At the present time the phenomenon of "fluorescence kindling," which is typical of asCP595 protein
and some of its mutants, is of great interest for the studies of intracellular traffic. The range of
changing of intracellular pH may be rather wide, however, the effect of pH on the fluorescent state
of this protein is poorly investigated. Our studies have revealed that the fluorescence intensity of
asCP595 increases significantly in alkaline conditions. In addition, the observed change in the
position of excitation, emission, and absorption maxima indicates the appearance of new spectral
forms of the protein. These conformers are characteristics of new fluorescent states. The changes in
the absorption spectrum are indicative of a new dark form of the protein at alkaline pH.
Practically all fluorescent proteins from corals are oligomerized or aggregated in solutions. For zFP538 it was demonstrated that intensity of fluorescence and pH-profile change upon dilution that was explained by the dissociation of aggregates. Particle size distribution of zFP538 aggregates adsorbed on the surface of graphite from solutions in the concentration range 10-7 - 10-9 M was studied by AFM. Multi-population size distribution was observed for all three protein concentrations. Within one concentration, size distribution is characterized by three populations: small, medium, and large. For 10-7M concentration, the largest size is 10 times less than that in the 10-5M solution or solid film. For 10-8M concentration, the maximum population is of medium size, but smaller in aggregation number than the minimal population for 10-7M. Aggregation numbers for small, medium, and large populations for 10-9M concentration are the same as for 10-8M, but the population is shifted to smaller aggregation number. Thus, upon dilution, an average aggregation number is gradually shifted to a smaller value. Difference in small, medium, and large particles is observed within 10-7 - 10-8M concentration range.
Our experiments show that fluorescence dependence of zFP538 on protein concentration is nonlinear over the range from 1.2•10-9 M to 5.5•10-7 M. Under pH from 5 to 9 it can be divided into two linear regions with different slope at concentration more and less than 1•10-7 M. One may conclude, there are at least two different forms of the yellow protein observed in the high (>1•10-7 M) and low (<1•10-7 M) concentration regions. These forms may be different aggregation states of the protein. We have obtained acid denaturation kinetics curves for zFP538 over pH range from 3.5 to 5.5 at different protein concentrations. Under all pH values relative residual fluorescence of the most concentrated solution (1.33•10-6 M) is higher than that for the solutions of lower protein concentration (1.36•10-7 M and 1.36•10-8 M). Fluorescence decrease under pH from 3.5 to 4.24 is three or two exponential. Under higher pH values kinetic curves have a plateau at first and then fluorescence drops in exponential way. The results obtained support the hypothesis that at higher protein concentrations zFP538 is more stable against acid denaturation.
We had previously reported about high aggregation number of the yellow fluorescent protein zFP538, as was shown by gel-filtration and dynamic light scattering. In this study we used atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) to image zFP538 in solid state. Height and phase measurements of the sample were taken using NT-MDT AFM operated in tapping mode. Height data provides three-dimensional topographical information on the sample while the phase data, which measures the phase shift in cantilever oscillation, responds to attractive and repulsive interactions between the cantilever tip and the sample. This signal can be related to the stiffness of the sample. Unlike EGFP, which crystallizes upon drying of water solution, zFP538 forms ring-like films due to its surfactant properties. According to AFM these films are comprised of ellipsoidal protein granules, with the major axis of the granules lying in the range from 50 to 300 nm and the minor axis - in the range from 30 to 130 nm. Average volume of these granules is about 19500 times higher than volume of zFP538 monomer, calculated as in the case of GFP molecule (a cylinder with a height of 4.2 nm and 2.4 nm diameter). NSOM with a 532 nm laser emission from CW Nd:YAG shows that fluorescence of zFP538 is retained in solid state. A ratio of maximum emission to shoulder at 580 nm is 0.65 for solid zFP538 and only 0.25 for water solution.
pH-dependent aggregation and dissociation of yellow fluorescent protein zFP538 were studied by gel-filtration, dynamic light scattering, and fluorescence spectroscopy. According to the gel-filtration data for low concentration of zFP538 the molecular weight of aggregates decreases upon changing pH from alkaline to neutral. Dynamic light scattering showed that zFP538 aggregates strongly in concentrated solutions. Aggregation influences heavily the pH-profile of fluorescence of zFP538 and stabilizes zFP538 against fluorescence quenching on acidification. Reduction of the protein concentration results in the shifting of pH profile to the alkaline region. Conclusion: aggregation of the yellow fluorescent protein zFP538 depends on pH; dilution of the protein solution is accompanied by dissociation of zFP538 aggregates under neutral and alkaline pH.
Fluorescence of the yellow fluorescent protein zFP538 strongly depends on concentration and starting pH form which pH profile is recorded. pH transitions typical for chromopeptides isolated from zFP538 can be observed for whole protein in diluted solutions. The quenching fluorescence of zFP538 is irreversible upon acidification or alkalization of the low concentrated soultions. In concentrated solutions, according to the data of dynamic light scattering, the protein zFP538 is strongly aggretaed (or oligomerized) and become more stable against acid denaturation. Spectral changes on pH are almost reversible both for fluorescence and absorbance. Two major chromopeptides obtained from zFP538 have different spectral properties and no similarity to the spectral properties of the chromopeptide obtained from GFP.