Protein Function Flashcards
Function of Enzyme
Enzyme facilitates conversion of a reactant into a product.
Given two curves, one for enzyme activity of a substrate and another of a substrate with a competitive inhibitor (binds to the active site). What is your expected trend or pattern in the graphs?
In a Michaelis Menten Graph, substrate+inhibitor curve would be lower than the substrate only.
In lineweaver burke, slope of substrate+inhibitor would be higher than substrate alone.
In Michaelis-Menten, what does Km symbolize?
Minimum concentration that will allow the substrate to proceed.
Methods of Protein-Protein Interaction Visualization
Fluorescence Resonance Energy Transfer (FRET)
Amplified Luminescent Proximity Homogenous Assay (ALPHA)
Co-immunoprecipitation Technique
Visualization method where when you excite one protein, the energy can transfer to another that emits its own light. Since different proteins resonate with different wavelengths, this causes different lights wavelengths to be emitted.
FRET
Instead of energy transfer, there is a specified donor and acceptor
Donor contains an enzyme or material that is needed by the other
Once transfer of needed material happens, light emits from acceptor
Bridge formed by Biotinylated P53 and GST-Tagged HDM2 form bridge
□ Streptavidin coated Alpha Donor - Binds to P53
□ Anti-GST conjugated Acceptor - Marked by antibody detecting HDM2
ALPHA
- Example you have X and Y and want to know if they interact
- Using protein A agarose bead, Antibody binds to pull down x in centrifuge
- Remove unbounded proteins using SDS
- See if X and Y are still there through western blot or mass spectrophotometry analysis
Co-immunoprecipitation Technique
In a western blot, with IR Beta, MHC I, Glu R1, and GAPDH in the lysate, after immunoprecipitating IR Beta, there are no bands for Glu R1, GAPDH, and a band in IR Beta for MHC I, what does this mean?
Since IR Beta precipitated along with MHC I (as shown by band), MHC I Physically interacts with IR Beta.
Two methods of studying Protein Function
Overexpression and Gene Knockout/Deletion
Explain what is Protein Overexpression.
Expression plasmid is introduced into the nucleus and will overexpress the protein (i.e. paparamihin) therefore compare it with a control then see the phenotypic changes
Ex. If we wanted to identify the function of a CDK protein using protein overexpression, what should be the expected result how and why?
CDK amplification, meaning faster cell cycle, do cytometry or methods to show the faster increase in cell count.
Explain gene knockdown to study protein/gene function in a cell.
Single stranded RNA is introduced which binds to the mRNA causing it to be dysfunctional and removed
Why is Gene Knockdown imperfect?
- Chance for different binding
- Chance for continued expression if transcription/translation is fast enough
- Chance for no change due to slow degradation
- Protein might be stable (cannot be knocked down)
Explain gene knockout to study protein/gene function in a cell.
Crispr Cas9 - Removal of start Protein, etc. Basically inhibiting expression or gene silencing (not expressed)
Compare with control to see change
Protein that can have multiple functions
Pleiotropic
T/F Denaturation with Urea is reversible?
True
T/F All denatured proteins cannot renature
False, depends on method (i.e. eggs that got fried will no longer be reversible but milk that was warmed can still return to its original conformation)
T/F Protein function is heavily related to structure
True
These mediate protein folding
Molecular Chaperones and chaperonins
Which amino acid residues often undergo posttranslational modification?
Ser, Thr, and Tyr are key residues for Phosphorylation due to present OH group. Important because usually leads to activation/inactivation
When an amino acid substitution copies the effect of a phosphorylated amino acid residue that activates or deactivates a protein
Phosphomimetics
Used to remove protein aggregation, this sets a cell to be targeted for degradation.
Ubiquitination
How is Western Blot used to compare differences in posttranslational modifications (e.g. phosphorylation) between two samples?
It can be used to observe protein interaction through knockdown or IP studies
What are examples of phosphomimetics in a protein?
Asp = Phospho-Ser
Glu = Phospho-Thr
Phosphorylation alters _____ and ______
conformation and activity
Check pdf for graph:
Cardiomyocytes transfected with non-targeting control siRNA or siRNA against protein phosphatase 4C (PP4C) were exposed to vehicle control (−) or H2O2 (+) for 24 h and immunoblotted for phospholemman (PLM) and phospho-PLM-Ser63
It was found that %Phosphorylation relative to control is significantly higher in the one treated with siPP4c with a new band forming udner PLM-pSer63, what does this mean?
PP4C knockdown increases phosphorylation of PLM at Ser63 in cardiomyocytes
Check pdf for the blot:
Western blot analysis of cultured adult rat ventricular myocytes exposed to the proteasome inhibitor MG132 (1 μM) for 0, 2, 4, 8, and 24 h using an anti-ubiquitin
antibody.
Blotting shows the band intensity increasing as time increases, what does this mean?
Proteasome inhibition prevents degradation of ubiquitinated proteins. (Ubiquitin Tagging still goes on but without proteasomes, tagged cells are not lysed)
