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Protein dynamics control proton transfer from bulk solvent to protein interior: A case study with a green fluorescent protein

¹ Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400 005, India
² National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore 565 065, India

(RECEIVED February 1, 2005; FINAL REVISION March 31, 2005; ACCEPTED March 31, 2005)

The kinetics of proton transfer in Green Fluorescent Protein (GFP) have been studied as a model system for characterizing the correlation between dynamics and function of proteins in general. The kinetics in EGFP (a variant of GFP) were monitored by using a laser-induced pH jump method. The pH was jumped from 8 to 5 by nanosecond flash photolysis of the "caged proton," o-nitrobenzaldehyde, and subsequent proton transfer was monitored by following the decrease in fluorescence intensity. The modulation of proton transfer kinetics by external perturbants such as viscosity, pH, and subdenaturing concentrations of GdnHCl as well as of salts was studied. The rate of proton transfer was inversely proportional to solvent viscosity, suggesting that the rate-limiting step is the transfer of protons through the protein matrix. The rate is accelerated at lower pH values, and measurements of the fluorescence properties of tryptophan 57 suggest that the enhancement in rate is associated with an enhancement in protein dynamics. The rate of proton transfer is nearly independent of temperature, unlike the rate of the reverse process. When the stability of the protein was either decreased or increased by the addition of co-solutes, including the salts KCl, KNO₃, and K₂SO₄, a significant decrease in the rate of proton transfer was observed in all cases. The lack of correlation between the rate of proton transfer and the stability of the protein suggests that the structure is tuned to ensure maximum efficiency of the dynamics that control the proton transfer function of the protein.

Keywords: green fluorescent protein; proton transfer; protein dynamics; Kramers’ theory; viscosity dependence; laser-induced pH jump; caged proton

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051391205.

Reprint requests to: Guruswamy Krishnamoorthy, Department of Chemical Sciences, Tata Institute of Fundamental Research, Homibhabha Road, Mumbai 400 005, India; e-mail: gk{at}tifr.res.in; fax: +91-22- 2280-4610.

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