bc0502762_si_001.pdf (184.42 kB)
Surface-Assisted Delivery of Fluorescent Groups to hGST A1-1 and a Lysine Mutant
journal contribution
posted on 2006-03-15, 00:00 authored by Johan Viljanen, Lotta Tegler, Jenny Larsson, Kerstin S. BrooHuman glutathione transferase (hGST) A1-1 and a lysine mutant (A216K) can both be rapidly and site-specifically
acylated on Y9 and K216, respectively, using a range of thiolesters of glutathione (GS-thiolesters) as modifying
reagents. The present investigation was aimed at developing a method with which to deliver a fluorescent acyl
group from a solid support under conditions compatible with standard protein purification schemes. A number of
fluorescent GS-thiolesters with modified peptide backbones were therefore prepared and tested for reactivity
toward hGST A1-1 and the A216K mutant. Substitutions at the α-NH2 part of the glutathione backbone were not
tolerated by the proteins. However, two fluorescent reagents that carry a biotin moiety at the C-terminal part of
glutathione were found through MALDI-MS experiments to react in solution with Y9 of the wild-type protein
and one reagent with K216 of A216K. The reaction can take place in the presence of glutathione and even in a
crude E. coli lysate of cells expressing A216K. Delivery of the fluorescent group to Y9 or K216 was possible
using NeutrAvidin (NA) beads that had been preincubated with biotinylated reagent. Alternatively, excess reagent
can be removed by a brief incubation with NA beads. We have thus now developed a system for protein labeling
with easy removal of excess and used up low-molecular weight reagent. This strategy can conceivably be utilized
in future protein purification and labeling experiments.