Systematic QM/MM Study for Predicting 31P NMR Chemical Shifts of Adenosine Nucleotides in Solution and Stages of ATP Hydrolysis in a Protein Environment.

NMR (nuclear magnetic resonance) spectroscopy allows for important atomistic insights into the structure and dynamics of biological macromolecules; however, reliable assignments of experimental spectra are often difficult. Herein, quantum mechanical/molecular mechanical (QM/MM) calculations can provide crucial support. A major problem for the simulations is that experimental NMR signals are time-averaged over much longer time scales, and since computed chemical shifts are highly sensitive to local changes in the electronic and structural environment, sufficiently large averages over representative structural ensembles are essential. This entails high computational demands for reliable simulations. For NMR measurements in biological systems, a nucleus of major interest is 31P since it is both highly present (e.g., in nucleic acids) and easily observable. The focus of our present study is to develop a robust and computationally cost-efficient framework for simulating 31P NMR chemical shifts of nucleotides. We apply this scheme to study the different stages of the ATP hydrolysis reaction catalyzed by p97. Our methodology is based on MM molecular dynamics (MM-MD) sampling, followed by QM/MM structure optimizations and NMR calculations. Overall, our study is one of the most comprehensive QM-based 31P studies in a protein environment and the first to provide computed NMR chemical shifts for multiple nucleotide states in a protein environment. This study sheds light on a process that is challenging to probe experimentally and aims to bridge the gap between measured and calculated NMR spectroscopic properties.

Characterizing ATP processing by the AAA+ protein p97 at the atomic level.

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of protein homeostasis and an impactful pharmacological target. The hexameric complex undergoes substantial conformational changes throughout its catalytic cycle. Here we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and molecular dynamics simulations. p97 populates a metastable reaction intermediate, the ADP·Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.

Observation of a Transient Reaction Intermediate Illuminates the Mechanochemical Cycle of the AAA-ATPase p97.

The human ATPase p97, also known as valosin containing protein or Cdc48, is a highly abundant AAA+ engine that fuels diverse energy-consuming processes in the human cell. p97 represents a potential target for cancer therapy and its malfunction causes a degenerative disease. Here, we monitor the enzymatic activity of p97 in real time via an NMR-based approach that allows us to follow the steps that couple ATP turnover to mechanical work. Our data identify a transient reaction intermediate, the elusive ADP.Pi nucleotide state, which has been postulated for many ATPases but has so far escaped direct detection. In p97, this species is crucial for the regulation of adenosine triphosphate turnover in the first nucleotide-binding domain. We further demonstrate how the enzymatic cycle is detuned by disease-associated gain-of-function mutations. The high-resolution insight obtained into conformational transitions in both protein and nucleotide bridges the gap between static enzyme structures and the dynamics of substrate conversion. Our approach relies on the close integration of solution- and solid-state NMR methods and is generally applicable to shed light on the mechanochemical operating modes of large molecular engines.

Comparison of Free Radical Levels in the Aerosol from Conventional Cigarettes, Electronic Cigarettes, and Heat-Not-Burn Tobacco Products.

Aerosols from electronic cigarettes and heat-not-burn tobacco products have been found to contain lower levels of almost all compounds from the list of Harmful and Potentially Harmful Constituents known to be present in tobacco products and tobacco smoke than smoke from conventional cigarettes. Free radicals, which also pose potential health risks, are not considered in this list, and their levels in the different product types have not yet been compared under standardized conditions. We compared the type and quantity of free radicals in mainstream aerosol of 3R4F research cigarettes, two types of electronic cigarettes, and a heat-not-burn tobacco product. Free radicals and NO in the gas phases were separately spin trapped and quantified by electron paramagnetic resonance (EPR) spectroscopy by using a smoking machine for aerosol generation and a flow-through cell to enhance reproducibility of the quantification. Particulate matter was separated by a Cambridge filter and extracted, and persistent radicals were quantified by EPR spectroscopy. Levels of organic radicals for electronic cigarettes and the heat-not-burn product, as measured with the PBN spin trap, did not exceed 1% of the level observed for conventional cigarettes and were close to the radical level observed in air blanks. The radicals found in the smoke of conventional cigarettes were oxygen centered, most probably alkoxy radicals, whereas a signal for carbon-centered radicals near the detection limit was observed in aerosol from the heat-not-burn product and electronic cigarettes. The NO level in aerosol produced by electronic cigarettes was below our detection limit, whereas for the heat-not-burn product, it reached about 7% of the level observed for whole smoke from 3R4F cigarettes. Persistent radicals in particulate matter could be quantified only for 3R4F cigarettes. Aerosols from vaping and heat-not-burn tobacco products have much lower free radical levels than cigarette smoke, however, the toxicological implications of this finding are as yet unknown.