NMR Experiments for Studies of Dilute and Condensed Protein Phases: Application to the Phase-Separating Protein CAPRIN1

Abstract

Intrinsically disordered proteins (IDPs) or regions of intrinsic disorder in otherwise folded proteins (IDRs) play important roles in many different biological processes, including formation of biological condensates via liquid–liquid phase separation. NMR spectroscopy is a powerful tool for obtaining site-specific structural and dynamical information on IDPs/IDRs, and recent efforts have focused on the development of experiments for atomic-resolution studies of these molecules. These include triple-resonance experiments that are based on 13CO-direct detection of magnetization, exploiting increased sensitivity of cryogenically cooled probes. In order to evaluate the different classes of experiment for studies of IDRs or IDPs in both dilute and phase-separated environments, in particular at neutral and higher pHs where many of these proteins phase separate, we compared 13CO-detect versus 1Hα-detect experiments, showing that significant sensitivity gains are achieved via proton detection under the conditions of our experiments. A suite of 1Hα-detect experiments was subsequently developed for studies of IDPs/IDRs and applied to the dilute phase of a 103-residue disordered region of CAPRIN1 that phase separates at neutral pH. Residue-specific chemical shifts derived from our study enable the accurate prediction of the importance of the N-terminal Arg-containing region of this construct for promoting phase separation relative to other Arg-rich stretches of sequence, subsequently confirmed by mutagenesis. Our study emphasizes that the sequence positions of key residues can be a critical factor in controlling phase separation and highlights the unique role of NMR in establishing the relations between amino acid sequence and phase-separation propensity.

Liquid–liquid phase separation of tau: From molecular biophysics to physiology and disease

“…Nuclear magnetic resonance (NMR) spectroscopy can provide residue‐specific or even atomic resolution structural insights into the conformational dynamics of biomolecules under physiological conditions. It is therefore optimally suited to dissect the conformational and dynamic properties of proteins and nucleic acids in the condensate state 144,145 . In addition, NMR spectroscopy has been critical for the determination of conformational ensembles of IDPs such as tau 72,146–149 .…”

Biomolecular phase separation through the lens of sodium‐23 NMR

“…NMR spectroscopy is a powerful technique allowing experimental access to biomolecular structure and dynamics at a broad range of time and length scales in different environments, including the liquid‐ and gel‐like biomolecular condensates 8,9 . Several recent studies have utilized high‐resolution NMR to monitor the structural dynamics of proteins and other biomolecules within condensed phases 10–13 .…”

HACANCOi: a new Hα-detected experiment for backbone resonance assignment of intrinsically disordered proteins

“…The coherence transfer efficiency is clearly superior to the corresponding intraresidual only iHA-CANCO experiment (Mäntylahti et al 2010;Tossavainen et al 2020) even for the smallest globular protein or complex. Recently, Wong et al proposed a suite of H α− detected experiments, including e.g., haCONHA and haNCOHA that yield H α (i), C′(i), N(i + 1), and H α (i + 1), C′(i), N(i + 1) (as well as H α (i), C′(i), N(i + 1)) correlations, respectively (Wong et al 2020). They found haNCONHA to be on average 3.5 times more sensitive than the 13 C-detected counterpart, haCON.…”

“…Also, assignment of consecutive prolines can readily be accomplished, making these experiments attractive alternatives to 13 C-detection based pulse schemes (Mäntylahti et al 2010(Mäntylahti et al , 2011Permi and Hellman 2012;Tossavainen et al 2020). Moreover, unless a 13 C-detection optimized probehead is used, these experiments offer superior sensitivity with respect to 13 C-detected experiments (Wong et al 2020), therefore enabling significant time savings. Previously we have successfully used a set of three 3D 1 H α -detected experiments, i.e.…”