Journal
CHEMBIOCHEM
Volume 10, Issue 7, Pages 1200-1207Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200900003
Keywords
analytical methods; fluorescent probes; local polarity detection; transition metals; tyrosine
Funding
- NSF of China [20525577, 90813032, 20875092]
- Chinese Academy of Sciences
- [2008AA02Z206]
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The design and synthesis of a novel long-wavelength polarity-sensitive fluorescence probe, 6-[9-(diethylamino)-5-oxo-5H-benzo[alpha]phenoxazin-2-yloxy]hex-2-enyl acetate, for the selective modification of tyrosine residues with the goal of providing local information on tyrosine domains in proteins, is reported. This probe comprises a polarity-sensitive Nile red fluorophore and an active pi-allyl group that can form pi-allyipalladium complexes and react selectively with tyrosine residues. The probe has the following features: 1) it has a long-wavelength emission of > 550nm, thanks to which interference from short-wavelength fluorescence from common biological matrixes can be avoided; 2) the maximum emission wavelength is sensitive only to polarity and not to pH or temperature; this allows the accurate determination of local polarity; and 3) it is a neutral, uncharged molecule, and does not disturb,the over-all charge of the labelled protein. With this probe the polarity and conformation changes of the Tyr108 domain in native and in acid- and heat-denatured bovine Cu/Zn superoxide dismutase were detected for the first time. It was found that the polarity of the Tyr108 domain hardly alters on acid denaturation between pH 4 and 9. However, heat denaturation caused the Tyr108 domain to be more hydrophobic, and was accompanied by an irreversible aggregation of the protein. In addition, the probe-binding experiments revealed that the surface of the protein becomes more hydrophobic after thermal denaturation; this can be ascribed to the formation of the more hydrophobic aggregates. This strategy might provide a general approach for studying the local environment changes of tyrosine domains in proteins under acid or heat denaturation conditions.
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