Population Branching in the Conical Intersection of the Retinal Chromophore Revealed by Multipulse Ultrafast Optical Spectroscopy

Zgrablić, Goran; Novello, Anna Maria; Parmigiani, F.

doi:10.1021/ja205763x

Cited by 47 publications

(57 citation statements)

References 56 publications

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“…Conversely, to enhance the isomerization efficiency instead of fluorescence, one needs to lower the excited-state energy barrier as well as tune the conical intersection toward more peaked topology. Similar to isomerization, the structure at the conical intersection has charge transfer character that could be stabilized via electrostatics, altering the shape of the conical intersection (24,82). However, because conical intersections can be higher-dimensional structures (e.g., seams), tuning them can also change their accessibility or channel the excited-state population into other unwanted degrees of freedom (49).…”

Section: Resultsmentioning

confidence: 99%

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Lin

Both

et al. 2017

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

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Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics-function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.split GFP | potential energy surface | photodissociation | cis-trans isomerization | photosensory protein O ptical techniques for investigating biological processes in vivo can achieve subcellular spatial and millisecond temporal resolution by using genetically encoded light-responsive proteins (1). Photoactivatable proteins are used as either imaging tools, such as reversibly photoswitchable fluorescent proteins (RSFPs), with fluorescence that can be modulated by light (2) or optogenetic switches that convert light input into physiological outputs, such as channelrhodopsins, which can regulate ion flow through membranes in response to light (3). Split GFPs have been widely applied for imaging as fluorescent reporters of cellular processes, because they are small (∼25 kDa), are stable in cytosol, produce chromophores autocatalytically, and are amenable to mutation and circular permutation (4). Typically, the protein is expressed as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another, offering readouts of protein-protein colocalizaton with low background and high specificity (5). However, this technique can generate misleading results, because the GFP complexes, after being formed and fluorescing, are bound irreversibly, which can be highly perturbative to the processes being studied, especially if the protein interaction being probed is ordinarily reversible (6). Although split GFP complexes essentially do not dissocia...

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Section: Resultsmentioning

confidence: 99%

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Lin

Both

et al. 2017

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

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“…Excited State Evolution. Both the transient dynamics of PSBAT in solution 123 and the PSB-KLE-CRABPII signals (see section 3.1) indicate that photoproducts are generated upon S 1 decay. To determine which bond could potentially isomerize in the PSB-KLE-CRABPII pocket, relaxed S 1 scans along both C10C11C12C13 and C12C13C14 C15 dihedrals were computed starting at the CT minimum of Figure 9D (corresponding to point 4 in Figure 9B) where the central double bonds have single bond character and are prepared for isomerization.…”

Section: Resultsmentioning

confidence: 99%

Toward an Understanding of the Retinal Chromophore in Rhodopsin Mimics

et al. 2013

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Recently, a rhodopsin protein mimic was constructed by combining mutants of the cellular retinoic acid binding protein II (CRABPII) with an all-trans retinal chromophore. Here, we present a combined computational quantum mechanics/molecular mechanics (QM/MM) and experimental ultrafast kinetic study of CRABPII. We employ the QM/MM models to study the absorption (λ(a)max), fluorescence (λ(f)max), and reactivity of a CRABPII triple mutant incorporating the all-trans protonated chromophore (PSB-KLE-CRABPII). We also study the spectroscopy of the same mutant incorporating the unprotonated chromophore and of another double mutant incorporating the neutral unbound retinal molecule held inside the pocket. Finally, for PSB-KLE-CRABPII, stationary fluorescence spectroscopy and ultrafast transient absorption spectroscopy resolved two different evolving excited state populations which were computationally assigned to distinct locally excited and charge-transfer species. This last species is shown to evolve along reaction paths describing a facile isomerization of the biologically relevant 11-cis and 13-cis double bonds. This work represents a first exploratory attempt to model and study these artificial protein systems. It also indicates directions for improving the QM/MM models so that they could be more effectively used to assist the bottom-up design of genetically encodable probes and actuators employing the retinal chromophore.

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“…If this is the case, the larger the velocity of the S 1 reactant moving toward the CI, the higher is the reaction quantum yield consistently with a Landau-Zener model (52). Although recent studies have denied the existence of such a correlation for the retinal chromophore in solution (53,54), this does not exclude that such correlation holds in the protein environment and when the S 1 lifetime is below a 100 fs threshold. In fact, this is in line with the more complete semiclassical treatment introduced by Weiss and Warshel (50) whose validity is also supported by the sudden change in charge distribution seen at decay in our models (SI Appendix, Fig.…”

Section: Significancementioning

confidence: 99%

Comparison of the isomerization mechanisms of human melanopsin and invertebrate and vertebrate rhodopsins

Rinaldi

Melaccio

Gozem

et al. 2014

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

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Comparative modeling and ab initio multiconfigurational quantum chemistry are combined to investigate the reactivity of the human nonvisual photoreceptor melanopsin. It is found that both the thermal and photochemical isomerization of the melanopsin 11-cis retinal chromophore occur via a space-saving mechanism involving the unidirectional, counterclockwise twisting of the =C11H-C12H= moiety with respect to its Lys340-linked frame as proposed by Warshel for visual pigments [Warshel A (1976) Nature 260 (5553):679-683]. A comparison with the mechanisms documented for vertebrate (bovine) and invertebrate (squid) visual photoreceptors shows that such a mechanism is not affected by the diversity of the three chromophore cavities. Despite such invariance, trajectory computations indicate that although all receptors display less than 100 fs excited state dynamics, human melanopsin decays from the excited state ∼40 fs earlier than bovine rhodopsin. Some diversity is also found in the energy barriers controlling thermal isomerization. Human melanopsin features the highest computed barrier which appears to be ∼2.5 kcal mol −1 higher than that of bovine rhodopsin. When assuming the validity of both the reaction speed/quantum yield correlation discussed by Warshel, Mathies and coworkers [Weiss RM, Warshel A (1979) J Am Chem Soc 101:6131-6133; Schoenlein RW, Peteanu LA, Mathies RA, Shank CV (1991) Science 254(5030):412-415] and of a relationship between thermal isomerization rate and thermal activation of the photocycle, melanopsin turns out to be a highly sensitive pigment consistent with the low number of melanopsincontaining cells found in the retina and with the extraretina location of melanopsin in nonmammalian vertebrates.F or a long time it was assumed that the human retina contains only two types of photoreceptor cells: the rods and cones responsible for dim-light and daylight vision, respectively. However, recent studies have revealed the existence of a small number of intrinsically photosensitive retinal ganglion cells (ipRGCs) that regulate nonvisual photoresponses (1). ipRGCs express an atypical opsin-like protein named melanopsin (2, 3) which plays a role in the regulation of unconscious visual reflexes and in the synchronization of endogenous physiological responses to the dawn/dusk cycle (circadian rhythms) (4, 5).Melanopsins are unique among vertebrate photoreceptors because their amino acid sequence shares greater similarity to invertebrate than vertebrate rhodopsin (i.e., the photoreceptor of rods) (6, 7). Like rhodopsins, melanopsins feature an up-down bundle architecture of seven transmembrane α-helices incorporating the 11-cis isomer of retinal as a covalently bound protonated Schiff base (PSB11 in Fig. 1A). Light-induced (i.e., photochemical) isomerization of PSB11 to its all-trans isomer (PSBAT) triggers an opsin conformational change that, ultimately, activates the receptor and signaling cascade (8, 9). However, similar to invertebrate and in contrast to vertebrate rhodopsins, melanopsins are bistable ...

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Population Branching in the Conical Intersection of the Retinal Chromophore Revealed by Multipulse Ultrafast Optical Spectroscopy

Cited by 47 publications

References 56 publications

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Toward an Understanding of the Retinal Chromophore in Rhodopsin Mimics

Comparison of the isomerization mechanisms of human melanopsin and invertebrate and vertebrate rhodopsins

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