Crystal Structure of the
            <b>
              <i>Aequorea victoria</i>
            </b>
            Green Fluorescent Protein

Ormö, Mats; Cubitt, A B; Kallio, Karen; Gross, Larry A.; Tsien, Roger Y.; Remington, S.J.

doi:10.1126/science.273.5280.1392

Cited by 2,155 publications

(1,750 citation statements)

References 31 publications

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“…This finding indicates that the recognition of the processed N-terminus, lacking the first three residues of the PEXEL (RxL), may be occurring at the PVM by a putative translocase and the inability of the parasite to traffic KAHRP(1-60)-GFP beyond the PVM could be due to a defect in this proposed recognition event. The crystal structure of GFP shows that the first nine amino acids are involved in the formation of an α-helix that plays an integral part in capping one end of the protein and may aid in folding events or in protecting the fluorophore [32,33]. It is possible that the close proximity of the processed PEXEL to this N-terminal α-helical cap of GFP obstructs the recognition of the processed N-terminus of the KAHRP chimera by a putative PVM translocase.…”

Section: Transport Across the Pvm Mediated By The Recognition Of The mentioning

confidence: 99%

N-terminal processing of proteins exported by malaria parasites

Chang

Falick

Carlton

et al. 2008

Molecular and Biochemical Parasitology

171

177

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Malaria parasites utilize a short N-terminal amino acid motif termed the Plasmodium export element (PEXEL) to export an array of proteins to the host erythrocyte during blood stage infection. Using immunoaffinity chromatography and mass spectrometry, insight into this signalmediated trafficking mechanism was gained by discovering that the PEXEL motif is cleaved and N-acetylated. PfHRPII and PfEMP2 are two soluble proteins exported by Plasmodium falciparum that were demonstrated to undergo PEXEL cleavage and N-acetylation, thus indicating that this Nterminal processing may be general to many exported soluble proteins. It was established that PEXEL processing occurs upstream of the brefeldin A-sensitive trafficking step in the P. falciparum secretory pathway, therefore cleavage and N-acetylation of the PEXEL motif occurs in the endoplasmic reticulum (ER) of the parasite. Furthermore, it was shown that the recognition of the processed N-terminus of exported proteins within the parasitophorous vacuole may be crucial for protein transport to the host erythrocyte. It appears that the PEXEL may be defined as a novel ER peptidase cleavage site and a classical N-acetyltransferase substrate sequence.

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Section: Transport Across the Pvm Mediated By The Recognition Of The mentioning

confidence: 99%

N-terminal processing of proteins exported by malaria parasites

Chang

Falick

Carlton

et al. 2008

Molecular and Biochemical Parasitology

171

177

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“…More importantly, mutants of GFP such as the S65T mutant [14] display enhanced brightness over the wild-type GFP [15] and are well characterized in terms of their photobleaching behavior [15,16]. The availability of the high-resolution structure of GFP [17] has enabled the prediction of the diffusion coefficient of GFP in a given medium using the Stokes-Einstein equation. The theoretically calculated diffusion coefficient of GFP in a viscous solution (equivalent to a 90% glycerol-water mixture) is 0.7 µm 2 s −1 [18].…”

Section: Introductionmentioning

confidence: 99%

Confocal Fluorescence Recovery After Photobleaching of Green Fluorescent Protein in Solution

Pucadyil

Chattopadhyay

2006

J Fluoresc

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Fluorescence recovery after photobleaching (FRAP) is one of the most widely used approaches to quantitatively estimate diffusion characteristics of molecules in solution and cellular systems. In general, comparison of the diffusion times (t 1/2 ) from a FRAP experiment provides qualitative estimates of diffusion rates. However, obtaining consistent and reliable quantitative estimates of mobility of molecules using FRAP is hindered by the lack of appropriate standards for calibrating the FRAP set-up (microscope configuration and data fitting algorithms) used in a given experiment. In comparison with other fluorescent markers, the green fluorescent proteins (GFP) possess characteristics that are ideal for use in such experiments. We have monitored the mobility of pure enhanced green fluorescent protein (EGFP) in a viscous solution by confocal FRAP experiments. Our experimentally determined diffusion coefficient of EGFP in a glycerol-water mixture is in excellent agreement with the value predicted for GFP in a solution of comparable viscosity, calculated using the Stokes-Einstein equation. The agreement in the experimentally determined diffusion coefficient and that predicted from theoretical framework improves significantly when one takes into account the effective size of the bleached spot in such experiments. Our results therefore validate the use of GFP as a convenient standard for FRAP experiments. Importantly, we present a simple method to correct for artifacts in the accurate determination of diffusion coefficient of molecules measured using FRAP arising due to the underestimation in the effective size of the bleached spot.

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“…The off-state structure was cryogenically trapped after full conversion of crystals with green illumination 5 . The chromophore of Dronpa is derived from the Cys-Tyr-Gly tripeptide and is present in a cis conformation in the on state 3,4 , as in the A. victoria GFP 7,8 . Substantial structural rearrangements of the protein environment were shown to coincide with the cis-trans photoisomerization of the chromophore.…”

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confidence: 99%

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

et al. 2013

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The reversible photoswitching between the 'on' and 'off' states of the fluorescent protein Dronpa involves photoisomerization as well as protein side-chain rearrangements, but the process of interconversion remains poorly characterized. Here we use time-resolved infrared measurements to monitor the sequence of these structural changes, but also of proton transfer events, which are crucial to the development of spectroscopic contrast. Light-induced deprotonation of the chromophore phenolic oxygen in the off state is a thermal ground-state process, which follows ultrafast (9 ps) trans-cis photoisomerization, and so does not involve excited-state proton transfer. Steady-state infrared difference measurements exclude protonation of the imidazolinone nitrogen in both the on and off states. Pump-probe infrared measurements of the on state reveal a weakening of the hydrogen bonding between Arg66 and the chromophore C ¼ O, which could be central to initiating structural rearrangement of Arg66 and His193 coinciding with the low quantum yield cis-trans photoisomerization

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Crystal Structure of the Aequorea victoria Green Fluorescent Protein

Cited by 2,155 publications

References 31 publications

N-terminal processing of proteins exported by malaria parasites

N-terminal processing of proteins exported by malaria parasites

Confocal Fluorescence Recovery After Photobleaching of Green Fluorescent Protein in Solution

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

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