Detection of Disordered Regions in Globular Proteins Using 13C-Detected NMR

Department of Pathology, University of Michigan, 1150 W. Medical Center Drive, MSRB1 4516, Ann Arbor, MI 48109
Protein Science (Impact Factor: 2.85). 12/2012; 21(12). DOI: 10.1002/pro.2174


Characterization of disordered regions in globular proteins constitutes a significant challenge. Here, we report an approach based on 13C-detected NMR 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.

Download full-text


Available from: Marcelo J Murai,
  • Source
    • "This happens regardless of residual secondary structure. Indeed, the presence of transient, low-populated a-helices or b-turns offers little protection against solvent exchange (Croke et al. 2008; Hsu et al. 2009; Gray et al. 2012). Even in the case of molten globule proteins, solvent exchange can thoroughly degrade the quality of 1 H– 15 N spectra (Loh et al. 1995). "
    [Show abstract] [Hide abstract]
    ABSTRACT: (1)H-(15)N HSQC spectroscopy is a workhorse of protein NMR. However, under physiological conditions the quality of HSQC spectra tends to deteriorate due to fast solvent exchange. For globular proteins only a limited number of surface residues are affected, but in the case of intrinsically disordered proteins (IDPs) HSQC spectra are thoroughly degraded, suffering from both peak broadening and loss of intensity. To alleviate this problem, we make use of the following two concepts. (1) Proton-decoupled HSQC. Regular HSQC and its many variants record the evolution of multi-spin modes, 2NxHz or 2NxHx, in indirect dimension. Under the effect of fast solvent exchange these modes undergo rapid decay, which results in severe line-broadening. In contrast, proton-decoupled HSQC relies on Nx coherence which is essentially insensitive to the effects of solvent exchange. Moreover, for measurements involving IDPs at or near physiological temperature, Nx mode offers excellent relaxation properties, leading to very sharp resonances. (2) Cross-polarization (1)H-to-(15)N transfer. If CP element is designed such as to lock both (1)H(N) and water magnetization, the following transfer is effected: [Formula: see text] Thus water magnetization is successfully exploited to boost the amount of signal. In addition, CP element suffers less loss from solvent exchange, conformational exchange, and dipolar relaxation compared to the more popular INEPT element. Combining these two concepts, we have implemented the experiment termed CP-HISQC (cross-polarization assisted heteronuclear in-phase single-quantum correlation). The pulse sequence has been designed such as to preserve water magnetization and therefore can be executed with reasonably short recycling delays. In the presence of fast solvent exchange, kex ~ 100 s(-1), CP-HISQC offers much better spectral resolution than conventional HSQC-type experiments. At the same time it offers up to twofold gain in sensitivity compared to plain proton-decoupled HSQC. The new sequence has been tested on the sample of drkN SH3 domain at pH 7.5, 30 °C. High-quality spectrum has been recorded in less than 1 h, containing resonances from both folded and unfolded species. High-quality spectra have also been obtained for arginine side-chain H(ε)N(ε) groups in the sample of short peptide Sos. For Arg side chains, we have additionally implemented (HE)NE(CD)HD experiment. Using (13)C-labeled sample of Sos, we have demonstrated that proton-to-nitrogen CP transfer remains highly efficient in the presence of solvent exchange as fast as kex = 620 s(-1). In contrast, INEPT transfer completely fails in this regime.
    Journal of Biomolecular NMR 02/2014; 58(3). DOI:10.1007/s10858-014-9815-5 · 3.14 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Menin is an essential co-factor of oncogenic MLL fusion proteins and the menin-MLL interaction is critical for development of acute leukemia in vivo. Targeting the menin-MLL interaction with small molecules represents an attractive strategy to develop new anticancer agents. Recent developments, including determination of menin crystal structure and development of potent small molecule and peptidomimetic inhibitors, demonstrate the feasibility of targeting the menin-MLL interaction. On the other hand, biochemical and structural studies revealed that MLL binds to menin in a complex bivalent mode engaging two MLL motifs, and therefore inhibition of this protein-protein interaction represents a challenge. This review summarizes the most recent achievements in targeting the menin-MLL interaction as well as discusses potential benefits of blocking menin in cancer.
    Future medicinal chemistry 03/2014; 6(4):447-62. DOI:10.4155/fmc.13.214 · 3.74 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Spin-state-selective methods to achieve homonuclear decoupling in the direct acquisition dimension of 13C detected NMR experiments have been one of the key contributors to converting 13C detected NMR experiments into really useful tools for studying biomolecules. We discuss here in detail the various methods that have been proposed, summarize the large array of new experiments that have been developed and present applications to different kinds of proteins in different aggregation states.
    Progress in Nuclear Magnetic Resonance Spectroscopy 11/2014; 84-85C. DOI:10.1016/j.pnmrs.2014.10.001 · 7.24 Impact Factor