Photochromic variants of fluorescent proteins are opening the way to a number of opportunities for high-sensitivity regioselective studies in the cellular environment and may even lead to applications in information and communication technology. Yet, the detailed photophysical processes at the basis of photoswitching have not been fully clarified. In this paper, we used synthetic FP chromophores to clarify the photophysical processes associated with the photochromic behavior. In particular, we investigated the spectral modification of synthetic chromophore analogues of wild-type green fluorescent protein (GFP), Y66F GFP (BFPF), and Y66W GFP (CFP) upon irradiation in solutions of different polarities. We found that the cis-trans photoisomerization mechanism can be induced in all the chromophores. The structural assignments were carried out both by NMR measurements and DFT calculations. Remarkably, we determined for the first time the spectra of neutral trans isomers in different solvents. Finally, we calculated the photoconversion quantum yields by absorption measurements under continuous illumination at different times and by a nanosecond laser-flash photolysis method. Our results indicate that cis-trans photoisomerization is a general mechanism of FP chromophores whose efficiency is modulated by the detailed mutant-specific protein environment.

To understand why the classical two-state allosteric model of Monod, Wyman, and Changeux explains cooperative oxygen binding by hemoglobin but does not explain changes in oxygen affinity by allosteric inhibitors, we have investigated the kinetic properties of unstable conformations transiently trapped by encapsulation in silica gels. Conformational trapping reveals that after nanosecond photodissociation of carbon monoxide a large fraction of the subunits of the T quaternary structure has kinetic properties almost identical to those of subunits of the R quaternary structure. Addition of allosteric inhibitors reduces both the fraction of R-like subunits and the oxygen affinity of the T quaternary structure. These kinetic and equilibrium results are readily explained by a recently proposed generalization of the Monod-Wyman-Changeux model in which a preequilibrium between two functionally different tertiary, rather than quaternary, conformations plays the central role.T he two-state allosteric model of Monod, Wyman, and Changeux (1) represented a conceptual breakthrough in explaining the cooperative and regulated behavior of multisubunit proteins, with application to a wide range of biological systems (2-5). Monod, Wyman, and Changeux proposed that ligands control protein function by altering a preexisting equilibrium between high (R) and low (T) reactivity conformations that differ in intersubunit bonding (quaternary structure) and not by inducing conformational changes that are propagated to neighboring subunits as in a sequential model (6, 7). Enzyme activation, for example, results from preferential binding of ligands to the R quaternary structure, whereas inhibitors preferentially bind to T. However, a long-known serious deficiency in the application of the Monod-Wyman-Changeux (MWC) model to hemoglobin, the paradigm of allosteric proteins, is that inhibitors may also change oxygen (O 2 ) affinity without a change in quaternary structure (8-11). To understand this phenomenon, we have investigated the ligand binding kinetics and equilibria of hemoglobin encapsulated in silica gels in either the T or R quaternary structure (Fig. 1).Previous studies of hemoglobin encapsulated in silica gels showed greatly simplified equilibrium properties, compared with those in solution, because quaternary conformational changes are markedly slowed by the constraints of the surrounding cross-linked polymer (12-16). In sharp contrast to hemoglobin free in solution, O 2 binding to gel-encapsulated hemoglobin, like O 2 binding to the hemoglobin crystal (17-19), is noncooperative (Fig. 2). Encapsulation as the fully deoxygenated molecule traps hemoglobin in the low-affinity T quaternary structure, whereas encapsulation as the fully oxygenated molecule traps hemoglobin in the high-affinity R structure (12). Moreover, the affinity of the deoxy-encapsulated molecule is lowered by inhibitor ligands (called negative heterotropic allosteric effectors) such as protons, chloride ions, inositol hexaphosphate, and bezafibrate (Fig. 2) in the ...

The nonsymbiotic hemoglobins, AHb1 and AHb2, have recently been isolated from Arabidopsis thaliana. Using steady-state and time-resolved spectroscopic methods, we show that Fe2+ AHb1 contains a mixture of penta- and hexacoordinated heme, while Fe2+ AHb2 is fully hexacoordinated. In the CO complexes, polar interactions and H-bonds with the ligand are stronger for AHb1 than for AHb2. The ligand binding kinetics are substantially different, reflecting the distribution between the penta- and hexacoordinated species, and indicate that protein dynamics and ligand migration pathways are very specific for each of the two proteins. In particular, a very small, non-exponential geminate rebinding observed in AHb1 suggests that the distal heme cavity is connected with the exterior by a relatively open channel. The large, temperature-dependent geminate rebinding observed for AHb2 implies a major role of protein dynamics in the ligand migration from the distal cavity to the solvent. The structures of AHb1 and AHb2, modeled on the basis of the homologous rice hemoglobin, exhibit a different cavity system that is fully compatible with the observed ligand binding kinetics. Overall, these kinetic and structural data are consistent with the putative NO-dioxygenase activity previously attributed to AHb1, whereas the role of AHb2 remains elusive.