A comparative study of electrochemically and fluorometrically addressed molecular reporter groups: effects of protein microenvironment.
To probe the effects of protein microenvironment on electrochemically and fluorometrically addressed molecular reporter groups, genetically engineered apo-cytochrome c peroxidase derivatives W51C, A174C, K243C, and S246C, each containing a single cysteine residue, were labeled at identical sites with two kinds of microenvironment sensitive reporters, either an electrochemically active sulfhydryl-reactive reagent, [Ru(II)(NH(3))(4)(1,10-phenanthroline-5-maleimide)](PF(6))(2) [RuPA4] or a fluorescent 6-acryloyl-2-dimethylaminonaphthalene [acrylodan] probe. Two types of sites were labeled with each probe based on their predicted solvent accessibilities from the known structure for holo-cytochrome c peroxidase. One set of sites (K243C and S246C) was selected to be completely solvent exposed, while the other two sites (W51C and A174C) were less accessible, residing in or near the heme binding site. Spectroscopic properties of the fluorescent probe were consistent with predictions for relative solvent accessibilities; however, even the less solvent accessible probes reported a quite polar environment, suggesting that this region of the apo-protein is either substantially solvent exposed or undergoes significant dynamic motion. A linear correlation was observed between the lambda(max) of the metal to ligand charge-transfer (MLCT) absorption band of the RuPA4 complex and the acrylodan emission maximum for the four labeled apo-protein variants. The same trend occurred for the formal potential of RuPA4 versus the acrylodan emission maximum, with the exception of electrochemical probe behavior at position 174, possibly due to specific probe-protein interactions.
Trammell
Scott A
SA
Center for Bio/Molecular Science and Engineering, Code 6900, US Naval Research Laboratory, Overlook Avenue SW, Washington, DC 20375, USA.
Jhaveri
Sulay D
SD
LaBrenz
Steven R
SR
Mauro
J Matthew
JM
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D004563:Electrochemistry / Q000379:methods*; D004777:Environment; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D017930:Genes, Reporter; D015335:Molecular Probes / Q000032:analysis / Q000737:chemistry*; D009942:Organometallic Compounds / Q000032:analysis / Q000737:chemistry*; D011506:Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D015335:Molecular Probes; D009942:Organometallic Compounds; D011506:Proteins; D011994:Recombinant Proteins; D012996:Solutions; C488381:tetraammine(1,10-phenanthroline-5-maleimide)ruthenium(II); C038518:acrylodan; D015081:2-Naphthylamine