Detection of disordered regions in globular proteins using ¹³C‐detected NMR

Abstract

Characterization of disordered regions in globular proteins constitutes a significant challenge. Here, we report an approach based on 13 C-detected nuclear magnetic resonance experiments for the identification and assignment of disordered regions in large proteins. Using this method, we demonstrate that disordered fragments can be accurately identified in two homologs of menin, a globular protein with a molecular weight over 50 kDa. Our work provides an efficient way to characterize disordered fragments in globular proteins for structural biology applications.

BMI1 regulates PRC1 architecture and activity through homo- and hetero-oligomerization

“…To define the boundaries of the globular central domain we employed carbon detected NMR experiments to efficiently identify flexible regions. We assigned disordered fragments in BMI1 106-240 by employing a combination of 13 C-detected 2D CACO, CBCACO and CANCO experiments using a previously published protocol39. We identified residues 106–120 and 236–240 as being highly flexible in solution (Supplementary Fig.…”

“…Previous attempts at structural studies of the BMI1 UBL domain were hampered by protein instability41. To design constructs of BMI1 and PHC2 suitable for structural studies we employed 13 C-detected NMR experiments to identify disordered protein fragments39. Using such an approach we efficiently mapped the minimal folded fragments of BMI1 and PHC2 involved in this interaction highlighting the utility of this methodology in designing constructs for structural biology39.…”

“…To design constructs of BMI1 and PHC2 suitable for structural studies we employed 13 C-detected NMR experiments to identify disordered protein fragments39. Using such an approach we efficiently mapped the minimal folded fragments of BMI1 and PHC2 involved in this interaction highlighting the utility of this methodology in designing constructs for structural biology39. Furthermore, structure determination of the BMI1–PHC2 complex solely by either X-ray crystallography or solution NMR was hampered by additional challenges, such as crystallographic artifacts from the crystal packing, low protein solubility and high propensity for aggregation.…”

The “Sticky Patch” Model of Crystallization and Modification of Proteins for Enhanced Crystallizability

“…The functional core units can also be identified experimentally, following limited proteolysis, by mass spectrometry [113]. Finally, experimental methods can be used to identify the disordered regions directly, such as deuterium-hydrogen exchange coupled to mass spectrometry (DXMS) [114–116], or NMR [117]. Unfortunately, many target variants may have to be screened to identify one amenable to crystallization, because in some cases short disordered fragments may even help[118].…”

Challenges and Opportunities in Targeting the Menin–MLL Interaction

“…The size of the human menin is 615 amino acids, and it varies between different homologs from 539 ( Nematostella vectensis ) to 763 ( Drosophila melanogaster ) amino acids. Bioinformatics analysis and NMR studies revealed that menin is a globular protein with several disordered fragments (Figure 2A) [66]. Truncations of these flexible fragments turned out to be essential for successful crystallization of menin.…”