P.F.S.R.L. - AP Mutants


mutants

Displayed above is raw data collected from our PMT during Phosphorescence experiments with WT-AP and several mutants. The different slopes to the decay curves indicate different RTP lifetimes. The insert shows tryptophan 109 and the local of the amino acids mutated in our lab.

In order to test theories about the correlation between local environmental effects and the RTP lifetime of tryptophan residues (and more generally about the correlation between internal structure and global stability) we made several amino acid mutations within the core of AP near the phosphorescing Tryptophan (W).

Tryosine (Y) amino acids are known quenchers of phosphorescence of NATA in solution6 and Y84 is very close to W109 in AP. Changing this Tyrosine to a Phenylalanine (F) residue, which is roughly the same size as Tyrosine but not as strong a quencher increased the RTP lifetime (the Y84F mutant) relative to the wild type (WT) protein. If the Tyrosine was replaced with a Glycine (G), which is not an effective quencher, but is much smaller that either Tyrosine or Phenylalanine, the RTP lifetime (the Y84G mutant) is decreased relative to WT. This can be attributed to a decrease in the local rigidity of W109's environment because of the increased size of the cavity caused by the small glycine.

The Glutamine residue (G) at position 320 is thought to hydrogen bond to W109 and therefore stabilize it. This stabilization is though to increase the RTP lifetime by making the environment of W109 more rigid. If this residue is replaced by a Leucine (L), which is roughly the same size as Glutamine but can't hydrogen bond to W109, the RTP lifetime is decreased compared to WT. If this residue is replace by a Glycine the lifetime drops even further, which can, as before, be attributed to an increase in the cavity size around W109.

While changes in the RTP lifetime can be drastically altered by these mutations, enzyme activity and thermodynamic stability of the holo enzymes are less affected.


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