Download Questions for Discussion or Assignment to Accompany the Ubiquitin

Questions for Discussion or Assignment to Accompany the Ubiquitin Laboratory
1a. Explain why no 1H signal due to the protein could be observed with the pulse-acquire
experiment.
1b. Compare the concentration of protons in water to the concentration of the protein.
What does this mean for a 16-bit receiver?
2. (a) What was your 90-degree proton pulse length, PW1, and the associated power level,
PL1, in dB? Also state the power level used for presaturation (PL2). (b) Find PW2, the
length of the 90-degree pulse at PL2 , using the following relation
PL2  PL1  20 log
PW1
PW 2
This assumes that the amplifier is linear and assumes that PL2 and PL1 represent
attenuations (i.e. higher attenuation is less power). (c) Now calculate the 360 degree
pulse at PL2. (d) Assuming a presaturation pulse of 4 seconds, how many revolutions
does the water magnetization vector undergo if the pulse is perfect? (e) Assume that pulse
imperfections cause the magnetization vector to dephase by 2% of its magnitude on each
complete rotation. What is the residual water magnetization?
3a. Count the number of signals in the two-dimensional 15N(1H) HSQC.
3b. Given that the 15N(1H) HSQC selects only for signals from R2NH groups, suggest
why the number of signals is less than the number of amino acids.
4a. Use concepts of protein secondary and tertiary structure to explain why the
‘constellation of stars’ distribution of signals that you observe in the hsqc is highly
characteristic of a folded protein. (hint : it may help to make a comparison to the case of a
denatured/unfolded protein)
4b. It is found that proteins that are largely alpha-helical show less dispersion in the 1H15
N HSQC than proteins with substantial beta-sheet composition. Suggest an
explanation.
5. By comparison to the known assignments, locate the signal due to ILE36 and
comment upon its 1H and 15N chemical shifts. You should download the structure file
from the protein data bank and examine it with Rasmol (pdbid : 1D3Z). Make a print out
of the protein with ILE36 clearly indicated, such as with a different color.
6. Investigate the literature for ubiquitin to find which region/s of the protein are
responsible for its function. Can you make any correlations with the HSQC?