Abstract
Today, the sedimentation of proteins into a magic-angle spinning (MAS) rotor gives access to fast and reliable sample preparation for solid-state Nuclear Magnetic Resonance (NMR), and this has allowed for the investigation of a variety of non-crystalline protein samples. High protein concentrations on the order of 400 mg/mL can be achieved, meaning that around 50–60% of the NMR rotor content is protein; the rest is a buffer solution, which includes counter ions to compensate for the charge of the protein. We have demonstrated herein the long-term stability of four sedimented proteins and complexes thereof with nucleotides, comprising a bacterial DnaB helicase, an ABC transporter, an archaeal primase, and an RNA polymerase subunit. Solid-state NMR spectra recorded directly after sample filling and up to 5 years later indicated no spectral differences and no loss in signal intensity, allowing us to conclude that protein sediments in the rotor can be stable over many years. We have illustrated, using an example of an ABC transporter, that not only the structure is maintained, but that the protein is still functional after long-term storage in the sedimented state.
Highlights
Using solid-state Nuclear Magnetic Resonance (NMR), a wide variety of biological materials can be studied, such as micro- or nanocrystals, fibrillar aggregates, and proteins embedded in membranes
We describe our experimental observations regarding the stability of four sedimented samples from different protein systems: (i) the bacterial helicase DnaB from Helicobacter pylori in a complex with ADP:AlF4− and DNA (Wiegand et al, 2019), (ii) the archaeal pRN1 primase from Sulfolobus islandicus in a complex with DNA and ATP (Boudet et al, 2019), (iii) the membrane protein BmrA, an ABC transporter from Bacillus subtilis (Lacabanne et al, 2019b), and (iv) the two RNA polymerase subunits Rpo4/7∗ from Methanocaldococcus jannaschii
We show that the sedimented samples investigated are stable over several months to years in an NMR rotor
Summary
Using solid-state Nuclear Magnetic Resonance (NMR), a wide variety of biological materials can be studied, such as micro- or nanocrystals, fibrillar aggregates, and proteins embedded in membranes. The molecular mass of the protein determines the success of sedimentation, and proteins, such as the RNA polymerase subunits Rpo4/7∗ (the ∗ indicates that the Rpo unit is uniformly 13C/15N labeled) with a molecular weight of 34 kDa (Torosyan et al, 2019), the pRN1 primase with 40 kDa (Boudet et al, 2019), the neonatal Fc receptor with 40 kDa (Stöppler et al, 2018) as well as the human superoxide dismutase with 32 kDa (Fragai et al, 2013), the bacterial helicase DnaB with 708 kDa (Gardiennet et al, 2012), the iron-storage protein ferritin with 480 kDa (Bertini et al, 2012), a variety of supramolecular assemblies (Lecoq et al, 2018; Gauto et al, 2019), and PEGylated proteins (Ravera et al, 2016), were shown to form sediments suitable for solid-state NMR This is for some of these systems probably a consequence of oligomerization that has been induced by the high protein concentrations (Bertini et al, 2013; Fragai et al, 2013). We show that the sedimented samples investigated are stable over several months to years in an NMR rotor
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
