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Kinetics of proton transfer in a green fluorescent protein: A laser-induced pH jump study
Authors:Roop Mallik   Jayant B. Udgaonkar  G. Krishnamoorthy
Affiliation:(1) National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, 560 065 Bangalore, India;(2) Department of Chemical Sciences, Tata Institute of Fundamental Research, 400 005 Mumbai, India;(3) Present address: Department of Developmental & Cell Biology, University of California Irvine, 2302 Natural Sciences 1, 92697 Irvine, CA, USA
Abstract:The sub-millisecond protonation dynamics of the chromophore in S65T mutant form of the green fluorescent protein (GFP) was tracked after a rapid pH jump following laser-induced proton release from the caged photolabile compoundo-nitrobenzaldehyde. Following a jump in pH from 8 to 5 (which is achieved within 2 μs), the fluorescence of S65T GFP decreased as a single exponential with a time constant of ∼90 μs. This decay is interpreted as the conversion of the deprotonated fluorescent GFP chromophore to a protonated non-fluorescent species. The protonation kinetics showed dependence on the bulk viscosity of the solvent, and therefore implicates bulk solvent-controlled protein dynamics in the protonation process. The protonation is proposed to be a sequential process involving two steps: (a) proton transfer from solvent to the chromophore, and (b) internal structural rearrangements to stabilize a protonated chromophore. The possible implications of these observations to protein dynamics in general is discussed
Keywords:Green fluorescent protein  proton transfer  pH-jump  caged proton  protein dynamics
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