Folding Study of Venus Reveals a Strong Ion Dependence of Its Yellow Fluorescence under Mildly Acidic Conditions

Abstract

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. 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. By following hydrogen-deuterium exchange of 15 N-labeled Venus using NMR spectroscopy over 13 months, residue-specific free energies of unfolding of some highly protected amide groups have been determined. Exchange rates of less than one per year are observed for some amide groups. A super-stable core is identified for Venus and compared with that previously reported for green fluorescent protein. These results are discussed in terms of the stability and folding of fluorescent proteins. Under mildly acidic conditions, we show that Venus undergoes a drastic decrease in yellow fluorescence at relatively low concentrations of guanidinium chloride. A detailed study of this effect establishes that it is due to pH-dependent, nonspecific interactions of ions with the protein. 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. Instead, chemical shift perturbations are observed for many residues primarily located in both lids of the ␤-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.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). 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). The FP family of proteins is now arguably the most versatile and commonly used biomarker employed in a myriad of applications in the Life Sciences (1, 4 -6). The impact that GFP and FPs have had is clearly illustrated by the award of the 2008 Nobel Prize in Chemistry to M. Chalfie, R. Y. Tsien, and O. Shimomura.The maturation of GFP and its variants involves the initial folding of the polypeptide chain into a native-like conformation that then undergoes an autocatalytic cyclization and dehydration of the tripeptide encompassing residues 65-67. This is then followed by a rate-limiting oxidation step to form the chromophore whose color is determined by the side-chain composition of the tripeptide as well as its local environment (2, 7, 8). In the case of yellow fluorescent protein (YFP), a yellow variant of GFP, a number of mutations give rise to the redshifted excitation and emission spectra (1). These include the following: (i) S65G, whi...

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Protein knotting through concatenation significantly reduces folding stability

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