Unit 3 Exam Flashcards
What are the two large categories of protein-protein interaction methods?
- In the organism
- Immunoprecipitations → Separate proteins using antibodies
- Biotinylation → tag proteins with biotin
- FRET → Fluorescence based approach
- BIFC → Fluorescence based approach - In an unrelated organism system or in a test tube
- GST-Pulldown → test binding in a test tube
- Two-hybrid → bacteria or yeast
What do electrophoresis and western blot allow us to do with proteins?
They allow us to detect proteins in a sample.
Generally:
- Separate proteins by size
- Use a dye that reveals all proteins
- Use antibodies to detect specific proteins
What do Co-immunoprecipitation experiments detect? List methodology. And then connect to Western-blot.
Co-immunoprecipitation experiments detect protein-protein interactions
Methodology:
1. Antibody recognize bait protein
2. Beads take the proteins out of solution (they sink)
3. Remove the unbound stuff
4. The beads in the bottom contain the bait protein with all the interacting proteins
5. Reveal the proteins using western blot
Key - Connection to western blot:
- cdc2 binds cyclinB
- One target at a time, time-consuming
What if there is no antibody available for the protein to fuse to?
If there is no antibody available, fuse the protein to a tag
Antibodies for tags are always available
Ex: His and FLAG
Proximity biotinylation is a new method that involves what?
Proximity biotinylation is a new method that uses a Biotin ligase to mark proteins that are close
Methodology:
1. Fuse the bait protein to a promiscuous biotin ligase (BirA)
2. BirA add biotin (Vit. B7) to exposed lysines on neighboring proteins
- the addition of biotin is called biotinylation
How can we tell where in the cell an interaction is occurring? What are the disadvantages and advantages of FRET & BIFC?
We can use fluorescence methods tell where the interaction happens
- Ex: Split-GFP, FRET (uses 2 proteins)
(-) Disadvantages of BIFC and FRET
- Only two proteins at a time
- Time-consuming
- Need a fluorescence microscope
(+) Advantages of BIFC and FRET
- Single cell resolution or better
- Temporal resolution
A yeast two-hybrid (Y2H) experiment detects the physical interactions of what? Does the yeast change color? Why?
What?
- detects the physical interactions of proteins through the downstream activation of a reporter gene.
Color-change?
- Reporter gene makes yeast blue
- Pick the blue colored yeast
- Sequence the blue colonies to reveal the interacting proteins.
Using knowledge of the yeast two-hybrid (Y2H) experiment, explain how the GAl4 transcription factor protein is used together and apart.
- The GAl4 protein is a transcription factor binds to the UAS sequence
- This protein is cut into 2 parts: AD and BD
- One is fused to the prey protein
- The other is fused to the bait protein
- If together, a functional Gal4 protein will form and activate transcription of the reporter gene
What is a neuron? What’s its task? Why is it discrete?
- Every neuron consists of a cell body (containing the nucleus) with usually one long axon and several shorter, branching dendrites
- Task of a neuron, or nerve cell, is to receive, integrate, conduct, and transmit signals
- Neuronal signals are transmitted from cell to cell at specialized sites of
contact known as synapses - Neurons are discrete cells as they choose to transmit info or not, they also undergo changes in membrane potential
When a neuron is not active, its membrane potential is called resting potential. Explain this concept, compared to action potential.
Resting Potential
- Excitatory signals open cation channels, depolarizing the membrane
- Inhibitory signals open either Cl- channels or K+ channels, suppressing firing and depolarizing the membrane.
Action Potential
- The changes in the Na+ channels, K+ channels, and current flows give rise to a traveling action potential.
Explain how membrane potential propagates through the membrane.
- Voltage-gated Na+ channels transmit signals in a wave through the nervous system.
- High difference = tightly closed
- Low difference = open and ions pass
- Voltage gated Na+ channel starts the depolarization phase by obv. depolarizing the membrane
How are membranes repolarized?
- Repolarization of the membrane requires opening of additional channels, the delayed K+ channels.
- These channels also open in response to membrane depolarization (that is, they are also voltage-gated), but have slower kinetics.
- Voltage gated K+ channel
- Ends the depolarization phase
Explain the 4-step (summarized) process of how neurons communicate at CHEMICAL synapses.
- When an action potential arrives at the presynaptic site, the depolarization of the membrane opens voltage-gated Ca2+ channels that are clustered in the presynaptic membrane.
- Ca2+ influx triggers the release into the cleft of small signal molecules known as neurotransmitters that are stored in membrane-enclosed synaptic vesicles and released by exocytosis
- The neurotransmitter diffuses rapidly across the synaptic cleft and provokes an electrical change in the postsynaptic cell by binding to and opening ligand-gated ion channels
- When the incoming action potential activates a Ca2+ channel in the T-tubule membrane, it triggers the opening of a Ca2+-release channel in the closely associated sarcoplasmic reticulum membrane
intracellular signaling molecules vs extracellular signaling molecules?
Second messengers are intracellular signaling molecules released by the cell in response to exposure to extracellular signaling molecules (aka first messengers)
Explain G-Protein-Coupled Receptors (GPCR)? What is the process of activating a G-protein?
GPCR
- Act by hydrolysis of GTP
- Molecules bind to their ligands, then transmit this signal across the membrane to heterotrimeric G proteins.
Process
- When the GPCR binds its ligand it binds and “activate” the G protein
- The activated G proteins then trigger a cascade of signals inside the cell
- G proteins are only active for a very short period of time
- Ex: some G proteins activate Adenylyl cyclase
Overall: GPCRs act through PKA or IP3
What is cAMP’s role in G-Protein-Coupled Receptors (GPCR)? How does it activate cAMP-dependent protein kinases?
- cAMP is a second messenger of some G proteins
- cAMP activates PKA (a cAMP-dependent protein kinase)
Activation of PKA?
- cAMP binds the regulatory subunits and removes them
- Protein Kinase A (PKA) then phosphorylates many target proteins
Other than cAMP, GPCRs also use ______ as a second messenger. Explain what this messenger activates.
- CPCRs use IP3 as a second messenger
- IP3 activates a Ca2+ channel at the ER
- IP3 receptors = ligand-gated ion channel
Explain enzyme-coupled receptors/protein kinase receptors?
- Activation induces a conformational change exposing the kinases
- Insulin Receptor is an example of tyrosine kinase receptor (has an extracellular binding pocket for insulin, a transmembrane domain and intracellular tyrosine kinases)
The last type of receptor is a nuclear receptor. Explain what steroid hormones are, and what these receptors directly interact with. Use the example of molting hormones in bugs.
- steroid hormone is a steroid that acts as a hormone
- Nuclear receptors often directly interact with DNA
- Nuclear hormone receptors are acting as transcription factors in the cell nucleus. They regulate gene expression of hormonal regulated target genes. The role of hormone in the transcriptional process is to modulate and change the nuclear receptor functionality.
- Ecdysone is a steroid hormone that controls insect molting
Explain a quick overview of the cell cycle.
- In the G1 phase, the cell synthesizes all of the proteins, ribosomes, and other machinery that it will need.
- In S phase DNA is replicated.
G2 is a gap phase After a gap phase called G2 - In M phase the cell divides
In S phase the chromosomes are_______, while in M phase, duplicated chromosomes are ______ into a pair of ________________.
Duplicated, segregated, daughter nuclei
What is flow cytometry? What can it measure? Importance?
- Flow cytometry is a laser-based technique used to detect and analyze the chemical and physical characteristics of cells or particles
- Measures fluorescently labeled cells (forward and side light) and the amount of DNA using a fluorescent dye
Importance?
- The amount of DNA in each cell tells the cycle phase
The control system can arrest the cell cycle at specific checkpoints. List some reasons why cells may be stopped. How do these checkpoints operate generally?
Reasons
1. Incomplete DNA replication
- Entry into mitosis is prevented
- Chromosomes not properly attached the mitotic spindle
- Chromosome separation in mitosis is delayed
General operation:
- Checkpoints generally operate through NEGATIVE INTRACELLULAR signals
