| title: |
Kinetics and Thermodynamics of Protein Structural Conversions |
| reg no: |
ETF5546 |
| project type: |
Estonian Science Foundation research grant |
| subject: |
1.1-1.5. Exact Sciences
|
| status: |
accepted |
| institution: |
Institute of Physics at University of Tartu |
| head of project: |
Koit Mauring |
| duration: |
01.01.2003 - 31.12.2006 |
| description: |
The aim of the present work is to elucidate the strength and dynamics of the hydrogen bonds between the chromophore and its surrounding amino acids in Green Fluorescent Protein (GFP). The experiments performed by the author in his home laboratory and in Stanford University have shown that the fluorescent form of GFP differs from the nonfluorescent one by the presence of hydrogen bond. In its absence the torsional deformation of the chromophore in the excited state quenches the fluorescence. From fluorescence temperature dependence we will determine the equilibrium between the protein forms and reveal the change in enthalpy (H) and entropy (S) of bond formation. The balance between H and S in the Gibbs free energy is related to the analogous trade-off between enthalpy and entropy in the protein folding process what is of prime interest. The hydrostatic pressure applied to GFP also changes the equilibrium between hydrogen bonded and nonbonded species. From the concomitant fluorescence intensity changes we will determine the elasticity parameters of protein.By inserting the GFP molecules into the matrixes of different viscosity we will tune the mobility of the beta-can, which surrounds the chromophore. This way we can modify the conversion time between the protein conformational substates. Using spectral measurements, will investigate the influence of the solvent shell on the protein inner dynamics and on the photoisomerization rate of the chromophore.From single molecule fluorescence experiments it is known that GFP molecules blink but there is no consistent explanation of this effect. We hypothesize that the origin of blinking is fluctuations in strength of hydrogen bonding. We will attempt to repeat this experiment on a CCD-equipped microscope and to find from the temperaure dependence of blinking rate the thermodynamic parameters of this process. |