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

Saxena, Anoop; Udgaonkar, Jayant B.; Krishnamoorthy, G.

doi:10.1110/ps.051391205

Cited by 29 publications

(35 citation statements)

References 69 publications

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“…EGFP was obtained from MC4100 Escherichia coli ( E. coli ) cells containing pEGFP (Clontech), as described earlier27. The plasmid pRSETA harboring mEos2 was transformed into E. coli BL21DE3 cells and mEos2 was obtained from the protein purification facility at the Centre for Cellular and Molecular Platforms (C-CAMP, Bangalore, India).…”

Section: Methodsmentioning

confidence: 99%

Light driven ultrafast electron transfer in oxidative redding of Green Fluorescent Proteins

Saha

Verma

Rakshit

et al. 2013

Sci Rep

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Fluorescent proteins undergoing green to red (G/R) photoconversion have proved to be potential tools for investigating dynamic processes in living cells and for photo-localization nanoscopy. However, the photochemical reaction during light induced G/R photoconversion of fluorescent proteins remains unclear. Here we report the direct observation of ultrafast time-resolved electron transfer (ET) during the photoexcitation of the fluorescent proteins EGFP and mEos2 in presence of electron acceptor, p-benzoquinone (BQ). Our results show that in the excited state, the neutral EGFP chromophore accepts electrons from an anionic electron donor, Glu222, and G/R photoconversion is facilitated by ET to nearby electron acceptors. By contrast, mEos2 fails to produce the red emitting state in the presence of BQ; ET depletes the excited state configuration en route to the red-emitting fluorophore. These results show that ultrafast ET plays a pivotal role in multiple photoconversion mechanisms and provide a method to modulate the G/R photoconversion process.

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Section: Methodsmentioning

confidence: 99%

Light driven ultrafast electron transfer in oxidative redding of Green Fluorescent Proteins

Saha

Verma

Rakshit

et al. 2013

Sci Rep

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“…Saxena et al . 25 studied the kinetics of proton transfer in green fluorescent protein (GFP) using o -NBA, as a model system for characterizing the correlation between dynamics and function of proteins in general. Each of these examples illustrates the advantages of using a rapid pH jump to study pH-dependent kinetic processes.…”

Section: Introductionmentioning

confidence: 99%

Influenza Virus-Membrane Fusion Triggered by Proton Uncaging for Single Particle Studies of Fusion Kinetics

et al. 2012

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We report a method for studying membrane fusion, focusing on influenza virus fusion to lipid bilayers, which provides high temporal resolution through the rapid and coordinated initiation of individual virus fusion events. Each fusion event proceeds through a series of steps, much like multi step chemical reaction. Fusion is initiated by a rapid decrease in pH that accompanies the `uncaging' of an effector molecule from o-nitrobenzaldehyde, a photoisomerizable compound that releases a proton to the surrounding solution within microseconds of long-wave ultraviolet irradiation. In order to quantify pH values upon UV irradiation and uncaging, we introduce a simple silica nanoparticle pH sensor, useful for reporting the pH in homogeneous nanoliter volumes under conditions where traditional organic dye-type pH probes fail. Subsequent single-virion fusion events are monitored using total internal reflection fluorescence microscopy. Statistical analysis of these stochastic events uncovers kinetic information about the fusion reaction. This approach reveals that the kinetic parameters obtained from the data are sensitive to the rate at which protons are delivered to the bound viruses. Higher resolution measurements can enhance fundamental fusion studies and aid anti-viral anti-fusogenic drug development.

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“…Fluctuations in protein structure appear not to be random. They may be directed toward enhancing function (3)(4)(5)(6)(7), but their role in preferentially directing protein folding and unfolding reactions on to specific pathways is poorly understood (8). Even less is known about how the thermal fluctuations that lead to protein unfolding are perturbed by denaturants such as guanidine hydrochloride (GdnHCl) and urea.…”

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confidence: 99%

Native state dynamics drive the unfolding of the SH3 domain of PI3 kinase at high denaturant concentration

Wani

Udgaonkar²

2009

Proc. Natl. Acad. Sci. U.S.A.

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Little is known about the role of protein dynamics in directing protein unfolding along a specific pathway and about the role played by chemical denaturants in modulating the dynamics and the initiation of unfolding. In this study, deuterium-hydrogen exchange (HX) detected by electrospray ionization mass spectrometry (ESI-MS) was used to study the unfolding of the SH3 domain of the PI3 kinase. Unfolding on the principal unfolding pathway occurs in 2 steps, both in the absence and in the presence of 1.8 M guanidine hydrochloride (GdnHCl). In both cases, the first step leads to the formation of an intermediate, I N, with 5 fewer protected amide hydrogen sites than in N. In the second step, IN loses the structure protecting the remaining 14 amide hydrogen sites from HX as it unfolds completely. ESI-MS analysis of fragments of the protein created by proteolytic digestion, after completion of the HX reaction, shows that I N has lost protection against HX in the same segments of native structure during unfolding in the absence and presence of 1.8 M GdnHCl. Hence, GdnHCl does not appear to play a direct active role in the initiation of unfolding. However, at higher GdnHCl concentrations, a second unfolding pathway is shown to compete effectively with the N 7 IN 7 U pathway. In this way, the denaturant modulates the energy landscape of unfolding.hydrogen exchange ͉ mass spectrometry ͉ protein unfolding ͉ partially unfolded conformation ͉ native-state exchange F olded proteins possess dynamic structures, and the lowest energy conformation of a folded protein exists at equilibrium with many high energy conformational substates, which are Boltzmann-distributed in the folded protein ensemble (1-4). Fluctuations in protein structure appear not to be random. They may be directed toward enhancing function (3-7), but their role in preferentially directing protein folding and unfolding reactions on to specific pathways is poorly understood (8). Even less is known about how the thermal fluctuations that lead to protein unfolding are perturbed by denaturants such as guanidine hydrochloride (GdnHCl) and urea. Protein denaturants may act indirectly by disrupting the structure of water, thereby making hydrophobic groups more readily solvated (9, 10), or directly by interacting more strongly than water with the protein backbone and side chains (11-13). To understand how denaturants act, it is necessary to determine whether the mechanism of unfolding of a protein, as well as the thermal fluctuations that enable unfolding, are the same in the absence of a chemical denaturant and in the presence of a high concentration of the denaturant.Structural characterization of protein unfolding in the presence, as well as absence, of denaturant becomes possible when the hydrogen exchange (HX) experiment is carried out in conjunction with NMR spectroscopy or MS. HX-NMR experiments have been carried out on many different proteins under conditions where HX is rate-limited by the intrinsic exchange rate constant (k int ) of the fully solvent-exposed a...

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

Cited by 29 publications

References 69 publications

Light driven ultrafast electron transfer in oxidative redding of Green Fluorescent Proteins

Light driven ultrafast electron transfer in oxidative redding of Green Fluorescent Proteins

Influenza Virus-Membrane Fusion Triggered by Proton Uncaging for Single Particle Studies of Fusion Kinetics

Native state dynamics drive the unfolding of the SH3 domain of PI3 kinase at high denaturant concentration

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