Neuronal Signaling Three Critical Steps for Neuronal Signaling Step one: neuron (with a negative resting membrane potential) receives a depolarizing stimulus Step two: depolarizing stimulus exceeds threshold potential and activates voltage-gated Na+ channels (action potential) Step three: sodium channels open long enough to activate neighboring sodium channels (propagation); signal travels forward towards nerve terminal  Neuronal Signaling Neurotransmitters can either excite (increase probability of action potential) or inhibit (decrease the probability of an action potential) postsynaptic neuron Excitatory Neurotransmitters Acetylcholine and glutamate are excitatory neurotransmitters that activate ion channels which transport positive ions (Na +) Influx of positive ions pushes membrane potential towards threshold for action potential Inhibitory Neurotransmitters GABA and glycine are inhibitory neurotransmitters that activate ion channels which transport Cl- ions Influx of negative ions pushes membrane potential away from threshold for action potential Protein Sorting Eukaryotic cells have many different membrane-enclosed organelles which provide intracellular compartments to perform a variety of chemical reactions Lipid bilayers on organelles provide selectively permeable barriers that allow the transport of specific molecules Each organelle contains a unique set of proteins which allows it to perform a specific function Protein sorting: mechanisms that direct proteins to their appropriate destination within or outside of the cell Proteins are initially sorted by ribosome used for translation: free ribosomes or membrane-bound ribosomes  Protein Sorting Free ribosomes: mitochondria, peroxisomes, and the interior of the nucleus receive proteins form the cytosol Membrane-bound ribosomes: Golgi apparatus, lysosomes, and plasma membrane receive proteins from ER Sorting signal: amino acid sequence in protein used to direct movement of protein inside cell Sorting signal can exist as a linear sequence of amino acids on polypeptide chain (signal sequence) Different organelles use different signal sequences Signal sequence is usually attached to N-terminus and removed once sorting process is complete Signal sequence is necessary and sufficient for protein sorting Sorting signal can also exist as a three-dimensional arrangement of amino acids (signal patch) Proteins that do not have signal sequence or signal patch are cytosolic proteins (not sorted to organelle)  Protein Sorting Free ribosomes: mitochondria, peroxisomes, and the interior of the nucleus receive proteins form the cytosol Membrane-bound ribosomes: Golgi apparatus, lysosomes, and plasma membrane receive proteins from ER Sorting signal: amino acid sequence in protein used to direct movement of protein inside cell; Sorting signals are recognized by sorting receptors that interact with membranes of organelles Sorting receptor Nuclear Transport Nuclear envelope encloses linear chromosomes and has two membranes (lipid bilayers) Inner nuclear membrane interacts with nuclear lamina Outer nuclear membrane is continuous with ER membrane Nuclear pores bypass both membranes in nuclear envelope and facilitate bidirectional traffic between cytosol and nucleus Nucleus imports histones, DNA polymerases, RNA polymerases, transcription factors, and RNA processing proteins Nucleus exports ribosomes, mRNAs, rRNAs, tRNAs and miRNAs  Nuclear Transport Nuclear pores are huge; composed of 1000 proteins (nuclear pore complex) 50 kDa nucleus ONLY if they have a sorting signal Nuclear Transport Large proteins must have correct signal sequence to enter nucleus (nuclear localization signal or NLS) NLS can be at N or C terminus of protein Nuclear proteins are translated by free ribosomes Nuclear Transport Large proteins must have correct signal sequence to enter nucleus (nuclear localization signal or NLS) Nuclear import receptor binds to nuclear localization signal on cargo protein in cytoplasm Nuclear import receptors disrupt interactions between nucleoporin proteins; can pass through using diffusion  Nuclear Transport Ran binds to nuclear receptor and provides directionality for nuclear import and export Ran is a GTP-binding protein that binds GTP and hydrolyzes it to GDP; Ran is small (