New Approach for Local Structure Analysis of the Tyrosine Domain in Proteins by Using a Site-Specific and Polarity-Sensitive Fluorescent Probe
✍ Scribed by Suming Chen; Xiaohua Li; Huimin Ma
- Publisher
- John Wiley and Sons
- Year
- 2009
- Tongue
- English
- Weight
- 273 KB
- Volume
- 10
- Category
- Article
- ISSN
- 1439-4227
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✦ Synopsis
Abstract
Designer label: A newly developed polarity‐sensitive fluorescent probe (DBHA) was combined with a tyrosine‐specific labelling method that uses transition metal catalysis, and was successfully used in local structural analysis of the Tyr108 domain in Cu/Zn superoxide dismutase (SOD; see scheme). The strategy presented here provides a new approach for studying the local polarity and conformation changes of this tyrosine domain in SOD under acid or heat denaturation conditions.magnified image
The design and synthesis of a novel long‐wavelength polarity‐sensitive fluorescence probe, 6‐[9‐(diethylamino)‐5‐oxo‐5__H‐benzo[α]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 π‐allyl group that can form π‐allylpalladium complexes and react selectively with tyrosine residues. The probe has the following features: 1) it has a long‐wavelength emission of >550 nm, 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 overall 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.__