Protein Function

Question 1

Function of Enzyme

Answer 1

Enzyme facilitates conversion of a reactant into a product.

Question 2

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?

Answer 2

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.

Question 3

In Michaelis-Menten, what does Km symbolize?

Answer 3

Minimum concentration that will allow the substrate to proceed.

Question 4

Methods of Protein-Protein Interaction Visualization

Answer 4

Fluorescence Resonance Energy Transfer (FRET)
Amplified Luminescent Proximity Homogenous Assay (ALPHA)
Co-immunoprecipitation Technique

Question 5

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.

Answer 5

FRET

Question 6

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

Answer 6

ALPHA

Question 7

1. Example you have X and Y and want to know if they interact
2. Using protein A agarose bead, Antibody binds to pull down x in centrifuge
3. Remove unbounded proteins using SDS
4. See if X and Y are still there through western blot or mass spectrophotometry analysis

Answer 7

Co-immunoprecipitation Technique

Question 8

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?

Answer 8

Since IR Beta precipitated along with MHC I (as shown by band), MHC I Physically interacts with IR Beta.

Question 9

Two methods of studying Protein Function

Answer 9

Overexpression and Gene Knockout/Deletion

Question 10

Explain what is Protein Overexpression.

Answer 10

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

Question 11

Ex. If we wanted to identify the function of a CDK protein using protein overexpression, what should be the expected result how and why?

Answer 11

CDK amplification, meaning faster cell cycle, do cytometry or methods to show the faster increase in cell count.

Question 12

Explain gene knockdown to study protein/gene function in a cell.

Answer 12

Single stranded RNA is introduced which binds to the mRNA causing it to be dysfunctional and removed

Question 13

Why is Gene Knockdown imperfect?

Answer 13

• 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)

Question 14

Explain gene knockout to study protein/gene function in a cell.

Answer 14

Crispr Cas9 - Removal of start Protein, etc. Basically inhibiting expression or gene silencing (not expressed)

Compare with control to see change

Question 15

Protein that can have multiple functions

Answer 15

Pleiotropic

Question 16

T/F Denaturation with Urea is reversible?

Answer 16

True

Question 17

T/F All denatured proteins cannot renature

Answer 17

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)

Question 18

T/F Protein function is heavily related to structure

Answer 18

True

Question 19

These mediate protein folding

Answer 19

Molecular Chaperones and chaperonins

Question 20

Which amino acid residues often undergo posttranslational modification?

Answer 20

Ser, Thr, and Tyr are key residues for Phosphorylation due to present OH group. Important because usually leads to activation/inactivation

Question 21

When an amino acid substitution copies the effect of a phosphorylated amino acid residue that activates or deactivates a protein

Answer 21

Phosphomimetics

Question 22

Used to remove protein aggregation, this sets a cell to be targeted for degradation.

Answer 22

Ubiquitination

Question 23

How is Western Blot used to compare differences in posttranslational modifications (e.g. phosphorylation) between two samples?

Answer 23

It can be used to observe protein interaction through knockdown or IP studies

Question 24

What are examples of phosphomimetics in a protein?

Answer 24

Asp = Phospho-Ser
Glu = Phospho-Thr

Question 25

Phosphorylation alters _____ and ______

Answer 25

conformation and activity

Question 26

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?

Answer 26

PP4C knockdown increases phosphorylation of PLM at Ser63 in cardiomyocytes

Question 27

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?

Answer 27

Proteasome inhibition prevents degradation of ubiquitinated proteins. (Ubiquitin Tagging still goes on but without proteasomes, tagged cells are not lysed)