Abstract
NMR represents a key spectroscopic technique that contributes to the emerging field of highly flexible, intrinsically disordered proteins (IDPs) or protein regions (IDRs) that lack a stable three-dimensional structure. A set of exclusively heteronuclear NMR experiments tailored for proline residues, highly abundant in IDPs/IDRs, are presented here. They provide a valuable complement to the widely used approach based on amide proton detection, filling the gap introduced by the lack of amide protons in proline residues within polypeptide chains. The novel experiments have very interesting properties for the investigations of IDPs/IDRs of increasing complexity.
Highlights
Invisible in X-ray studies of protein crystals, intrinsically disordered regions (IDRs) of complex proteins have for a long time been considered to be passive linkers connecting functional globular domains and are, often ignored in structural biology studies
NMR experiments were acquired at 288 K and at 283 K with a 16.4 T Bruker AVANCE NEO spectrometer operating at 700.06 MHz 1H, 176.05 MHz 13C, and 70.97 MHz 15N HSQC 800 (15N) frequencies, equipped with a 5 mm cryogenically cooled probe head optimized for 13C direct detection (TXO)
Detection and assignment of proline-rich regions of highly flexible intrinsically disordered proteins allows us to have a glimpse of the ways in which proline residues encode specific properties in IDRs/IDPs by tuning their distribution along the primary sequence
Summary
Invisible in X-ray studies of protein crystals, intrinsically disordered regions (IDRs) of complex proteins have for a long time been considered to be passive linkers connecting functional globular domains and are, often ignored in structural biology studies. Intrinsic disorder and high flexibility have very relevant effects for NMR investigations, such as reduction in chemical shift dispersion and efficient exchange processes with the solvent due to the open conformations that, when approaching physiological pH and temperature, broaden amide proton resonances beyond detection. In particular for proteins that are largely exposed to the solvent, it may be interesting to study their near-physiological pH and temperature conditions (Gil et al, 2013). In this context, 13C direct detection NMR developed into a valuable alternative
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