The frustrated Lewis pair tBu 3 P/B(C 6 F 5 ) 3 (1) readily adds SO 2 to yield the zwitterionic adduct tBu 3 P + -S(O)-C 6 H 10 (5)] add SO 2 at À78 C to yield the corresponding six-membered addition products 4a, 4b, 6. The adducts contain a chiral sulfur center. The [B]-O-(O)S-[P] addition products 3, 4b and 6 were characterized by X-ray diffraction. Scheme 1 Reactions of inter-and intramolecular FLPs with SO 2 .

DnaB helicases are motor proteins that couple ATP-hydrolysis to the loading of the protein onto DNA at the replication fork and to translocation along DNA to separate double-stranded DNA into single strands during replication. Using a network of conformational states, arrested by nucleotide mimics, we herein characterize the reaction coordinates for ATP hydrolysis, DNA loading and DNA translocation using solid-state NMR spectroscopy. AMP-PCP is used as pre-hydrolytic, ADP:AlF4− as transition state, and ADP as post-hydrolytic ATP mimic. 31P and 13C NMR spectra reveal conformational and dynamic responses to ATP hydrolysis and the resulting DNA loading and translocation with single amino-acid resolution. This allows us to identify residues guiding the DNA translocation process and to explain the high binding affinities for DNA observed for ADP:AlF4−, which turns out to be optimally preconfigured to bind DNA.

Protein aggregation into amyloid fibrils is associated with multiple neurodegenerative diseases, including Parkinson’s disease. Kinetic data and biophysical characterization have shown that the secondary nucleation pathway highly accelerates aggregation via the absorption of monomeric protein on the surface of amyloid fibrils. Here, we used NMR and electron paramagnetic resonance spectroscopy to investigate the interaction of monomeric α-synuclein (α-Syn) with its fibrillar form. We demonstrate that α-Syn monomers interact transiently via their positively charged N terminus with the negatively charged flexible C-terminal ends of the fibrils. These intermolecular interactions reduce intramolecular contacts in monomeric α-Syn, yielding further unfolding of the partially collapsed intrinsically disordered states of α-Syn along with a possible increase in the local concentration of soluble α-Syn and alignment of individual monomers on the fibril surface. Our data indicate that intramolecular unfolding critically contributes to the aggregation kinetics of α-Syn during secondary nucleation.

Covalent bonding interactions between the Lewis acid and Lewis base functionalities have been probed in a series of "frustrated Lewis pairs" (FLPs) (mainly substituted vinylene linked intramolecular phosphane−borane adducts), using solid-state nuclear magnetic resonance techniques and accompanying DFT calculations. Both the 11 B NMR isotropic chemical shifts and nuclear electric quadrupolar coupling parameters turn out to be extremely sensitive experimental probes for such interactions, revealing linear correlations with boron−phosphorus internuclear distances. The principal component V zz of the 11

Paramagnetic metal ions deliver structural information both in EPR and solid-state NMR experiments, offering a profitable synergetic approach to study bio-macromolecules. We demonstrate the spectral consequences of Mg / Mn substitution and the resulting information contents for two different ATP:Mg -fueled protein engines, a DnaB helicase from Helicobacter pylori active in the bacterial replisome, and the ABC transporter BmrA, a bacterial efflux pump. We show that, while EPR spectra report on metal binding and provide information on the geometry of the metal centers in the proteins, paramagnetic relaxation enhancements identified in the NMR spectra can be used to localize residues at the binding site. Protein engines are ubiquitous and the methods described herein should be applicable in a broad context.

The conjugated frustrated phosphane/borane Lewis pairs formed by 1,1-carboboration of a substituted diphenylphosphino acetylene, undergo a synergistic 1,1-addition reaction to n-butyl isocyanide with formation of new B-C and P-C bonds to the former isonitrile carbon atom. Using tert-butyl isocyanide dynamic behaviour between the isocyanide-[B] adduct and the 1,1-addition product formation was observed in solution. The different modes of isocyanide binding to the FLPs in the solid state were characterized using X-ray crystal structure analyses and comprehensive 11 B and 31 P solid-state magicangle-spinning (MAS-) NMR experiments. The free FLP, the Lewis adduct at the borane group, and the cyclic product resulting from isocyanide addition to both reaction centers, can be differentiated via 11 B and 31 P isotropic chemical shifts, 11 B nuclear electric quadrupole coupling constants, isotropic indirect 11 B-31 P spin-spin coupling constants, and 11 B/ 31 P internuclear distances measured by rotational echo double resonance.

Graphical Abstract Highlights d Liquid-and solid-state NMR structure of a primase HBD bound to DNA and two ATPs d ATP binding triggers the sequence-specific DNA recognition of the primase HBD d In binding nucleotides and template, the HBD prepares dinucleotide synthesis d This preparatory role of the HBD is conserved in archaeoeukaryotic primases

ATP-binding-cassette (ABC) transporters are molecular pumps that translocate molecules across the cell membrane by switching between inward-facing and outward-facing states. To obtain a detailed understanding of their mechanism remains a challenge to structural biology, as these proteins are notoriously difficult to study at the molecular level in their active, membrane-inserted form. Here we use solid-state NMR to investigate the multidrug ABC transporter BmrA reconstituted in lipids. We identify the chemical-shift differences between the inward-facing, and outward-facing state induced by ATP:Mg 2+ :Vi addition. Analysis of an X-loop mutant, for which we show that ATPase and transport activities are uncoupled, reveals an incomplete transition to the outward-facing state upon ATP:Mg 2+ :Vi addition, notably lacking the decrease in dynamics of a defined set of residues observed in wild-type BmrA. This suggests that this stiffening is required for an efficient transmission of the conformational changes to allow proper transport of substrate by the pump.