The Role of the Protein Matrix in Green Fluorescent Protein Fluorescence

Maddalo, Scott L.; Zimmer, Marc

doi:10.1562/2005-04-11-ra-485

Cited by 79 publications

(92 citation statements)

References 49 publications

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“…Although the chromophore is able to isomerize unhindered in solution without the structural restraints of the surrounding barrel, isomerization is limited in the folded state unless there is a hula twist (22,23,27). This conformational transition is responsible for the slow search toward the second basin {N iso }.…”

Section: Discussionmentioning

confidence: 99%

“…Our coarse-grained model captures some structural aspects of the chromophore that are important in describing folding through a noncanonical kink in the central ␣-helix. Recent work on GFP-family chromophore isomerization using crystallography and molecular dynamics has revealed chromophore isomerization in fluorescent proteins (19)(20)(21) and major differences in the interior hydrogen-bond networks between chromophore isomerizations (22)(23)(24).…”

Section: Structure Of Gfpmentioning

confidence: 99%

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The dual-basin landscape in GFP folding

Andrews

Gosavi

Finke

et al. 2008

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

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Recent experimental studies suggest that the mature GFP has an unconventional landscape composed of an early folding event with a typical funneled landscape, followed by a very slow search and rearrangement step into the locked, active chromophore-containing structure. As we have shown previously, the substantial difference in time scales is what generates the observed hysteresis in thermodynamic folding. The interconversion between locked and the soft folding structures at intermediate denaturant concentrations is so slow that it is not observed under the typical experimental observation time. Simulations of a coarse-grained model were used to describe the fast folding event as well as identify native-like intermediates on energy landscapes enroute to the fluorescent native fold. Interestingly, these simulations reveal structural features of the slow dynamic transition to chromophore activation. Experimental evidence presented here shows that the trapped, native-like intermediate has structural heterogeneity in residues previously linked to chromophore formation. We propose that the final step of GFP folding is a ''locking'' mechanism leading to chromophore formation and high stability. The combination of previous experimental work and current simulation work is explained in the context of a dual-basin folding mechanism described above. molecular dynamics ͉ multicanonical method ͉ protein folding ͉ topological frustration ͉ folding funnel G FP has become a common reagent in biochemistry laboratories because of its ability to fold and form a visually fluorescent chromophore through autocatalytic cyclization and dehydration/oxidation reactions (1). Recent studies on the folding of GFP have shown multiple kinetic phases (2, 3) and high barriers to folding that lead to a slow ''drift'' of the unfolding equilibrium transition curve (4). In fact, a ''superfolder'' variant of GFP (sfGFP) (5) has an apparent hysteresis in equilibrium folding that is observed in the experiment (6, 7) shown in Fig. 1A. Considering the apparent nonthermodynamic behavior observed in experiment, can we use simulation to probe the free-energy landscape and explain these results?Proteins whose folding has been optimized by evolution have a sufficiently smooth, funneled energy landscape, with an ensemble of folding routes directed toward the native structural basin (8-10). Coarse-grained models with potentials designed to bias the protein toward the native basin, structure-based (Go ) models (11), have been able to capture the main structural features of the folding mechanism observed in experiments. Because these models are energetically unfrustrated, the agreement to experiments demonstrate that typical well folding proteins have such small amounts of residual energetic frustration that the observed structural heterogeneity observed in folding is mostly determined by geometrical properties of the native structure.Although it is a good folder, GFP has a folding landscape that differs from most proteins because of a structural rearrangement ne...

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Section: Discussionmentioning

confidence: 99%

Section: Structure Of Gfpmentioning

confidence: 99%

The dual-basin landscape in GFP folding

Andrews

Gosavi

Finke

et al. 2008

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

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“…Presumably, the protein must inhibit displacement along all bridge torsion coordinates in order to preserve the emitting state. This "multiple pathway problem" has been highlighted by Zimmer and co-workers, [26][27][28][29] However, these models cannot address the twisted intramolecular charge-transfer ͑TICT͒ character that accompanies excited-state torsion. 23,24 Straightforward extension of theories of enzymatic catalysis 30 would suggest that electrostatic interactions between the TICT states and the protein may contribute to suppression or control of the photoisomerization.…”

Section: Why Is This State Not Observed In Chromophores Outside Of Thmentioning

confidence: 99%

A diabatic three-state representation of photoisomerization in the green fluorescent protein chromophore

Olsen

McKenzie

2009

The Journal of Chemical Physics

122

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We give a quantum chemical description of the photoisomerization reaction of green fluorescent protein (GFP) chromophores using a representation over three diabatic states. Photoisomerization leads to non-radiative decay, and competes with fluorescence in these systems. In the protein, this pathway is suppressed, leading to fluorescence. Understanding the electronic states relevant to photoisomerization is a prerequisite to understanding how the protein suppresses it, and preserves the emitting state of the chromophore. We present a solution to the state-averaged complete active space problem, which is spanned at convergence by three fragment-localized orbitals. We generate the diabatic-state representation by block diagonalization transformation of the Hamiltonian calculated for the anionic chromophore model HBDI with multi-reference, multi-state perturbation theory. The diabatic states are charge-localized and admit a natural valence-bond interpretation. At planar geometries, the diabatic picture of the optical excitation reduces to the canonical two-state charge transfer resonance of the anion. Extension to a three-state model is necessary to describe decay via two possible pathways associated with photoisomerization of the (methine) bridge. Parametric Hamiltonians based on the three-state ansatz can be fit directly to data generated using the underlying active space. We provide an illustrative example of such a parametric Hamiltonian.

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“…236 In addition, the surrounding protein cavity (steric hindrance) can also influence the fluorescence of the fluorophores. 237 The major advantages of optical imaging techniques are their relatively low operational costs, simple imaging procedures, absence of ionization radiation exposure, quick image acquisition, and ability to image multiple animals repetitively over a long period of time. Recent development of fluorophores in the NIR range has allowed for imaging of tissues as deep as 7–14 cm.…”

Section: Optical Imaging Techniquesmentioning

confidence: 99%

Whole animal imaging

Sandhu

Solorio

Broome

et al. 2010

WIREs Mechanisms of Disease

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Translational research plays a vital role in understanding the underlying pathophysiology of human diseases, and hence development of new diagnostic and therapeutic options for their management. After creating an animal disease model, pathophysiologic changes and effects of a therapeutic intervention on them are often evaluated on the animals using immunohistologic or imaging techniques. In contrast to the immunohistologic techniques, the imaging techniques are noninvasive and hence can be used to investigate the whole animal, oftentimes in a single exam which provides opportunities to perform longitudinal studies and dynamic imaging of the same subject, and hence minimizes the experimental variability, requirement for the number of animals, and the time to perform a given experiment. Whole animal imaging can be performed by a number of techniques including x-ray computed tomography, magnetic resonance imaging, ultrasound imaging, positron emission tomography, single photon emission computed tomography, fluorescence imaging, and bioluminescence imaging, among others. Individual imaging techniques provide different kinds of information regarding the structure, metabolism, and physiology of the animal. Each technique has its own strengths and weaknesses, and none serves every purpose of image acquisition from all regions of an animal. In this review, a broad overview of basic principles, available contrast mechanisms, applications, challenges, and future prospects of many imaging techniques employed for whole animal imaging is provided. Our main goal is to briefly describe the current state of art to researchers and advanced students with a strong background in the field of animal research.

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The Role of the Protein Matrix in Green Fluorescent Protein Fluorescence

Cited by 79 publications

References 49 publications

The dual-basin landscape in GFP folding

The dual-basin landscape in GFP folding

A diabatic three-state representation of photoisomerization in the green fluorescent protein chromophore

Whole animal imaging

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