Preparation of DNA Substrates and Functionalized Glass Surfaces for Correlative Nanomanipulation and Colocalization (NanoCOSM) of Single Molecules.

Simultaneous nanomanipulation and colocalization of single molecules (NanoCOSM) provides a unique opportunity to correlate the mechanical properties and activities of biomolecules with their conformational states or states of assembly as part of dynamic macromolecular complexes. This opens the door to real-time single-molecule analysis of the correlations between structure, function, and composition of large multicomponent protein complexes.

Copper(I)-photocatalyzed trifluoromethylation of alkenes.

Using the photoreducible Cu(II) precatalyst 2, trifluoromethylation reactions of alkenes are conducted effectively at low copper loading (0.1-0.5 mol%) on exposing the reaction mixture to sunlight/ambient light.

Multi-frequency high-field EPR studies on metal-substituted xylose isomerase.

The bacterial enzyme D-xylose isomerase (XI) catalyses the conversion of D-xylose to D-xylulose. Each subunit of the homotetrameric protein contains a bimetallic active centre requiring divalent metal ions such as Mg2+, Mn2+ or Co2+ for catalytic activity. We report here on XI in which the metal binding site 1 is specifically loaded with EPR active Mn2+, while binding site 2 is occupied by Co2+ or Cd2+, rendering a catalytically active or inactive species respectively. The Q-band (34 GHz) EPR spectra of these mixed-metal samples (Co2+/Mn2+ and Cd2+/Mn2+ XI) show a clear influence of the metal in site 2 on the Mn2+ EPR parameters. Likewise, a systematic increase of the zero field splitting parameters (zfs) of Mn2+ in site 1 upon incubation with the inhibitor xylitol or substrates for both mixed-metal samples is found. For Co2+/Mn2+ XI complexed with substrate, a drastic line broadening of the central -1/2 <--> +1/2 transition is observed in Q-band EPR, which was not amenable to analysis so far. By means of a multi-frequency approach at frequencies beyond Q-band, the relevant zfs parameters were derived from spectral simulations of EPR spectra measured at 94, 285 and 670 GHz. It is shown that parallel to the increase of the D-value its distribution also grows considerably in going from free Co2+/Mn2+ XI to the species complexed with inhibitor or substrate. For XI with bound substrate, D-values in the range of 70-90 mT and a distribution of about 30 mT were found from simulation trials. The large distribution in zfs values is thought to be correlated to the structural disorder induced by the shift of the metal ion of site 2 into a location necessary for the isomerisation reaction. The results are discussed with respect to high-resolution crystal data.