BIO250 Midterm I
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Transcript of BIO250 Midterm I
Cell Biology 250
9.1.15
Lysozome
We discussed the lysosome where proteins come and eventually get degraded.
There are a few different ways to put stuff into the lysosome: autophagy, endocytosis and phagocytosis
Tay-sachs disease we have a deficiency in one of the enzymes (beta heaminodase A) where the protein has a different structure than it’s supposed to so it can’t get into
Enzyme b hemanidosa a: degrades gangliosidesWhat we can do: screen for drugs that can alter enzyme/protein shape so that it can get transported inside the lysosome.
Cell
Has polarity (in a direction), going in one way. None of this is by accident.
How do these movements occur: cytoskeleton. Gives polarity.
In EM, we would see vesicles containing proteins going outside, etc.
Trans golgi is what step? Last! Step
Nucleus: what does it do?: regulate movement of molecules into cytoplasm/ outside.
Mitochondria:
disease angle (mutation in mitochondrial DNA)green mitochondria, red lysosomewhere cell gains ATPdefects in mitochondria: human diseases associated with defectsDoug Wallace: discovery of human disease with mitochondrial
Loss of function in oxidative phosphorylationTissues that require energy, like retina, leads to blindness. Not making enough ATP: Major issue
What is being done: Ways to do an end-run in mitochondrial defect. Mitochondrial donor!!!Pro-nuclear transfer
Remove nucleus from donor egg. Take donor oocyte enucleated . but you can have carryover of bad mitochondria, when she goes on to have children, some have defective
Fluorescence Microscopy
Tagging Proteins: GFPPick up an order of magnited
9-12: track a fluorescent protein. Actin hooked up to GFP. Use fluorescenceTag a protein with GFP. Nuclear envelope protein, lamin: Regulatory DNA. Create an animal with an extra gene with same lamin sequence with fluorescence. It’s where lamin is expressed. In other words: you can add a gene (GFP, fluorescent protein). Could affect the type of protein that you are making. Make a very different structure, sending it to the wrong place of the cell. Tag a protein: Can have consequences in the cell.GFP: gene that can glow green, protein coded by a single gene. Inserted correctly, rna can initiate transcription
Fluorescence: where is the protein found\function of protein is not changed by fusion. See localization of the GFP tagged protein, but view distribution in a living cell over time.
Tagging Protein: TetraCysteine tag of protein
Connexin (Gap junction connect cell), tag on connexin cell. After binding of a dye, can we see where it localizes. Tetracysteine tag. Done localization and process to EM,Tetracysteine allow you to create electron dense precipitate too. Can see EM of the exact same structure. Higher EM magnitude.
Localized and then in finer detail
Immunofluorescence microscopy:
Purify protein1. Produce antibody2. Localize to tissue (incubate with tissue, want to3. Fix or immobilize (formaldehyde, cross-linking agents)- stop enzymatic activity. 4. Make tissue permeable. 5. Primary antibody could be fluoresced (Easy). Fluorescent tag6. generate second antibody (Goat Ab) amplified response, get localization
What if you can’t reproduce Ab? Some proteins suck! Some ways around it, we would have to tag the protein probs. Create GLUT with tagp. 409
Detect specific proteins in fixed cells. Antibodies are proteins secreted by WBC bid with high affinity to their antigen
Drosophila w. fluorescence microscopyWg localized with red fluorescenceCo-localization. Express proteins at the same time, at the point of their intersection, can see whereve they overlap and they eliminate.You can see the point of co-localization.Red independent proteins are localized
Localized, where are the proteins interacting all with fluorescence microscopy
book: Limitation of fluorescence
fluorescent light emitted plane of focus and molecules above and below, blurry pic. Difficult for determine actual molecular arragenemnts.
Confocal Fluorescence Microscopy
Look at small plane. Obtain images from a specific focal plane and exclude light from others 3d can be seen. NO BLURRING from other background fluorescence.
Blocks all light except where you are focused.
Deconvolution Fluorescence Microscopy
Get rid of light, without pinhole? Remove fluorescence remove how much fluorescen it contributes. Detail without Gets reassigns out of focus photons to their site of origin.
Tirf Microscopy
Internal reflection. Only looking at an excitable at an angle, so light barely pierces the crystal. Happens at the contact point between cell and right at contact. We can look at proteins at adjesion sites ad look at kinetics with microtubules.
Electron Microscopy
Magnets condense beams. Electrons pass through. Metal binds tightly to tissue. Not blocking electrons. Tissues are dead. Localize enzyme in very fine detail. Localization Immuno + EMLocalize catalysae, generate an antibody. And with EM, find electron dense staining. Use gold molecules. To protein A. and get very fine localization.
Bridge chem + bio with these techniques
Summary: Major functions of cell that we’lll discuss. 1. Tay-Sachs Disease: gangliosides, B-Hex A; make drugs that allow enzyme to change structure to go into lysosome.2. Cell has polarity: cytoskeleton, EM shows us this,3. Mitochondria, defects in human disease; oxid phosphor; pro-nuclear transfer for mitochondrial
9.4.15
Electron MicroscopyTransmission, block throughScanning- sample coated with metal and reflects electrons, so we get a surface view, structure of head of drosophila etc. different levels of organization.
TEM with collagenSEM: whole cell, fairly fine level at cellular level (not sub-cellular)
FIX SAMPLE, DEHYDRATE, IMPREGNATE WITH PLASTIC, CUT SECTIONS STAINED WITH HEAVY METAS. THIN SLICE OF CELL. Electron dense marker
FACS
Live cell, incubate with AB, and fluorescence. Sort cell by level of fluorescence. Look at multiple markers (Red and green aB), select for cells with diff proteins
TRANS-GENIC FISH, neural protein that can make it glow. Make brain cells. Cell mixture incubated to a dye, linked to an antibodyAll about intensity and different Cool graph with FACS
Proteins
Secondary structure: do something different, structure and functionAlpha helix: face on one face, periodicity where R groups are. Beta sheet: pleated, kinked series of steps, R group in DIFFERET DIRECTIONS
Hydrogen bond across sheets (looks like dry wall). Cells due use this to their advantage to make channels or pores
Alpha helix: create a face with hydrophobic amino acids. In solution, they come together, alpha helix, tightly bonded. Highly charged surface too.
Beta turns: structure and reverse direction (Transcription factors) have helices that bind together tightly. Attached to other domain. Helix turn and another domain. DNA bound tightly to dimer. ]
Motifs (Little)
Charged groups face outside
Coil-coil motif and we create a dimer. Charge on one side and other amipathic helix, dimerizes with hydrophobic residues. Leucine zipper (Transcription factor) high frequency on one side. One motif.
Calcium sensingEF hand/helix-loop-helix motifInteracts with calcium. Super important signaling molecules. Want low Ca in cytosol. Come from outside or within cellCalmodulin- interacts with EF hand motif, interacts with AA. Coordinate with calcium, changing shape of protein!Calcium + calmodulin -> protein diff conformation, different areas.
Zinc-finger motifDna binding proteins regulate transcription
Domains (Big protein)Flu-virus protein that mediates infection. What types of domain that interacts with receptor?Globular domain
Trans-membrane domain, specificityTwo domains separated: fibrous domain, not highly structured (length)
Often site of recombination, re-assortment of domains.
Quaternary Structure
all bout the combination of proteins together!
3.2 Protein Folding
ChaperonesMost stable configuration, why do we end up with one structure. CELL helps nascent proteins. Chaperone proteins help to make folds correctly.
HSP 70HSP 70 HEAT Shock protein?
Traumatize a protein at a high temp denature and it might not fold right back up the same way Every organism has a heat shock response to refold proteins, fold nascent proteins. Two domains
1 atp, AND GROWING INTERACTS at atp form, when it hydrolses. Hydrophobic pocket occupies resides and helps it find most stable hydrophobic associations.
Proteins can crash to each other. Chamber where AA slowly tested without negative effects. Chamber where you can form proteins.
Protein folding fails, sometimes. Even with normal proteins equence fails, or a mutation in a polypeptide sequence, which predisposes protein to misfold during the process or with chaperon.
Alzheimer’sAlzheimer’s plaque: proteins misfold. Neurodegenerative disease,
Wild type: alpha- helix, Mutation or spontaneous conform. Change. Second conformation it can multimerize, main issues with disease, or intermediates are toxic
Protein: change in conformation, crash out of solation.
Three ways to prevent or treat neurodegenerative disease:
1. Drug one: slow or prevent alpha helix flipping into beta sheet2. Between beta sheet structures and prevent them from forming. The multimers
3. TOO Late: Multimers in a disease state, consider: split apart or DISAGGREGATE. One possible mechanism.
Read abstract: HSP 70, exploit chaperons up regulate HSP 70. We would reverse Multimeric assemblies with and without HSP. Up regulate in tissue, fibrils begin to degrade
FIRST STEP TOWARDS TREATMENT OF NEURODEGENERATIVE DISEASE
Degradation
Regulate protein: how much synthesized, degraded, activity level of enzyme
Aberration in protein. Protein mutant shape, aberrated: recognized and destroyed. Regulate at a certain or critical level.
Main players: Ubiquitin and Proteasome
Attach Ubiquitin at a polypeptide Lysine residue. Ubiquitin going to add on to that protein. Also has lysine residues. Red flag on protein. Poly ubiquination.
Goes to proteasome that has a cap on two ends that recognize polyubiquitin, once it sees it and binds to protein through ubiquitin. Cut after basic amino acids
Normal protein not folded by chaperon- ubiquiting ligase target proteins and efficient at recognizing motifs and domains. Needs ubiquitin on
Lysine residues.
Nascent-> cytoplasmic protein goes to proteasome.
9.8.15
How to isolate proteins; purify.
Centrifugation
Separate Organelles and proteins: centrifugation. Nuclei go to down. Supernatant on top. Or separate them in a gradient.
SDS Gel
Use SDS: turns monomers, opens up a polypeptide to stretch out through lengthDetergent SDS binds hydrophobic areas on protein and add negative charges
SDS -> NEGATIVE charges. Got rid of multimers. FROM NEGATIVE TO POSITIVESeparate by SIZE.Many times we grind up a cell.
Identify a single polypeptide on a WESTERN blot.
Two-dimensional gel electrophoresis Isoelectric focusing, protein moves under charge.
Two polypeptides with exact same length. One positively charged, one neg. charged. Separate by charge not weight. Create a gel without SDS, but use ampholytes with a ph Gradient. Isoelectric focusing, protein moves under charge.
Separate proteins and by size
Grind up cell lysate w. a complex mix of proteins1. run isoelectric focusing separating acidic vs. basic class2. attach to sds, layering the first physically on top of first3. separate by size.
Get a lot of detail. Mutant vs. wildtype: do spots appear or disappear? Sequency polypeptide in spot and sequence that dna sequence to isolate gene, and you know what gene is used.
Liquid Chromatography/ gel filtration/ column
Large and small. Gell filtration With beads and diff sizes. Large proteins fly through column and ignore the beadsSmall want to be with beads Separate by size. Big guys quickly, sds they are slow
Ion Charges
opposite charges attract. With electrostatic.Adding a higher concentration of sodium that outcompetes a neg charged and collect different fractions. Like isoelectric focusing. Fine separation.
Antibody-Affinity Chromatography
A little easier to purify or isolate a complex the multimeric association. Grind up cell with a column. Bead with antibody. Separate protein recognized by antibody.
Membrane Structure
1. Proteins through membrane: integral (outside and in) attached to membrane2. Lipid-anchor protein: doesn’t go through completely. 3. Peripheral membrane: outside or inside, and come off easily from membrane. Add salt they are removed easily (Electrostatic interactions)
HIV, buds off from lymphocytes from cell membrane. The more we know, huge considerations.
Not memorize structures.
LipidsMembrane lipids, amphipathic:
phosphoglycerides, are in bilayer with carged head groups (face extracellular side) acyl hydrocarbon chain. Don’t have any OH,
Sphingolipids will bond better to other lipids. Make the protein be less mobileIn general: these are stiffer (less fluid) in the membrane. The chemistry depends how fluyd. produced in golgi and face extracellular domain in plasma membrane.Tay0sachs can’t clear out sphingolipids (glycolipid)
Cholesterol- highly or less likely fluid
Ampipathic, OH on side of aqueous. Key constituent of membranes.
Three classes of lipids
Saturations: are important
Liposome1. use to deliver drugs (Fuse with cells) Can see what you can alter with liposome.
Purify proteins out of membranes with an organic solvent.
VesicleBi-directional. Whatever is in the lumen is extracellular Something is endocytosis, cytoplasmic whatever is from outside the cell is in the lumen.
Modifications (Enzymatic level) is done in the lumen.
Fluidity
Temperature dependent, can be measured.
FRAP (fluorescence recovery after photobleaching)Determine rate of recovery and extent of proteins or lipidsLabeling membrane proteins with a fluorescent tag on it. How fluid is membraneBleach area and see how efficiently bleach molecules turn back to fluorescence.
Results: never really achieve 50% of the fluorescence. Liposome with only fluorescent protein we get 100%Proteins and lipids in cell membranes don’t flow around, there are restrictions to flows.
What modifies fluidity? (Temp, sphingolipids, cholesterol, how long the tails are, saturated or unsaturated, and interactios of proteins)
Lipids RaftsIdea that cell membranes aren’t a mix. Within cell membrane, area distinguishable by less mobile (lipids rafts)
Enrichment of sphingomyelin(lipids) and cholesterol.Those two make you a lot slower and denser in plasma membrane. These areas are where proteins in cell signaling are enriched.
FRET: MEASURING DISTANCE
If molecules proteins are close to each other, you can emit a wavelength that is higher.
When we add calcium binds to calmodulin and CTP and YFP go to FRETGoing to measure calcium concentrations. Look to see if you get FRET or not.Extent of calcium conc. A lot of 535.
Cell signaling + lipid rafts + fret
Experiment: calmodulin + RGS4 protein responds to calcium. Targeted to specific part of membrane or not?ECFP (Attaches to calmodulin) Raise calcium, calmodulin changes shape and interacts with RSG4CFP and Venus are close enough to make a FRET reactionsCFP and YFP brought within 10 nm. Calmoduin interacts with protein it regulates.
You can deplete lipid rafts!!!!!
If you deplete lipid rafts you should see less activity. Calmodulin + rgs4 want to find each other.
Another experimentCAV1 protein binds to cholesterol
Notch sits in rafts. It’s not everywhere. Signaling molecules function within lipid raft regions of membranes. Occurs in a subset of cell membrane.
9.10.15
Complexity of membrane structure. Disproportion. Domains of activity in key places in cell.
Phospholipids, susceptible to enzymatic activityPhospholipases C cuts.
Enzyme cuts used to degrade. Particular classes are signaling molecules. Enzymatic. Occupy. Not just structural.
Membrane Protein Components and Function
33% gene encode a membrane protein,
1. Proteins through membrane: integral (outside and in) attached to membrane2. Lipid-anchor protein: doesn’t go through completely. 3. Peripheral membrane: outside or inside, and come off easily from membrane. Add salt they are removed easily (Electrostatic interactions)
Transmembrane proteinDistinct run of uninterrupted hydrophobic amino acids
Talk about ampipathic (coil coil)Hydrophobic residues, leucine zippers. Transmembrane to exist in lipid bilayer, transmembrane hydrophobic surfaces on both sides. Hydrophobic on both side
Glycophorin: integral membrane proteinProtein can pass by membrane many times