Concerted Asynchronous Hula‐Twist Photoisomerization in the S65T/H148D Mutant of Green Fluorescent Protein

Zhang, Qiangqiang; Chen, Xuebo; Cui, Ganglong; Fang, Wei‐Hai; Thiel, Walter

doi:10.1002/anie.201405303

Cited by 36 publications

(46 citation statements)

References 58 publications

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“…Furthermore, they postulated a relaxation process in the excited state that would follow after the proton transfer extends into the picosecond time scale. This relaxation process has been recently theoretically studied by Thiel and co-workers by means of relaxed potential energy profiles, 19 which also led to a possible deactivation process via the hulatwist torsion of the chromophore, similar to that previously found by Olivucci and co-workers for the anionic form of the GFP chromophore. 20 In our previous theoretical work based on dynamic studies, we were able to precisely explain the reasons for the proton transfer and reproduce the time for the transfer (<50 fs) via quantum dynamics calculations.…”

Section: Introductionsupporting

confidence: 65%

New insights into the structure–spectrum relationship in S65T/H148D and E222Q/H148D green fluorescent protein mutants: a theoretical assessment

et al. 2014

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The green fluorescent protein (GFP) variant S65T/H148D recovers the A-band fluorescence lost in the single mutant S65T, and it has been established that Asp148 is the alternate proton acceptor for the excited state proton transfer (ESPT). This mutant has been widely studied and presents unique spectroscopic properties, such as an ultrafast rise in the fluorescence (<50 fs). Also it exhibits a red-shift of the A absorption band of 20 nm with respect to wt-GFP's. The double mutant E222Q/H148D presents a very similar behaviour, at least within the experimental data available (which is scarcer than those of S65T/H148D). By means of dynamic theoretical studies we have been able to (1) reproduce and thoroughly analyse the red-shifted absorption spectra of both mutants and (2) predict the structure that the variant E222Q/H148D (for which there is no X-ray-resolved structure available) most probably adopts in water at room temperature. Our results deepen the understanding of the way GFP variants work and give some new insights into the rational design of fluorescent proteins and biological photosystems in general.

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Section: Introductionsupporting

confidence: 65%

New insights into the structure–spectrum relationship in S65T/H148D and E222Q/H148D green fluorescent protein mutants: a theoretical assessment

et al. 2014

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“…The loss of RF in H181W may be attributed to the loss of stability because loosening the core would cause the chromophore to be more flexible and therefore more likely to decay nonradiatively from the excited state. 14 For the same reasons, this mutation may have caused inefficient chromophore maturation. In other studies, the CM reaction depends strongly on the stability of the folded protein (K. Fraser and C. Bystroff, unpublished data).…”

Section: Resultsmentioning

confidence: 99%

Mispacking and the Fitness Landscape of the Green Fluorescent Protein Chromophore Milieu

et al. 2017

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The autocatalytic maturation of the chromophore in green fluorescent protein (GFP) was thought to require the precise positioning of the side chains surrounding it in the core of the protein, many of which are strongly conserved among homologous fluorescent proteins. In this study, we screened for green fluorescence in an exhaustive set of point mutations of seven residues that make up the chromophore microenvironment, excluding R96 and E222 because mutations at these positions have been previously characterized. Contrary to expectations, nearly all amino acids were tolerated at all seven positions. Only four point mutations knocked out fluorescence entirely. However, chromophore maturation was found to be slower and/or fluorescence reduced in several cases. Selected combinations of mutations showed nonadditive effects, including cooperativity and rescue. The results provide guidelines for the computational engineering of GFPs.

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“…41 However, our new view and the broader understanding of GFP quenching is that solvent exposure is irrelevant; rather, folding is required to stabilize the planar conformation of the excited state of the chromophore by blocking the “asynchronous hula twist” rotation of the p -hydroxybenzylidene moiety that leads to overlap of the ground state and excited state energy landscapes and triggers nonradiative decay. 17 We note in retrospect that computationally designed sequence libraries were screened for fluorescence, not directly for peptide binding, on the assumption that fluorescence would correlate with peptide binding. That assumption turned out to be wrong.…”

Section: Discussionmentioning

confidence: 99%

“…Fluorescence is quenched in the unfolded state via hula-twist motions in the excited state of the liberated CRO. 17 …”

mentioning

confidence: 99%

Toward Computationally Designed Self-Reporting Biosensors Using Leave-One-Out Green Fluorescent Protein

Huang

Banerjee²,

Crone³

et al. 2015

Biochemistry

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Leave-one-out green fluorescent protein (LOOn-GFP) is a circularly permuted and truncated GFP lacking the nth β-strand element. LOO7-GFP derived from the wild-type sequence (LOO7-WT) folds and reconstitutes fluorescence upon addition of β-strand 7 (S7) as an exogenous peptide. Computational protein design may be used to modify the sequence of LOO7-GFP to fit a different peptide sequence, while retaining the reconstitution activity. Here we present a computationally designed leave-one-out GFP in which wild-type strand 7 has been replaced by a 12-residue peptide (HA) from the H5 antigenic region of the Thailand strain of H5N1 influenza virus hemagglutinin. The DEEdesign software was used to generate a sequence library with mutations at 13 positions around the peptide, coding for approximately 3 × 105 sequence combinations. The library was coexpressed with the HA peptide in E. coli and colonies were screened for in vivo fluorescence. Glowing colonies were sequenced, and one (LOO7-HA4) with 7 mutations was purified and characterized. LOO7-HA4 folds, fluoresces in vivo and in vitro, and binds HA. However, binding results in a decrease in fluorescence instead of the expected increase, caused by the peptide-induced dissociation of a novel, glowing oligomeric complex instead of the reconstitution of the native structure. Efforts to improve binding and recover reconstitution using in vitro evolution produced colonies that glowed brighter and matured faster. Two of these were characterized. One lost all affinity for the HA peptide but glowed more brightly in the unbound oligomeric state. The other increased in affinity to the HA peptide but still did not reconstitute the fully folded state. Despite failing to fold completely, peptide binding by computational design was observed and was improved by directed evolution. The ratio of HA to S7 binding increased from 0.0 for the wild-type sequence (no binding) to 0.01 after computational design (weak binding) and to 0.48 (comparable binding) after in vitro evolution. The novel oligomeric state is composed of an open barrel.

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Concerted Asynchronous Hula‐Twist Photoisomerization in the S65T/H148D Mutant of Green Fluorescent Protein

Cited by 36 publications

References 58 publications

New insights into the structure–spectrum relationship in S65T/H148D and E222Q/H148D green fluorescent protein mutants: a theoretical assessment

New insights into the structure–spectrum relationship in S65T/H148D and E222Q/H148D green fluorescent protein mutants: a theoretical assessment

Mispacking and the Fitness Landscape of the Green Fluorescent Protein Chromophore Milieu

Toward Computationally Designed Self-Reporting Biosensors Using Leave-One-Out Green Fluorescent Protein

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