Q2. How can the authors determine the frequency of amino acids in the sequence?
From any one-dimensional sequence of amino acids of length l, a numerical sequence f(k) can be derived, by assigning numerical values, for example hydrophobicity scores, to the amino acids in the sequence.
Q3. What is the underlying sequence alignment required for the Fourier transform analysis of periodicity?
The underlying multiple sequence alignment, also required for the Fourier transform analysis of periodicity, indicated that the hydrophobic regions around the 7 GXXG motifscommence near the C-terminal end of each of the 7 inner helices of the α-toroid and continue tothe N-terminal region of the helix.
Q4. What is the effect of insertions between hydrophobic regions in a sequence?
Where insertions are made between hydrophobic regions in a sequence, and the length of the sequence is not kept constant, the effect would be to increase the number of wavelengths that could be fitted into the sequence as a whole.
Q5. How many random sequences were used to test the significance of the results?
In order to test the significance of the result from the olfactory protein DNA data, the RRM method was applied to 10 000 sets of 12 random DNA sequences.
Q6. What is the significance of Fourier transform methods?
In an initial test, Fourier transform methods are applied to random protein sequences to study previously reported significance levels.
Q7. How have periodicities been found in the DNA sequences?
Statistically significant periodicities have also been found through the application of Fourier transform methods to DNA sequences [5-8].
Q8. How many insertions were made at random points in the sequence?
random insertions made at random points in the sequence lowered the frequency at which periodicity wasAC CEP TED MAN USC RIP Tfound, but periodicity was still recoverable at high rates of mutation of up to 83% (here residues were removed from the end of the sequence to maintain the fixed length of 300 residues).
Q9. What is the important fact about the GCR2 periodicity?
GCR2 does indeed have 7fold hydrophobic periodicity that resides in the inner helical regions of the α-barrel and this wasidentified more strongly by the RRM method than the corresponding property in other wellcharacterized 7TM proteins such as bacteriorhodopsin and rhodopsin.
Q10. What is the frequency of the bacteriorhodopsin sequences?
The frequency of 7 corresponds to the 7 hydrophobic alpha helices in the bacteriorhodopsin structures, thus demonstrating a clear link between the Fourier transform results and structure.
Q11. How many amino acids are needed to infer periodicity?
Thus to infer periodicity in the EIIP values with a 95% certainty for a set of 20 proteins of length 300 amino acids, a single to noise ratio in excess of 98.2 is required.
Q12. What is the effect of non-periodic insertions to the periodic sequence?
The authors suggest that in this case, non-periodic insertions to the periodic sequence have distorted the frequency at which periodicity is found.
Q13. What are the results of the method?
The results highlight potential pitfalls of the method, and suggest that previous predictions of periodicity may have been over-interpreted, though they also illustrate cases in which the method can be very useful, for example in uncovering genuine low frequency periodicities.
Q14. What is the reason why GCR2 was proposed as a GPCR?
Given the negative results from the BLAST search and the transmembrane prediction algorithms, it is difficult to see why GCR2 has been proposed as a GPCR, particularly given its alignment to the Lanthionine synthetase C-like protein family.
Q15. What is the significance level of the RRM method?
Applying the same method to the 10 000 sets of random proteins of the same length gave a 99% significance level of 107, indicating that the observed peak is significant.
Q16. How was the signal-to-noise ratio calculated for P(n)?
As a measure of the significance of the resultant signal, the signal-to-noise ratio, S/N, was calculated for P(n) using Equation 3.3.