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Nicotinamide adenine dinucleotide (NAD) is found in all living cells where the oxidized (NAD + ) and reduced (NADH) forms play important roles in many enzymatic reactions. However, little is known about NAD + and NADH conformational changes and kinetics as a function of the cell environment. In the present work, an analytical workflow is utilized to study NAD + and NADH dynamics as a function of the organic content in solution using fluorescence lifetime spectroscopy and in the gas-phase using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and infrared multiple photon dissociation (IRMPD) spectroscopy. NAD solution time decay studies showed a two-component distribution, assigned to changes from a “close” to “open” conformation with the increase of the organic content. NAD gas-phase studies using nESI-TIMS-MS displayed two ion mobility bands for NAD + protonated and sodiated species, while four and two ion mobility bands were observed for NADH protonated and sodiated species, respectively. Changes in the mobility profiles were observed for NADH as a function of the starting solution conditions and the time after desolvation, while NAD + profiles showed no dependence. IRMPD spectroscopy of NAD + and NADH protonated species in the 800–1800 and 3200–3700 cm −1 spectral regions showed common and signature bands between the NAD forms. Candidate structures were proposed for NAD + and NADH kinetically trapped intermediates of the protonated and sodiated species, based on their collision cross sections and IR profiles. Results showed that NAD + and NADH species exist in open, stack, and closed conformations and that the driving force for conformational dynamics is hydrogen bonding of the N–H–O and O–H–O forms with ribose rings.
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The effects of solution additives and gas‐phase modifiers on the molecular environment and conformational space of common heme proteins
RationaleThe molecular environment is known to impact the secondary and tertiary structures of biomolecules both in solution and in the gas phase, shifting the equilibrium between different conformational and oligomerization states. However, there is a lack of studies monitoring the impacts of solution additives and gas‐phase modifiers on biomolecules characterized using ion mobility techniques. MethodsThe effect of solution additives and gas‐phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100–500 ms) using nanoelectrospray ionization coupled to trapped ion mobility spectrometry with detection by time‐of‐flight mass spectrometry. Organic compounds used as additives/modifiers (methanol, acetonitrile, acetone) were either added to the aqueous protein solution before ionization or added to the ion mobility bath gas by nebulization. ResultsChanges in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) and the protein. In the presence of gas‐phase modifiers (i.e., N2doped with methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas‐modifier mass and size and changes in the relative abundances and number of IMS bands are observed. ConclusionsWe attribute the observed changes in the mobility profiles in the presence of gas‐phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.
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- Award ID(s):
- 1654274
- PAR ID:
- 10462419
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Rapid Communications in Mass Spectrometry
- Volume:
- 33
- Issue:
- 5
- ISSN:
- 0951-4198
- Format(s):
- Medium: X Size: p. 399-404
- Size(s):
- p. 399-404
- Sponsoring Org:
- National Science Foundation
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