Determine the residue number at location: C: D: E: F: G: H: I: As a side note, C

Question

Determine the residue number at location:

C:

D:

E:

F:

G:

H:

I:

As a side note, C is 220 and D is 240.

You are studying a multi-span membrane protein. Your goal is to determine the orientation of the protein in the plasma membrane. You have two antibodies, one specific for the N-terminus of the protein and the second specific the C-terminus. You obtain the following data: (1) A SDS PAGE gel of intact undigested protein gives the pattern shown in lane 1 of the gel pictured below. (2) A SDS PAGE gel of the protein after the intact cell is digested [from outside] with a protease is shown in lane 2 of the gel pictured below. (3) A SDS PAGE gel of the intact protein after in vitro translation in the presence of ER microsomes is shown in lane 3 of the gel pictured below (4) A SDS PAGE gel of the protein after in vitro translation in the presence of ER microsomes followed by protease treatment is shown in lane 4 of the gel pictured below (5) The corresponding Western blots of the same four samples (lanes 1-4) are shown below. The blots were probed with either the anti-N-terminus antibody or with the anti-C-terminus antibody 1 23 4 1 23 4 1 2 3 4 225 kD-> - 104 kD 54 kD 42 kD 31 kD SDS gel Anti-N-term antibody Anti-C-term antibody western blot western blot Based on these data, determine the arrangement of the protein in the plasma membrane: which side is each terminus on, and what are the amino acid residue numbers at the boundaries of the membrane spans? Assume that one amino acid has a MW of 100 Da (0.1 kDa), and that membrane-spanning domains are exactly 20 aa (2 kD). A diagram of this protein is illustrated below. The termini are labeled A and B, and the locations of landmark amino acid residue numbers are indicated by C-I extracellular cytoplasm

Explanation / Answer

N-terminus is at location A and the C terminus is at location B.

Determine the residue number at location:

C: 310

D: 316

E: 724

F: 731

G: 933

H: 940

I: 2250

The point I and B is equal. According to the given data, the total amino acids in the protein is 2250.

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