Abstract
Unidirectional coherence transfer is highly efficient in intrinsically disordered proteins (IDPs). Their elevated ps-ns timescale dynamics ensures long transverse (T2) relaxation times allowing sophisticated coherence transfer pathway selection in comparison to folded proteins. 1Hα-detection ensures non-susceptibility to chemical exchange with the solvent and enables chemical shift assignment of consecutive proline residues, typically abundant in IDPs. However, many IDPs undergo a disorder-to-order transition upon interaction with their target protein, which leads to the loss of the favorable relaxation properties. Long coherence transfer routes now result in prohibitively large decrease in sensitivity. We introduce a novel 4D 1Hα-detected experiment HACANCOi, together with its 3D implementation, which warrant high sensitivity for the assignment of proline-rich regions in IDPs in complex with a globular protein. The experiment correlates 1Hαi, 13Cαi, 15Ni and ^{13} C^{prime}_{i} spins by transferring the magnetization concomitantly from 13Cαi to 15Ni and ^{13} C^{prime}_{i}. The B1 domain of protein G (GB1), and the enteropathogenic E.coli EspF in complex with human SNX9 SH3, serve as model systems to demonstrate the attainable sensitivity and successful sequential assignment.
Highlights
Disordered proteins (IDPs), proteins with intrinsically disordered regions (IDRs) or modular proteins containing intrinsically disordered linkers (IDLs) have raised great interest in the scientific community in the last two decades
Whereas prohibitively fast transverse relaxation is the biggest hurdle in the structure determination of larger folded proteins and complexes, resonance overlap constitutes the major hindrance in the chemical shift assignment and structural characterization of disordered systems
Our group is actively working with Intrinsically disordered proteins (IDPs) harboring SH3 domain targeting short linear motifs (SLiMs)
Summary
Disordered proteins (IDPs), proteins with intrinsically disordered regions (IDRs) or modular proteins containing intrinsically disordered linkers (IDLs) have raised great interest in the scientific community in the last two decades. Because these proteins or regions have been tightly linked to disease related biology, and because they have significantly influenced our perception of mechanistic structural biology in general. Resonance overlap substantially limits the feasibility of the well-established amide proton (HN) detection based experiments which rely on Cα/Cβ shifts to obtain sequential connections, commonly used for the assignment of folded proteins Combination of 13C′ dispersion with the high sensitivity and robustness of the HN-detection based experiments, exploiting their compatibility with the TROSY and BEST
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