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Ultrafast proton shuttling in Psammocora cyan fluorescent protein.
J Phys Chem B 2013; 117(38):11134-43JP

Abstract

Cyan, green, yellow, and red fluorescent proteins (FPs) homologous to green fluorescent protein (GFP) are used extensively as model systems to study fundamental processes in photobiology, such as the capture of light energy by protein-embedded chromophores, color tuning by the protein matrix, energy conversion by Förster resonance energy transfer (FRET), and excited-state proton transfer (ESPT) reactions. Recently, a novel cyan fluorescent protein (CFP) termed psamFP488 was isolated from the genus Psammocora of reef building corals. Within the cyan color class, psamFP488 is unusual because it exhibits a significantly extended Stokes shift. Here, we applied ultrafast transient absorption and pump-dump-probe spectroscopy to investigate the mechanistic basis of psamFP488 fluorescence, complemented with fluorescence quantum yield and dynamic light scattering measurements. Transient absorption spectroscopy indicated that, upon excitation at 410 nm, the stimulated cyan emission rises in 170 fs. With pump-dump-probe spectroscopy, we observe a very short-lived (110 fs) ground-state intermediate that we assign to the deprotonated, anionic chromophore. In addition, a minor fraction (14%) decays with 3.5 ps to the ground state. Structural analysis of homologous proteins indicates that Glu-167 is likely positioned in sufficiently close vicinity to the chromophore to act as a proton acceptor. Our findings support a model where unusually fast ESPT from the neutral chromophore to Glu-167 with a time constant of 170 fs and resulting emission from the anionic chromophore forms the basis of the large psamFP488 Stokes shift. When dumped to the ground state, the proton on neutral Glu is very rapidly shuttled back to the anionic chromophore in 110 fs. Proton shuttling in excited and ground states is a factor of 20-4000 faster than in GFP, which probably results from a favorable hydrogen-bonding geometry between the chromophore phenolic oxygen and the glutamate acceptor, possibly involving a short hydrogen bond. At any time in the reaction, the proton is localized on either the chromophore or Glu-167, which implies that most likely no low-barrier hydrogen bond exists between these molecular groups. This work supports the notion that proton transfer in biological systems, be it in an electronic excited or ground state, can be an intrinsically fast process that occurs on a 100 fs time scale. PsamFP488 represents an attractive model system that poses an ultrafast proton transfer regime in discrete steps. It constitutes a valuable model system in addition to wild type GFP, where proton transfer is relatively slow, and the S65T/H148D GFP mutant, where the effects of low-barrier hydrogen bonds dominate.

Authors+Show Affiliations

Department of Physics and Astronomy and LaserLaB, Faculty of Sciences, Vrije Universiteit , De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

23534404

Citation

Kennis, John T M., et al. "Ultrafast Proton Shuttling in Psammocora Cyan Fluorescent Protein." The Journal of Physical Chemistry. B, vol. 117, no. 38, 2013, pp. 11134-43.
Kennis JT, van Stokkum IH, Peterson DS, et al. Ultrafast proton shuttling in Psammocora cyan fluorescent protein. J Phys Chem B. 2013;117(38):11134-43.
Kennis, J. T., van Stokkum, I. H., Peterson, D. S., Pandit, A., & Wachter, R. M. (2013). Ultrafast proton shuttling in Psammocora cyan fluorescent protein. The Journal of Physical Chemistry. B, 117(38), pp. 11134-43. doi:10.1021/jp401114e.
Kennis JT, et al. Ultrafast Proton Shuttling in Psammocora Cyan Fluorescent Protein. J Phys Chem B. 2013 Sep 26;117(38):11134-43. PubMed PMID: 23534404.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Ultrafast proton shuttling in Psammocora cyan fluorescent protein. AU - Kennis,John T M, AU - van Stokkum,Ivo H M, AU - Peterson,Dayna S, AU - Pandit,Anjali, AU - Wachter,Rebekka M, Y1 - 2013/04/19/ PY - 2013/3/29/entrez PY - 2013/3/29/pubmed PY - 2014/4/20/medline SP - 11134 EP - 43 JF - The journal of physical chemistry. B JO - J Phys Chem B VL - 117 IS - 38 N2 - Cyan, green, yellow, and red fluorescent proteins (FPs) homologous to green fluorescent protein (GFP) are used extensively as model systems to study fundamental processes in photobiology, such as the capture of light energy by protein-embedded chromophores, color tuning by the protein matrix, energy conversion by Förster resonance energy transfer (FRET), and excited-state proton transfer (ESPT) reactions. Recently, a novel cyan fluorescent protein (CFP) termed psamFP488 was isolated from the genus Psammocora of reef building corals. Within the cyan color class, psamFP488 is unusual because it exhibits a significantly extended Stokes shift. Here, we applied ultrafast transient absorption and pump-dump-probe spectroscopy to investigate the mechanistic basis of psamFP488 fluorescence, complemented with fluorescence quantum yield and dynamic light scattering measurements. Transient absorption spectroscopy indicated that, upon excitation at 410 nm, the stimulated cyan emission rises in 170 fs. With pump-dump-probe spectroscopy, we observe a very short-lived (110 fs) ground-state intermediate that we assign to the deprotonated, anionic chromophore. In addition, a minor fraction (14%) decays with 3.5 ps to the ground state. Structural analysis of homologous proteins indicates that Glu-167 is likely positioned in sufficiently close vicinity to the chromophore to act as a proton acceptor. Our findings support a model where unusually fast ESPT from the neutral chromophore to Glu-167 with a time constant of 170 fs and resulting emission from the anionic chromophore forms the basis of the large psamFP488 Stokes shift. When dumped to the ground state, the proton on neutral Glu is very rapidly shuttled back to the anionic chromophore in 110 fs. Proton shuttling in excited and ground states is a factor of 20-4000 faster than in GFP, which probably results from a favorable hydrogen-bonding geometry between the chromophore phenolic oxygen and the glutamate acceptor, possibly involving a short hydrogen bond. At any time in the reaction, the proton is localized on either the chromophore or Glu-167, which implies that most likely no low-barrier hydrogen bond exists between these molecular groups. This work supports the notion that proton transfer in biological systems, be it in an electronic excited or ground state, can be an intrinsically fast process that occurs on a 100 fs time scale. PsamFP488 represents an attractive model system that poses an ultrafast proton transfer regime in discrete steps. It constitutes a valuable model system in addition to wild type GFP, where proton transfer is relatively slow, and the S65T/H148D GFP mutant, where the effects of low-barrier hydrogen bonds dominate. SN - 1520-5207 UR - https://www.unboundmedicine.com/medline/citation/23534404/Ultrafast_proton_shuttling_in_Psammocora_cyan_fluorescent_protein_ L2 - https://dx.doi.org/10.1021/jp401114e DB - PRIME DP - Unbound Medicine ER -