Scuola di Dottorato di Scienze. Corso di Dottorato di Ricerca ... tions in plants ,
structure dynamics , reporter gene technology , cell biology  and in drug ...
` DEGLI STUDI DI MILANO BICOCCA UNIVERSITA
Scuola di Dottorato di Scienze Corso di Dottorato di Ricerca in Fisica e Astronomia
Chiara Bosisio Matricola 033245
Protonation dynamics and chemical unfolding of GFP Mutants by time resolved fluorescence spectroscopy
Tutore Prof. Maddalena Collini Coordinatore Prof. Claudio Destri
Ciclo XXI 2005-2008
Summary In the last twenty years green fluorescent protein (GFP) has changed from a nearly unknown protein to a commonly used tool in molecular biology, medicine, and cell biology, leading to the 2008 chemistry Nobel prize. GFP is particularly useful due to the fact that its chromophore is formed in an autocatalytic cyclization that does not require a cofactor. This has enabled researchers to use GFP in living systems, and it has led to GFP ’s widespread use in cell dynamics and development studies as well as in applications in plants , structure dynamics , reporter gene technology , cell biology  and in drug discovery . Because of the rapidly increasing number of applications of green fluorescent protein and its mutants as noninvasive fluorescent markers in molecular biology  , considerable interest has been developed in its biochemical and optical properties. Understanding its photophysics on the basis of recently solved crystal structures   should allow the design of mutants tailored to specific needs. The study of the internal photodynamics of this class of visible emitting proteins is interesting for at least two reasons. The first is that the efficient use of the protein as a cellular marker in microscopy is directly related to the minimization of fluorescence flickering and/or switching. For bulk measurements the fluorescence fluctuations induce a reduction in the effective quantum yield, while for single molecule techniques the fluorescence flickering limits the possibility to use GFP as a molecular tracer. Secondly, the internal photodynamics can be used as a sensor tool for proton concentration within the cell : proton detection  and redox measurements have been reported with particular mutants . A technique that is particularly suitable for the investigation of the molecular photodynamics is the Fluorescence Correlation Spectroscopy (FCS). This technique is sensitive to fluctuations in fluorescence intensity observed from a small open volume element (on the order of 10−15 liter) containing only a few molecules. These fluctuations are due to molecules diffusing in and out of the volume or to chemical transitions between fluorescent and nonfluorescent states. The method of FCS is able to extract kinetic and thermodynamic information on these processes from the temporal autocorrelation of the intensity fluctuations. In the first part of this work we have applied FCS to study the photodynamics induced by protonation-deprotonation processes of GFPMut2 class. Since Serine 65, Threonine 203, Glutamate 222 and Histidine 148 have been indicated as the key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of the H148 and E222 residues by studying the fluorescence dynamics of GFPMut2 (S65A, V68L, S72A) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components were found in the fluorescence autocorrelation functions of these mutants: a
3 10-100 µs, pH dependent, component and a 100 -500 µs laser power dependent component. The comparison of the pH dependent component of the three mutants has shown that the mutation of H148 to glycine induces a three-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the H148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, has been ascribed to an excited state process and is probably related to conformational rearrangements of the chromophore after the photo-excitation more than to the chromophore excited state proton transfer, as previous works have suggested . GFP stability against pH or chemical denaturant is essential to its application in cellular biophysics and nano-technology due to the close relation between the fluorescence quantum yield and protein structure. Despite the potentially useful characteristics of GFP, rather little is known about the detailed mechanisms of the unfolding of GFP itself in vitro and to date there has been no report on rapid folding kinetics starting from GFP in an extensively unfolded state, neither has the detailed mechanism of GFP folding been described in the literature. In the second part of this work we have then investigated GFPmut2 and Mut2G in terms of chemical denaturation induced by Guanidinium Chloride, GuHCl in order to gain insight into the denaturant effects on the fluorescent emission during the unfolding. Since the unfolding reactions of GPF family are reported to be very slow , and the equilibrium is reached after several days under conditions of medium GuHCl concentration, we have focused our studies on the kinetic behaviour after the addition of GuHCl. We have followed the kinetics of unfolding at different pH monitoring the chromophore fuorescence in bulk experiments. GFPMut2 is found to be significantly more stable than Mut2G at high pH values in presence of denaturant and the action of H148 as shutter which limits the accessibility of the denaturant to the chromophore is found to be modulated by the pH. Different spectroscopic techniques such as fluorescence lifetime, fluorescence polarization anisotropy, and FCS have been applied to investigate the conformational changes and the protein fluctuations between the folded/unfolded states. Conformational rearrangements of the β-barrel induced by GuHCl, already reported in CD measurements , have been detected by the finding of ’soft’ region in the protein. The characteristic time of the fluctuations between the folded and the unfolded state have been detected by modifying the traditional FCS setup in a scanning version operating on immobilized (non diffusing) substrates. In this way a characteristic time of 600 µs has appended in the GFP sample after GuHCl addition which was previously hidden by the diffusional brownian process in solution.
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