These proteins have been developed further using protein engineering techniques to obtain a variety of FPs with broad fluorescence emission spectra, ranging from blue to far-red ( 3). Since the identification of the GFP 7 from the jellyfish Aequorea victoria in the early 1990s, a large number of fluorescent proteins (FPs) have been isolated from natural sources, primarily from marine animals and corals ( 1, 2). Instead, chemical shift perturbations are observed for many residues primarily located in both lids of the β-barrel structure, which suggests that small scale structural rearrangements occur on increasing ionic strength under mildly acidic conditions and that these are propagated to the chromophore resulting in fluorescence quenching. In contrast to previous studies on enhanced green fluorescence protein variant S65T/T203Y, which showed a specific halide ion-binding site, NMR chemical shift mapping shows no evidence for specific ion binding. A detailed study of this effect establishes that it is due to pH-dependent, nonspecific interactions of ions with the protein. Under mildly acidic conditions, we show that Venus undergoes a drastic decrease in yellow fluorescence at relatively low concentrations of guanidinium chloride. These results are discussed in terms of the stability and folding of fluorescent proteins. A super-stable core is identified for Venus and compared with that previously reported for green fluorescent protein. Exchange rates of less than one per year are observed for some amide groups. By following hydrogen-deuterium exchange of 15N-labeled Venus using NMR spectroscopy over 13 months, residue-specific free energies of unfolding of some highly protected amide groups have been determined. Here, we present a detailed study of the stability and folding of Venus in the pH range from 6.0 to 8.0 using chemical denaturants and a variety of spectroscopic probes. Venus is a yellow fluorescent protein that has been developed for its fast chromophore maturation rate and bright yellow fluorescence that is relatively insensitive to changes in pH and ion concentrations.
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