Electroanalysis of amino acid substitutions in bioengineered acetylcholinesterase.
This study reports the electrochemical profiling of Nippostrongylus brasiliensis acetylcholinesterase (AChE) wild-type and mutant proteins. An irreversible oxidation signal of electro-active tyrosine (Y), tryptophan (W) and cysteine (C) residues in five mutant proteins along with the wild-type AChE were detected using square-wave voltammetry (SWV) on screen-printed carbon electrodes. Significant differences were observed in the W303L, T65Y and M301W substituted proteins showing a 25-35% higher peak current intensity compared to the Y349Y and F345Y mutants. It was predicted that AChE substituted with electrochemically active residues would produce the greatest signals and this trend was observed in the T65Y, M301W and Y349L mutants. However, conformational changes in the proteins structure as a result of the substitutions appeared to be most influential on peak current intensities. This was demonstrated by the W303L and F345Y mutant enzymes. The current intensity of W303L was greatest despite the removal of its electro-active W residue whereas the F345Y mutant had the lowest peak value despite the addition of an electro-active Y residue. The preliminary results of this study demonstrate that SWV provides a promising tool to probe the presence of electro-active amino acid residues on the surface of a protein produced through bioengineering.
Dept. of Physical and Environmental Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario, Canada M1C 1A4.
Mehdi
M
Somji
Vladimir
V
Dounin
Susanne B
SB
Muench
Holger
H
Schulze
Till T
TT
Bachmann
Kagan
K
Kerman
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics*; D019943:Amino Acid Substitution; D000818:Animals; D002244:Carbon / Q000737:chemistry; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004566:Electrodes; D008024:Ligands; D008958:Models, Molecular; D009559:Nippostrongylus / Q000201:enzymology; D011487:Protein Conformation; D015202:Protein Engineering
D008024:Ligands; D002244:Carbon; D000110:Acetylcholinesterase