Abstract
Asparagine and glutamine side-chains can form hydrogen-bonded ladders which contribute significantly to the stability of amyloid fibrils. We show, using the example of HET-s(218–289) fibrils, that the primary amide side-chain proton resonances can be detected in cross-polarization based solid-state NMR spectra at fast magic-angle spinning (MAS). J-coupling based experiments offer the possibility to distinguish them from backbone amide groups if the spin-echo lifetimes are long enough, which turned out to be the case for the glutamine side-chains, but not for the asparagine side-chains forming asparagine ladders. We explore the sensitivity of NMR observables to asparagine ladder formation. One of the two possible asparagine ladders in HET-s(218–289), the one comprising N226 and N262, is assigned by proton-detected 3D experiments at fast MAS and significant de-shielding of one of the NH2 proton resonances indicative of hydrogen-bond formation is observed. Small rotating-frame 15N relaxation-rate constants point to rigidified asparagine side-chains in this ladder. The proton resonances are homogeneously broadened which could indicate chemical exchange, but is presently not fully understood. The second asparagine ladder (N243 and N279) in contrast remains more flexible.
Highlights
The terminal primary amide group of asparagine and glutamine side-chains can function as both, hydrogen-bond donor via the NH2 group as well as acceptor via the carbonyl group and these side-chains are important for protein stability (Vernet et al, 1995; Worth and Blundell, 2010), protein-protein (Van Melckebeke et al, 2010; Talavera et al, 2011; Wälti et al, 2016), protein-ligand (Higman et al, 2004; Raymond et al, 2004) and protein RNA/DNA interactions (Luscombe et al, 2001; Lejeune et al, 2005)
We show that fast magicangle spinning (MAS) (Agarwal et al, 2014; Andreas et al, 2015, 2016; Böckmann et al, 2015; Sternberg et al, 2018; Stöppler et al, 2018; Malär et al, 2019; Penzel et al, 2019; Vasa et al, 2019) can detect asparagine and glutamine side-chains in cross-polarization (CP) solid-state nuclear magnetic resonance (NMR) proton spectra and, sometimes, in J-coupling based experiments, in particular INEPT (Morris and Freeman, 1979) experiments, which can be employed to distinguish the two NH2 side-chain proton resonances from amide NH protons
The side-chain assignment is based on a NCOCX 3D experiment performed on a fully protonated HET-s(218–289) sample shown in Supplementary Figure S2 which yields the 15N side-chain resonances
Summary
The terminal primary amide group of asparagine and glutamine side-chains can function as both, hydrogen-bond donor via the NH2 group as well as acceptor via the carbonyl group and these side-chains are important for protein stability (Vernet et al, 1995; Worth and Blundell, 2010), protein-protein (Van Melckebeke et al, 2010; Talavera et al, 2011; Wälti et al, 2016), protein-ligand (Higman et al, 2004; Raymond et al, 2004) and protein RNA/DNA interactions (Luscombe et al, 2001; Lejeune et al, 2005). Solid-state nuclear magnetic resonance (NMR) spectroscopy is a suitable technique to identify asparagine/glutamine ladders in amyloid fibrils: the 1H chemical-shifts are sensitive reporters and less-shielded 1H resonances indicate a participation of the proton in a hydrogen bond (Wagner et al, 1983) whereas relaxation properties can describe dynamical effects Such spectroscopic information extends the structure information from NMR and cryo-EM (Gremer et al, 2017; Guerrero-Ferreira et al, 2018, 2019; Kollmer et al, 2019; Schmidt et al, 2019; Hervas et al, 2020). We discuss NMR spectral and relaxation parameters determined for asparagine side-chains forming asparagine ladders, such as highfrequency shifted proton resonances and small rotating-frame 15N relaxation-rate constants
Sign-in/Register to view highlights & summary 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.
