Question

can anyone help me with this cell bio question?

In Figure 2 part A (Non-denaturing). Lane 1 NEP( E403C) , 2 = WT ECE, , 4= WT= NEP, 9= ECE(C416E). Figure 2 part B. The same as part A, but under denaturing conditions. (note; for simplicity, many lanes are ignored) 1 2 3 5 67 89 10 a -213 -123 -85 -50 b kDa -213 -123 85 -50 Figure 2 Immunoblot analyses of wild-type and cysteine mulants of NEP and ECE / 2 2 in this studay, the autnors were expioring the Simiiarity/aITTerences betweeri two proteins that were both zinc metalloproteases: neutral endopeptidase (NEP) and endothelial-converting enzyme (ECE). ECE has a disulfide located at CYS412, whereas NEP equivalent position has a GLU408 that does not produce a disulfide bond. The authors used a genetic manipulation tool called mutagenesis to change the CYS in ECE at position 416 to GLU (C416E) and the GLU in NEP at position 408 to CYS (E403C). They then used a technique called western blot to verify the change and then tested the enzyme kinetics of the mutagenized NEP (E403C). *A change of an amino acid at a particular position is designated as follows: change the CYS (C) in ECE at position 416 to GLU (E) = ECE (C416E). A Western blot involves separating proteins on a native (non-denaturing) or SDS (denaturing) -PAGE gel, transferring the separated proteins to blot paper and then using antibodies specific for your protein of interest to locate the proteins. Questions (figure 2): Answer on a separate paper 1. What unique bond can form when you have two cysteine residues in a protein? What is the difference between Figure 2 part A versus part B? Why was this purpose of showing 2. this? For figure 2a, which are the control lanes? Which are the experimental lanes? 3. By looking at figure 2a and b, how would you know whether a protein had a disulfide 4. bond or not? 5. Were the authors successful (see question 1)?

Answer 1

Answer 1:-

1. The amino acid called cysteine is responsible for formation of disulfide bond. This bond is formed between the two sulfur atoms present between the two molecules. It is a strong covalent bond.

Answer 2:-

1. NEP (Neprilysin) and ECE(Endothelin converting enzyme) have been stated in the above immunoblot.
2. They have a motif at Carboxy-terminal which shows the presence of cysteine amino acid as a part of conserved sequence.
3. This is involved in protein folding. Site directed mutagenesis is used to create mutant.
4. In figure A, immunoblot assay is done with the wild type and in figure B, immunoblot assay is done with the mutant. This is carried out to deal with the functional aspect of the motif.