Computational De Novo Design and Characterization of a Protein That Selectively Binds a Highly Hyperpolarizable Abiological Chromophore

This work reports the first example of a single-chain protein computationally designed to contain four α-helical segments and fold to form a four-helix bundle encapsulating a supramolecular abiological chromophore that possesses exceptional nonlinear optical properties. The 109-residue protein, designated SCRPZ-1, binds and disperses an insoluble hyperpolarizable chromophore, ruthenium(II) [5-(4\u27-ethynyl-(2,2\u27;6\u27,2″-terpyridinyl))-10,20-bis(phenyl)porphinato]zinc(II)-(2,2\u27;6\u27,2″-terpyridine)(2+) (RuPZn) in aqueous buffer solution at a 1:1 stoichiometry. A 1:1 binding stoichiometry of the holoprotein is supported by electronic absorption and circular dichroism spectra, as well as equilibrium analytical ultracentrifugation and size exclusion chromatography. SCRPZ-1 readily dimerizes at micromolar concentrations, and an empirical redesign of the protein exterior produced a stable monomeric protein, SCRPZ-2, that also displayed a 1:1 protein:cofactor stoichiometry. For both proteins in aqueous buffer, the encapsulated cofactor displays photophysical properties resembling those exhibited by the dilute RuPZn cofactor in organic solvent: femtosecond, nanosecond, and microsecond time scale pump-probe transient absorption spectroscopic data evince intensely absorbing holoprotein excited states having large spectral bandwidth that penetrate deep in the near-infrared energy regime; the holoprotein electronically excited triplet state exhibits a microsecond time scale lifetime characteristic of the RuPZn chromophore. Hyper-Rayleigh light scattering measurements carried out at an incident irradiation wavelength of 1340 nm for these holoproteins demonstrate an exceptional dynamic hyperpolarizabilty (β1340 = 3100 × 10(-30) esu). X-ray reflectivity measurements establish that this de novo-designed hyperpolarizable protein can be covalently attached with high surface density to a silicon surface without loss of the cofactor, indicating that these assemblies provide a new approach to bioinspired materials that have unique electro-optic functionality

Laser-controlled fluorescence in two-level systems

The ability to modify the character of fluorescent emission by a laser-controlled, optically nonlinear process has recently been shown theoretically feasible, and several possible applications have already been identified. In operation, a pulse of off-resonant probe laser beam, of sufficient intensity, is applied to a system exhibiting fluorescence, during the interval of excited- state decay following the initial excitation. The result is a rate of decay that can be controllably modified, the associated changes in fluorescence behavior affording new, chemically specific information. In this paper, a two-level emission model is employed in the further analysis of this all-optical process; the results should prove especially relevant to the analysis and imaging of physical systems employing fluorescent markers, these ranging from quantum dots to green fluorescence protein. Expressions are presented for the laser-controlled fluorescence anisotropy exhibited by samples in which the fluorophores are randomly oriented. It is also shown that, in systems with suitably configured electronic levels and symmetry properties, fluorescence emission can be produced from energy levels that would normally decay nonradiatively. © 2010 American Chemical Society

Evolution of Linear Absorption and Nonlinear Optical Properties in V-Shaped Ruthenium(II)-Based Chromophores

In this article, we describe a series of complexes with electron-rich cis-{Ru^(II)(NH_3)_4}^(2+) centers coordinated to two pyridyl ligands bearing N-methyl/arylpyridinium electron-acceptor groups. These V-shaped dipolar species are new, extended members of a class of chromophores first reported by us (Coe, B. J. et al. J. Am. Chem. Soc. 2005, 127, 4845−4859). They have been isolated as their PF_6− salts and characterized by using various techniques including ^1H NMR and electronic absorption spectroscopies and cyclic voltammetry. Reversible Ru^(III/II) waves show that the new complexes are potentially redox-switchable chromophores. Single crystal X-ray structures have been obtained for four complex salts; three of these crystallize noncentrosymmetrically, but with the individual molecular dipoles aligned largely antiparallel. Very large molecular first hyperpolarizabilities β have been determined by using hyper-Rayleigh scattering (HRS) with an 800 nm laser and also via Stark (electroabsorption) spectroscopic studies on the intense, visible d → π^* metal-to-ligand charge-transfer (MLCT) and π → π^* intraligand charge-transfer (ILCT) bands. The latter measurements afford total nonresonant β_0 responses as high as ca. 600 × 10^(−30) esu. These pseudo-C_(2v) chromophores show two substantial components of the β tensor, β_(zzz) and β_(zyy), although the relative significance of these varies with the physical method applied. According to HRS, β_(zzz) dominates in all cases, whereas the Stark analyses indicate that β_(zyy) is dominant in the shorter chromophores, but β_(zzz) and β_(zyy) are similar for the extended species. In contrast, finite field calculations predict that β_(zyy) is always the major component. Time-dependent density functional theory calculations predict increasing ILCT character for the nominally MLCT transitions and accompanying blue-shifts of the visible absorptions, as the ligand π-systems are extended. Such unusual behavior has also been observed with related 1D complexes (Coe, B. J. et al. J. Am. Chem. Soc. 2004, 126, 3880−3891)

Diquat Derivatives: Highly Active, Two-Dimensional Nonlinear Optical Chromophores with Potential Redox Switchability

In this article, we present a detailed study of structure−activity relationships in diquaternized 2,2′-bipyridyl (diquat) derivatives. Sixteen new chromophores have been synthesized, with variations in the amino electron donor substituents, π-conjugated bridge, and alkyl diquaternizing unit. Our aim is to combine very large, two-dimensional (2D) quadratic nonlinear optical (NLO) responses with reversible redox chemistry. The chromophores have been characterized as their PF_6^− salts by using various techniques including electronic absorption spectroscopy and cyclic voltammetry. Their visible absorption spectra are dominated by intense π → π^* intramolecular charge-transfer (ICT) bands, and all show two reversible diquat-based reductions. First hyperpolarizabilities β have been measured by using hyper-Rayleigh scattering with an 800 nm laser, and Stark spectroscopy of the ICT bands affords estimated static first hyperpolarizabilities β_0. The directly and indirectly derived β values are large and increase with the extent of π-conjugation and electron donor strength. Extending the quaternizing alkyl linkage always increases the ICT energy and decreases the E_(1/2) values for diquat reduction, but a compensating increase in the ICT intensity prevents significant decreases in Stark-based β_0 responses. Nine single-crystal X-ray structures have also been obtained. Time-dependent density functional theory clarifies the molecular electronic/optical properties, and finite field calculations agree with polarized HRS data in that the NLO responses of the disubstituted species are dominated by ‘off-diagonal’ β_(zyy) components. The most significant findings of these studies are: (i) β_0 values as much as 6 times that of the chromophore in the technologically important material (E)-4′-(dimethylamino)-N-methyl-4-stilbazolium tosylate; (ii) reversible electrochemistry that offers potential for redox-switching of optical properties over multiple states; (iii) strongly 2D NLO responses that may be exploited for novel practical applications; (iv) a new polar material, suitable for bulk NLO behavior

Human and murine fibroblast single cell transcriptomics reveals fibroblast clusters are differentially affected by ageing, and serum cholesterol

Aims Specific fibroblast markers and in-depth heterogeneity analysis are currently lacking, hindering functional studies in cardiovascular diseases (CVDs). Here, we established cell-type markers and heterogeneity in murine and human arteries and studied the adventitial fibroblast response to CVD and its risk factors hypercholesterolaemia and ageing. Methods and results Murine aorta single-cell RNA-sequencing analysis of adventitial mesenchymal cells identified fibroblast-specific markers. Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, whereas traditional markers such as the cluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiating along trajectories. Three trajectories, marked by CD55 (Cd55+), Cxcl chemokine 14 (Cxcl14+), and lysyl oxidase (Lox+), were reproduced in an independent RNA-seq dataset. Gene ontology (GO) analysis showed divergent functional profiles of the three trajectories, related to vascular development, antigen presentation, and/or collagen fibril organization, respectively. Trajectory-specific genes included significantly more genes with known genome-wide associations (GWAS) to CVD than expected by chance, implying a role in CVD. Indeed, differential regulation of fibroblast clusters by CVD risk factors was shown in the adventitia of aged C57BL/6J mice, and mildly hypercholesterolaemic LDLR KO mice on chow by flow cytometry. The expansion of collagen-related CXCL14+ and LOX+ fibroblasts in aged and hypercholesterolaemic aortic adventitia, respectively, coincided with increased adventitial collagen. Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens emphasized translational value as CD55+, CXCL14+ and LOX+ fibroblasts were observed in healthy and atherosclerotic specimens. Also, trajectory-specific gene sets are differentially correlated with human atherosclerotic plaque traits. Conclusion We provide two adventitial fibroblast-specific markers, PDGFRA and DPEP1, and demonstrate fibroblast heterogeneity in health and CVD in humans and mice. Biological relevance is evident from the regulation of fibroblast clusters by age and hypercholesterolaemia in vivo, associations with human atherosclerotic plaque traits, and enrichment of genes with a GWAS for CVD

Developments in the Photonic Theory of Fluorescence

Conventional fluorescence commonly arises when excited molecules relax to their ground electronic state, and most of the surplus energy dissipates in the form of photon emission. The consolidation and full development of theory based on this concept has paved the way for the discovery of several mechanistic variants that can come into play with the involvement of laser input – most notably the phenomenon of multiphoton-induced fluorescence. However, other effects can become apparent when off-resonant laser input is applied during the lifetime of the initial excited state. Examples include a recently identified scheme for laser-controlled fluorescence. Other systems of interest are those in which fluorescence is emitted from a set of two or more coupled nanoemitters. This chapter develops a quantum theoretical outlook to identify and describe these processes, leading to a discussion of potential applications ranging from all-optical switching to the generation of optical vortices

Synthesis, Structures, and Optical Properties of Ruthenium(II) Complexes of the Tris(1-pyrazolyl)methane Ligand

Four new complex salts [Ru^(II)Cl(Tpm)(L^A)_2][PF_6]_n [Tpm = tris(1-pyrazolyl)methane; n = 1, L^A = pyridine (py) 1 or ethyl isonicotinate (EIN) 2; n = 3, L^A = N-methyl-4,4′-bipyridinium (MeQ^+) 3 or N-phenyl-4,4′-bipyridinium (PhQ^+) 4] have been prepared and characterized. Electronic absorption spectra show intense d → π^* metal-to-ligand charge-transfer (MLCT) absorption bands, while cyclic voltammetry reveals a reversible Ru^(III/II) wave, accompanied by quasireversible or irreversible L^A-based reductions for all except 1. Single crystal X-ray structures have been obtained for 1•Me_2CO, 2, and 3•Me_2CO. For 2–4, molecular first hyperpolarizabilities β have been measured in acetonitrile solutions via the hyper-Rayleigh scattering (HRS) technique at 800 nm. Stark (electroabsorption) spectroscopic studies on the MLCT bands in frozen butyronitrile allow the indirect estimation of static first hyperpolarizabilities β_0. The various physical data obtained for 3 and 4 are compared with those reported previously for related cis-{Ru^(II)(NH_3)_4}^(2+) species [Coe, B. J. et al. J. Am. Chem. Soc. 2005, 127, 4845]. TD-DFT calculations on the complexes in 1–4 confirm that their lowest energy absorption bands are primarily Ru^(II) → L^A MLCT in character, while Ru^(II) → Tpm MLCT transitions are predicted at higher energies. DFT agrees with the Stark, but not the HRS measurements, in showing that β_0 increases with the electron-accepting strength of L^A. The 2D nature of the chromophores is evidenced by dominant β_(xxy) tensor components