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 beta-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.
Highlights
The maturation of GFP and its variants involves the initial folding of the polypeptide chain into a native-like conformation that undergoes an autocatalytic cyclization and dehydration of the tripeptide encompassing residues 65– 67
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, which prevents the formation of a hydrogen bond between Ser-65 and Glu-222, and (ii) T203Y, which produces additional polarizability around the chromophore, whereas the - stacking between tyrosine fluorescence (Tyr)-203 and the chromophore phenol ring reduces the excited state energy
Together with cyan fluorescent protein, YFP is often used as a fluorescence acceptor-donor pair in Forster resonance energy transfer measurements [1, 4, 5, 9]
Summary
Plasmid and Reagents—A plasmid containing the gene encoding Venus was a kind gift from Prof. The largest difference in fluorescence between the native and denatured states was observed at 527 nm, and the emission at this wavelength was used in the subsequent analysis. Determination of the pKa of the Yellow Chromophore—The pKa of the p-hydroxybenzylidene imidazolidinone chromophore of Venus was determined by monitoring the fluorescence (527 nm) or absorbance (516 nm) of the protein as a function of pH in a low ionic strength 10 mM MOPS-MES buffer. The observed HDX rate constants of amide protons, kex, were used to calculate the free energy of unfolding, ⌬GHX, defined as shown in Equation 4,. Chloride/Nitrate Ion Titration by NMR Spectroscopy—200 l of 15N-labeled Venus, at a concentration of 140 M, containing 10% D2O (v/v), 0.1 mM dithiothreitol and buffered in 10 mM MES (pH 6.0) (for chloride ion titration) or 10 mM MES/MOPS (pH 6.0) (for nitrate ion titration), was used as a starting sample for chloride or nitrate ion titration at 37 °C. The titration was carried out using a 3-mm NMR tube to minimize the salt effect, which is pronounced for a high field NMR spectrometer equipped with a cryoprobe; in our case, a 700 MHz Bruker AVANCE NMR spectrometer was used for the titration experiments
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