NMR Spectroscopic Studies of the Conformational Ensembles of Intrinsically Disordered Proteins

Abstract

Intrinsically disordered proteins (IDPs) are characterized by substantial conformational flexibility and thus not amenable to conventional structural biology techniques. Given their inherent structural flexibility NMR spectroscopy offers unique opportunities for structural and dynamic studies of IDPs. The past two decades have witnessed significant development of NMR spectroscopy that couples advances in spin physics and chemistry with a broad range of applications. This chapter will summarize key advances in NMR methodology. Despite the availability of efficient (multi-dimensional) NMR experiments for signal assignment of IDPs it is discussed that NMR of larger and more complex IDPs demands spectral simplification strategies capitalizing on specific isotope-labeling strategies. Prototypical applications of isotope labeling-strategies are described. Since IDP-ligand association and dissociation processes frequently occur on time scales that are amenable to NMR spectroscopy we describe in detail the application of CPMG relaxation dispersion techniques to studies of IDP protein binding. Finally, we demonstrate that the complementary usage of NMR and EPR data provide a more comprehensive picture about the conformational states of IDPs and can be employed to analyze the conformational ensembles of IDPs.

15N transverse relaxation measurements for the characterization of µs–ms dynamics are deteriorated by the deuterium isotope effect on 15N resulting from solvent exchange

“…68,69 and non-local and local dynamics of IDPs using mainly15 N CPMG based relaxation dispersion experiments[15][16][17][18]67,[69][70][71][72][73][74][75][76][77][78][79][80] . Several experimental strategies have been designed to allow the recording of 201 H-15 N correlation spectra81,82 and CPMG relaxation experiments of IDPs under physiological conditions and obviate the influence of amide exchange43,83 , but the adverse impact of D 2 O through the isotope effect has to our knowledge escaped attention.…”

Multivalent interactions between eIF4G1, Pub1 and Pab1 drive the formation of protein condensates

“…Different methods have been used to calculate IDP conformational ensembles using experimental data (NMR, SAXS and others) and/or computational approaches [42][43][44][45][46][47] .…”

“…intermolecular NOEs) for calculation of the structure of the complex. To overcome this technical problem, we constructed recombinant chimeras of eIF4G1 [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49] and Pub1…”

Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome

“…The gradual appearance of the peaks is attributed to the unfolding of the corresponding region and all the analyses from here on rely on this assumption: if the resonance peak of a nonproline residue X was only observed in the 1 H-15 N HSQC at Y M urea, this is the urea concentration where this residue is now assumed to be in a fully unfolded structure. This assumption is valid, since these peaks are all located in a very narrow chemical shift region close to 8 ppm in the proton dimension, typical of unfolded structures and intrinsically disordered proteins [64].…”