Question

Shown following is the first 30 amino acid (one-letter code) region of this putative amino acid sequence for p18:

¹YFNPS     ^6TSDWPT    ¹¹LAPAN    ¹⁶YTFLF    ²¹FLARY    ²⁶WYINL³⁰

A) Based on the partial amino acid sequence provided, do you think that it is more likely that this region of p18 associates with a cellular membrane or is exposed to the cytosol?

B) Why or why not?  

C) Describe how you would experimentally demonstrate if p18 was associated with the membrane or the cytosol.

Answer 1

A. The P18 region of the protein associates with the interior of cell membrane as it has more hydrophobic amino acid. Hence, it will repel water.

B. ¹YFNPS     ^6TSDWPT    ¹¹LAPAN    ¹⁶YTFLF    ²¹FLARY    ²⁶WYINL³⁰

The amino acid sequence in 3 letter-code is

1 Tyr-Phe-Asn-Pro-Ser

6 Ser-Asp-Trp-Pro-Thr

11 Leu-Ala-Pro-Ala-Asn

16 Tyr-Thr-Phe-Leu-Phe

21 Phe-Leu-Ala-Arg-Tyr

26 Trp-Tyr- Ile-Asn-leu

1. 1 Tyr-Phe-Asn-Pro-Ser

Hydrophobic amino acids: 3

Hydrophillic amino acid=2

2. 6 Ser-Asp-Trp-Pro-Thr

Hydrophobic amino acids: 2

Hydrophillic amino acid=3

3. 11 Leu-Ala-Pro-Ala-Asn

Hydrophobic amino acids: 4

Hydrophillic amino acid=1

4. 16 Tyr-Thr-Phe-Leu-Phe

Hydrophobic amino acids: 4

Hydrophillic amino acid=1

5. 21 Phe-Leu-Ala-Arg-Tyr

Hydrophobic amino acids: 4

Hydrophillic amino acid=1

6. 26 Trp-Tyr- Ile-Asn-leu

Hydrophobic amino acids: 4

Hydrophillic amino acid=1

Total number of hydrophobic amino acids: 21

Total number of hydrophilic amino acids= 9

As the number of hydrophobic amino acids are more than hydrophilic amino acid , the protein will associate with the cell membrane.

C. Integral membrane proteins associate with the plasma membrane permanently. Peripheral membrane proteins face the cytosolic side and are transiently associated with the membrane and can be removed by altering pH or salt concentration. In order to determine whether the protein is integral or peripheral, the protein sequence is first expressed in a cell line. Expression of the protein can be detected by western blot or immunoflourescence technique.

The cells can be washed with cold PBS and harvested by scraping in 5 mM Tris/HCl pH 7.0 supplemented with protease inhibitor cocktail (PIC). Extracts are centrifuged at 4 °C for 5 min at 10,000 g and resuspended in buffer with PIC. Pellets can be dispersed by vortex and through a syringe. Nuclear fractions and other debri are removed by centrifugation at 4 °C for 5 min at 500 g. Homogenates can be subjected to ultracentrifugation at 4 °C for 1 h at 400,000 g to obtain cellular fractions. The pellet fraction containing the membrane fraction is resuspended extraction buffer with PIC. Precipitation can be done with chloroform:methanol (1:4 v/v). The precipitated cell membrane fraction can be treated with sodium carbonate at pH 11.54 °C for 40 min. Control samples are treated with same conditions, without the sodium carbonate with either pH 11.5 or pH 7.5. The fractions are repurified as membrane fraction and soluble fractions and proteins can be detected with western blot techniques using antibodies against p18.

If the protein is an integral membrane, soluble fraction after sodium carbonate treatment will not show a band. The membrane fraction should show a band. If the protein is peripheral, then the protein will not be seen in the membrane bound fraction but will be present in the soluble fraction.