Hidden motions and motion-induced invisibility: Dynamics-based spectral editing in solid-state NMR

Abstract

Solid-state nuclear magnetic resonance (ssNMR) spectroscopy enables the structural characterization of a diverse array of biological assemblies that include amyloid fibrils, non-amyloid aggregates, membrane-associated proteins and viral capsids. Such biological samples feature functionally relevant molecular dynamics, which often affect different parts of the sample in different ways. Solid-state NMR experiments' sensitivity to dynamics represents a double-edged sword. On the one hand, it offers a chance to measure dynamics in great detail. On the other hand, certain types of motion lead to signal loss and experimental inefficiencies that at first glance interfere with the application of ssNMR to overly dynamic proteins. Dynamics-based spectral editing (DYSE) ssNMR methods leverage motion-dependent signal losses to simplify spectra and enable the study of sub-structures with particular motional properties.

Heterochromatin Protein HP1α Gelation Dynamics Revealed by Solid‐State NMR Spectroscopy

“…Comparing the intensities of these spectra with the DP experiment, it also became clear that CP and INEPT together do not account for all the 13 Cs ignals in the sample.T hus,i nt he dense liquid droplet state there are pHP1a molecules or regions of each individual protein that exhibit motion in an intermediate dynamic range rendering them invisible in both scalar-based and dipolar-based MAS NMR spectroscopy experiments,s imilar to the behavior of other complex biological assemblies. [31] Expecting that pHP1a condensates will equilibrate into gels given sufficient time, [7] we continuously performed MAS and monitored the intensities of the CP and INEPT spectra at 12 hour intervals over the course of aw eek (Figure 1c and Supporting Information, Figure S4). Reaching agel state after 7days (Supporting Information, Figure S2), the total CP signal increased 3.3 fold, the INEPT signal decreased 1.…”

“…Zuschriften theless provide rich information about the overall dynamics in the condensate (Figure 1b). [31] First, the scalar-based 1D INEPT transfer experiment selects for highly mobile sites that have long transverse relaxation times similar to those detected under solution NMR conditions.S econd, rigid regions that exhibit strong dipole-dipole couplings can be selected with 1D cross-polarization (CP) experiments.A nd finally,t he 13 Cd irect polarization (DP) experiment can, in principle,b eu sed to detect all the 13 Cn uclei in the sample independent of dynamics.Atthe start of our experiments,the 1 H-13 CC Ps ignal was remarkably lower than the 1 H-13 C INEPT signal (Figure 1b). Comparing the intensities of these spectra with the DP experiment, it also became clear that CP and INEPT together do not account for all the 13 Cs ignals in the sample.T hus,i nt he dense liquid droplet state there are pHP1a molecules or regions of each individual protein that exhibit motion in an intermediate dynamic range rendering them invisible in both scalar-based and dipolar-based MAS NMR spectroscopy experiments,s imilar to the behavior of other complex biological assemblies.…”

Auto-regulatory J-domain interactions control Hsp70 recruitment to the DnaJB8 chaperone

“…However, strikingly, distinct subsets of narrow peaks are also detected, with 1D projections showing linewidths of 0.1 to 0.2 kHz (Figure 4C-D left). These experiments employ the cross-polarization (CP) technique, which means that the observable residues must be rigid or immobilized, lacking high flexibility 40 . This conclusion is reinforced by a lack of signals in INEPT-based ssNMR spectra that are selective for highly dynamic peptide segments 41–43 (more below).…”

“…We interpret this as indicating that the JD gains increased mobility due to disruption of its long-range electrostatic interactions but is still folded and partly immobilized by its covalent attachment to the overall assembly. As a consequence, the JD is invisible due to intermediate timescale dynamics 40, 47 . Since the broad signals from the other domains are preserved, it appears that the core architecture of the oligomers persists even at high ionic strength, such that it is primarily the JD that is dislodged as electrostatic long-range interactions are weakened.…”

“…We built 5000 predictive models of the CTD using ab initio ROSETTA which employed fragment libraries derived from experimental structures 40 . We calculated the R h for the ensemble using HYDROPPRO and the resulting values were consistent with the DLS measurement of 1.7nm (Figure 5E).…”