Algorithms for separable nonlinear least squares with application to modelling time-resolved spectra

The multiexponential analysis problem of fitting kinetic models to time-resolved spectra is often solved using gradient-based algorithms that treat the spectral parameters as conditionally linear. We make a comparison of the two most-applied such algorithms, alternating least squares and variable projection. A numerical study examines computational efficiency and linear approximation standard error estimates. A new derivation of the Fisher information matrix under the full Golub-Pereyra gradient allows a numerical comparison of parameter precision under variable projection variants. Under the criteria of efficiency, quality of standard error estimates and parameter precision, we conclude that the Kaufman variable projection technique performs well, while techniques based on alternating least squares have significant disadvantages for application in the problem domain.     

Detection of Asp371, Phe375, and Tyr376 Influence on GD-95-10 Lipase Using Alanine Scanning Mutagenesis

GD-95-10 and GD-95-20 lipases are modified GD-95 lipase variants, which lack 10 and 20 C-terminal amino acids, respectively. Previous analysis showed that GD-95-10 lipase has higher activity than GD-95 lipase, while GD-95-20 lipase almost completely loses its activity. Analysis in silico suggested three conservative amino acids at region between 369 and 378 amino acids which can be relevant to the activity of GD-95-10 lipase. These amino acids have direct contacts with residues involved in substrate binding, stabilization of the serine loop or form oxyanion hole. In this work, the role of Asp371, Phe375, and Tyr376 on activity, functionality, and structure of GD-95-10 lipase was analyzed by Ala scanning mutagenesis. We showed that even a single mutation can impact the main structure and activity of Geobacillus lipases. Our experiments provide new knowledge about lipases from thermophilic Geobacillus bacteria and are important for protein engineering and synthetic biology. These enzymes and their engineering can be basis for future biocatalysts applied in production of biofuel or other industrial esters.     

Ultrafast dynamics of isolated model photoactive yellow protein chromophores: "Chemical perturbation theory" in the laboratory

Pump-probe and pump-dump probe experiments have been performed on several isolated model chromophores of the photoactive yellow protein (PYP). The observed transient absorption spectra are discussed in terms of the spectral signatures ascribed to solvation, excited-state twisting, and vibrational relaxation. It is observed that the protonation state has a profound effect on the excited-state lifetime of p-coumaric acid. Pigments with ester groups on the coumaryl tail end and charged phenolic moieties show dynamics that are significantly different from those of other pigments. Here, an unrelaxed ground-state intermediate could be observed in pump-probe signals. A similar intermediate could be identified in the sinapinic acid and in isomerization-locked chromophores by means of pump-dump probe spectroscopy; however, in these compounds it is less pronounced and could be due to ground-state solvation and/or vibrational relaxation. Because of strong protonation-state dependencies and the effect of electron donor groups, it is argued that charge redistribution upon excitation determines the twisting reaction pathway, possibly through interaction with the environment. It is suggested that the same pathway may be responsible for the initiation of the photocycle in native PYP.     

Excited-state dynamics of carotenoids in light-harvesting complexes. 2. Dissecting pulse structures from optimal control experiments

Dispersed multipump-probe (PPP) spectroscopy was used to explore the role of saturation, annihilation, and structured pulses in recent coherent control experiments on the light-harvesting 2 complex from Rhodopseudomonas acidophila (Herek et al. Nature 2002, 417, 533). We discuss the complimentary aspects between the PPP technique and coherent control studies, in particular the ability to dissect complicated pulse structures and the utility in exploring incoherent mechanisms. With the aid of a simple multistate model involving only population dynamics, we illustrate how the optimized structured pulses may be explained in terms of an interplay between excited-state populations, saturation, and annihilation. Furthermore, we discuss the experimental conditions that are required for incoherent effects to contribute to control experimental signals, with particular emphasis on pulse intensities, and show that the optimization of a ratio of conservative signals (i.e., not modulated by external dynamics) is required to exclude saturation effects from coherent control studies.     

Energy transfer, excited-state deactivation, and exciplex formation in artificial caroteno-phthalocyanine light-harvesting antennas

We present results from transient absorption spectroscopy on a series of artificial light-harvesting dyads made up of a zinc phthalocyanine (Pc) covalently linked to carotenoids with 9, 10, or 11 conjugated carbon-carbon double bonds, referred to as dyads 1, 2, and 3, respectively. We assessed the energy transfer and excited-state deactivation pathways following excitation of the strongly allowed carotenoid S2 state as a function of the conjugation length. The S2 state rapidly relaxes to the S* and S1 states. In all systems we detected a new pathway of energy deactivation within the carotenoid manifold in which the S* state acts as an intermediate state in the S2-->S1 internal conversion pathway on a sub-picosecond time scale. In dyad 3, a novel type of collective carotenoid-Pc electronic state is observed that may correspond to a carotenoid excited state(s)-Pc Q exciplex. The exciplex is only observed upon direct carotenoid excitation and is nonfluorescent. In dyad 1, two carotenoid singlet excited states, S2 and S1, contribute to singlet-singlet energy transfer to Pc, making the process very efficient (>90%) while for dyads 2 and 3 the S1 energy transfer channel is precluded and only S2 is capable of transferring energy to Pc. In the latter two systems, the lifetime of the first singlet excited state of Pc is dramatically shortened compared to the 9 double-bond dyad and model Pc, indicating that the carotenoid acts as a strong quencher of the phthalocyanine excited-state energy.     

Use of ultrafast dispersed pump-dump-probe and pump-repump-probe spectroscopies to explore the light-induced dynamics of peridinin in solution

Optical pump-induced dynamics of the highly asymmetric carotenoid peridinin in methanol was studied by dispersed pump-probe, pump-dump-probe, and pump-repump-probe transient absorption spectroscopy in the visible region. Dispersed pump-probe measurements show that the decay of the initially excited S2 state populates two excited states, the S1 and the intramolecular charge-transfer (ICT) state, at a ratio determined by the excitation wavelength. The ensuing spectral evolution occurs on the time scale of a few picoseconds and suggests the equilibration of these states. Dumping the stimulated emission of the ICT state with an additional 800-nm pulse after 400- and 530-nm excitation preferentially removes the ICT state contribution from the broad excited-state absorption, allowing for its spectral characterization. At the same time, an unrelaxed ground-state species, which has a subpicosecond lifetime, is populated. The application of the 800-nm pulse at early times, when the S2 state is still populated, led to direct generation of the peridinin cation, observed for the first time in a transient absorption experiment. The excited and ground electronic states manifold of peridinin has been reconstructed using target analysis; this approach combined with the measured multipulse spectroscopic data allows us to estimate the spectra and time scales of the corresponding transient states.     

Excited-state dynamics of carotenoids in light-harvesting complexes. 1. Exploring the relationship between the S1 and S* states

Dispersed transient absorption spectra collected at variable excitation intensities in combination with time-resolved signals were used to explore the underlying connectivity of the electronic excited-state manifold of the carotenoid rhodopin glucoside in the light-harvesting 2 complex isolated from Rhodopseudomonas acidophila. We find that the S state, which was recently identified as an excited state in carotenoids bound in bacterial light-harvesting complexes, exhibits a different response to the increase of excitation intensity than the S(1) state, which suggests that the models used so far to describe the excited states of carotenoids are incomplete. We propose two new models that can describe both the time-resolved and the intensity-dependent data; the first postulates that S(1) and S* are not populated in parallel after the decay of the initially excited S(2) state but instead result from the excitation of distinct ground-state subpopulations. The second model introduces a resonantly enhanced light-induced transition during excitation, which promotes population to higher-lying excited states that favors the formation of S* over S(1). Multiwavelength target analysis of the time-resolved and excitation-intensity dependence measurements were used to characterize the involved states and their responses. We show that both proposed models adequately fit the measured data, although it is not possible to determine which model is most apt. The physical origins and implications of both models are explored.     

Synthesis of 1′,3,3′,4-tetrahydrospiro[chromene-2,2′-indoles] as a new class of ultrafast light-driven molecular switch

A new class of ultrafast light-driven molecular switch has been designed and synthesized. The reaction of 1-substituted 2-methylidene-3,3-dimethyl-2,3-dihydro-1H-indoles with 2-chloromethyl-4-nitrophenol afforded 1′,3,3′,4-tetrahydrospiro[chromene-2,2′-indoles], following a basic workup. The spectroscopic properties and nanosecond-resolved photoinduced dynamics of five compounds in the 1′,3,3′,4-tetrahydrospiro[chromene-2,2′-indole] family were investigated.