Denaturation studies reveal significant differences between GFP and blue fluorescent protein

Green fluorescent protein (GFP) is an unusually stable fluorescent protein that belongs to a family of related auto-fluorescent proteins (AFPs). These AFPs have been generated from jelly fish GFP by mutating the amino acids in the chromophore or its vicinity. Variants that emit light in the blue region (Blue Fluorescent Protein, BFP), red region, or yellow region are readily available and are widely used in diverse applications. Previously, we had used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with SDS, urea, and heat. Surprisingly, we found that SDS, urea or heat, did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5, however, at pH 6.5, the protein lost all fluorescence within a very short period of time. These results suggested that GFP undergoes a structural/stability shift between pH 6.5 and 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SIDS, urea, and heat. In the present study, we wanted to explore whether the stability or structure of the closely related BFP is also pH dependent. As expected, we found heat-induced denaturation and renaturation of BFP to be pH dependent, very much like GFP. However, when exposed to other denaturants like urea/heat or SDS we found BFP to behave very differently than GFP. Unlike GFP, which at pH 8.5 and 7.5 is very resistant to SDS-induced denaturation, BFP readily lost about 20% of its fluorescence at pH 8.5 and about 60% fluorescence at pH 7.5. Also, our denaturation and renaturation studies show that under certain conditions, BFP is more stable than GFR such that under conditions where GFP is completely denatured, BFP still retained significant fluorescence. Taken together, our preliminary results show that despite being very similar in both amino acid sequences and overall structures, there may be subtle and important structural/conformational differences between BFP and GFP. (C) 2009 Elsevier B.V. All rights reserved.