The difference between the spectra of the two populations suggests different demographic histories; the T. aestivum spectra resemble those typical of an expanding population whereas wild emmer spectra resemble a spectrum generated by a past bottleneck [ 36].
If the tryptophans did fluoresce independently of each other, the wild type spectrum should be identical to one-third of the sum of the four spectra of the single mutants [12] (Fig 1C).
The generated spectra were compared to the predicted mass-peaks of the sequence and several wild type spectra.
However, a positive peak at 230 nm in the wild-type spectrum was not observed in the W255G spectrum.
This result indicates that substitution of Tyr236 or Tyr229 specifically suppressed the other primary Tyr site in CcP, leaving just the minor species (also Tyr radical, <15% contribution to the wild-type spectrum).
In the wild-type spectrum the minor slow exchange state represented only 12% of the total population, whereas in the G89A variant the analogous set of cross-peaks yielded 67% of the protein signal.
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Base substitution biases were calculated using the Fisher exact test to compare with a wild-type mutation spectrum derived from the literature (Lynch et al. 2008; Lang et al. 2013).
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