Spectroscopic and Nanoscopic Studies of Anions, DNA, and Membranes

Abstract

Spectroscopy, the study of the change in light signal upon the interaction with the objects of interest, and microscopy, the study of the objects by directly imaging them with microscope have been widely used from elementary particle physics to bioscience. Numerous efforts have been made to combine these two fields to develop new observation tools even though they were usually used independently. Fluorescence microscopy made us to observe living species in real time without invasion through labeling of the fluorescent molecules. However, there is an intrinsic optical diffraction limit in far-field optical microscopy. Recently, super-resolution microscopy techniques have developed that overcome the diffraction limit using the photophysical properties like absorption and emission of the molecules. As a result, one can observe small objects such as organelles, proteins, DNAs that have smaller size than diffraction limit. A combination of dynamic molecular combing and STED nanoscopy was demonstrated to accurately and precisely measure the lengths of DNA ranging widely in size from 117 bp to 23,130 bp. A size difference of as small as 100 bp could be discriminated, fostering the prospect of this method being used in detecting copy number variation. With the combination of STED nanoscopy and alternating laser excitation (ALEX)-FRET single-molecule spectroscopy, we were able to detect a single molecule in a tightly confined volume that is 50-times smaller than the diffraction-limited confocal volume. We demonstrated the feasibility of this new technique for a dsDNA (20 bp) labeled by ATTO647N and DY-510XL. FRET between two dyes was acquired under both confocal and STED conditions. With this new technique, we were able to observe a single diffusing DNA molecule at up to 5 nM concentration, which is 100 times higher than the condition of typical single-molecule measurements. Model lipid membranes such as GUVs and tethered free-standing bilayers are excellent systems for studying trans-membrane proteins free from any surface interactions. However, membrane proteins in such systems are not very well studied because of the difficulty of incorporating proteins into GUVs or tethered membranes while still maintaining protein function. Inspired by previous work on DNA-mediated membrane fusion, we have applied this DNA-machinery to mediate fusion of small proteoliposomes containing the photosynthetic reaction center to either GUVs or DNA-tethered lipid membrane patches formed by GUV rupture onto DNA coated glass surfaces. The diffusion behavior of the delivered proteins was measured and compared with the diffusion of those in glass-supported bilayers. Also, the protein activity and orientation before and after fusion was analysed. We investigated electron attachment to three dihalobenzene molecules, bromochlorobenzene (BCB), bromoiodobenzene (BIB) and chloroiodobenzene (CIB) by molecular beam photoelectron spectroscopy (PES). The most prominent product of electron attachment in the anion mass spectra was the atomic fragment of the less electronegative halogen of the two, i.e., Br- for BCB and I- for BIB and CIB. Photoelectron spectroscopy and ab initio calculations suggested that the approaching electron prefers to attack the less electronegative atom, a seemingly counterintuitive finding but consistent with the mass spectrometric result. For the iodine-containing species BIB and CIB, the photoelectron spectrum consists of bands from both the molecular anion and atomic I-, the latter of which is produced by photodissociation of the former. Molecular orbital analysis revealed that a large degree of orbital energy reordering takes place upon electron attachment. These phenomena were shown to be readily explained by simple molecular orbital theory and the electronegativity of the halogen atoms. Hetero-dimer anions of naphthalene (Np), anthracene (An), phenanthrene (Ph) and pyrene (Py) were investigated by time-of-flight mass spectrometer (TOF-MS), anion photoelectron spectroscopy and theoretical calculation. Two possible geometries exist with their electron affinity (EA) difference, parallel displaced (PD) and T-shaped. Dispersion force plays a key role in PD structure with the formation of new anionic core while π-hydrogen interaction plays a key role in T-shaped structure with the monomer anionic core. Their optimized structures and charge distributions can be simply explained by the relative difference of EA.