Structure of isolated biomolecules by electron diffraction-laser desorption: uracil and guanine

We report the structure of isolated biomolecules, uracil and guanine, demonstrating the capability of a newly developed electron diffraction apparatus augmented with surface-assisted IR laser desorption. This UED-4 apparatus provides a pulsed, dense molecular beam, which is stable for many hours and possibly days. From the diffraction patterns, it is evident that the plume composition is chemically pure, without detectable background from ions, fragmentation products, or molecular aggregates. The vibrational temperature deduced is indeed lower than the translational temperature of the plume indicating that the molecules are intact on such short time scales. The structures of uracil and guanine were refined at the deduced internal temperatures, and we compare the results with those predicted by density functional theory. Such experimental capability opens the door for many other studies of the structure (and dynamics) of biomolecules

Ultrafast Electron Diffraction: Pulsed Laser Desorption Enables Time-Resolved Structural Determination of Thermally Labile Chromophores

The construction and utilization of the fourth-generation ultrafast electron diffraction apparatus, UED4, is the subject of this thesis. With UED4 and its novel and universal sample delivery method based on laser desorption, we were able to vaporize thermally labile molecular samples and determine their ground-state structures and the structures of their photochemical and photophysical reaction products. Each component part of the new UED4 apparatus is described, and the experimental and computational procedures used to extract structural information from the time-resolved diffraction patterns are presented. Several molecules were studied in their ground states and photoinduced excited states or product states on the time scale of picoseconds and nanoseconds. With UED3, nitrobenzene was shown to undergo intramolecular rearrangement prior to NO loss in an ultrafast fragmentation reaction. In indole, the chromophore of the amino acid tryptophan, the involvement of a dark structure, formed on the picosecond time scale, was revealed in the nonradiative decay pathway of the initially excited state. By determining the ground state structures of the thermally labile nucleobases uracil and guanine, the first use of surface-assisted laser desorption in a pulsed electron diffraction experiment was reported using the newly developed UED4 apparatus. The determined structures of the photochemically generated species of the photochromic molecule 6-nitro-BIPS further demonstrated the capability of laser desorption electron diffraction to function as a time-resolved experiment. Finally, the fragmentation reaction of the amino acid tryptophan upon UV laser irradiation was studied with UED4. The ability to deliver increasingly large and conformationally heterogeneous molecules into the gas phase now provides new challenges and opportunities of both experimental and theoretical nature for the field of ultrafast electron diffraction.</p

4D visualization of embryonic, structural crystallization by single-pulse microscopy

In many physical and biological systems the transition from an amorphous to ordered native structure involves complex energy landscapes, and understanding such transformations requires not only their thermodynamics but also the structural dynamics during the process. Here, we extend our 4D visualization method with electron imaging to include the study of irreversible processes with a single pulse in the same ultrafast electron microscope (UEM) as used before in the single-electron mode for the study of reversible processes. With this augmentation, we report on the transformation of amorphous to crystalline structure with silicon as an example. A single heating pulse was used to initiate crystallization from the amorphous phase while a single packet of electrons imaged selectively in space the transformation as the structure continuously changes with time. From the evolution of crystallinity in real time and the changes in morphology, for nanosecond and femtosecond pulse heating, we describe two types of processes, one that occurs at early time and involves a nondiffusive motion and another that takes place on a longer time scale. Similar mechanisms of two distinct time scales may perhaps be important in biomolecular folding

Direct Structural Determination of Conformations of Photoswitchable Molecules by Laser Desorption–Electron Diffraction

Anfractuous paths: Electron diffraction reveals the involvement of multiple structures in the complex photochemistry of photoswitchable nitro-substituted 1,3,3-trimethylindolinobenzospiropyran. The spiropyran-to-merocyanine isomerization due to ring opening produces primarily the cis–trans–cis structure (see picture; red O, blue N, yellow C), while competing nonradiative pathways lead to other structures, namely the closed forms in their triplet and singlet ground states

Ultrashort electron pulses for diffraction, crystallography and microscopy: theoretical and experimental resolutions

Pulsed electron beams allow for the direct atomic-scale observation of structures with femtosecond to picosecond temporal resolution in a variety of fields ranging from materials science to chemistry and biology, and from the condensed phase to the gas phase. Motivated by recent developments in ultrafast electron diffraction and imaging techniques, we present here a comprehensive account of the fundamental processes involved in electron pulse propagation, and make comparisons with experimental results. The electron pulse, as an ensemble of charged particles, travels under the influence of the space–charge effect and the spread of the momenta among its electrons. The shape and size, as well as the trajectories of the individual electrons, may be altered. The resulting implications on the spatiotemporal resolution capabilities are discussed both for the N-electron pulse and for single-electron coherent packets introduced for microscopy without space–charge

Ultrafast Electron Diffraction Reveals Dark Structures of the Biological Chromophore Indole

With UED, as detailed in the experimental section, we are able to determine both ground- and excited-state structures and obtain the temporal behavior for excitation of indole at 267 nm. For the ground-state structure (see Scheme SI1 in the Supporting Information), the experimental and the theoretical molecular scattering function, sM(s), together with the radial distributions, f(r), are shown in Figure 1. The refined structural parameters are listed in Table SI1 in the Supporting Information, together with values obtained from density functional theory (DFT) calculations. The satisfactory agreement between experiment and theory gives the refined structural parameters, which were found to have discrepancies at most within 0.007 Å and 0.18° for bond lengths and angles, respectively

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging. Photoconvertible proteins occupy two color channels thereby limiting multicolour localisation microscopy applications. Here the authors present UnaG, a new green-to-dark photoswitching fluorescent protein for super-resolution imaging, whose activation is based on a noncovalent binding with bilirubin

Histone acetylation-independent transcription stimulation by a histone chaperone

Histone chaperones are thought to be important for maintaining the physiological activity of histones; however, their exact roles are not fully understood. The physiological function of template activating factor (TAF)-I, one of the histone chaperones, also remains unclear; however, its biochemical properties have been well studied. By performing microarray analyses, we found that TAF-I stimulates the transcription of a sub-set of genes. The transcription of endogenous genes that was up-regulated by TAF-I was found to be additively stimulated by histone acetylation. On performing an experiment with a cell line containing a model gene integrated into the chromosome, TAF-I was found to stimulate the model gene transcription in a histone chaperone activity-dependent manner additively with histone acetylation. TAF-I bound to the core histones and remodeled the chromatin structure independent of the N-terminal histone tail and its acetylation level in vitro. These results suggest that TAF-I remodel the chromatin structure through its interaction with the core domain of the histones, including the histone fold, and this mechanism is independent of the histone acetylation status