Bridge over troubled proline: assignment of intrinsically disordered proteins using (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H experiments concomitantly with HNCO and i(HCA)CO(CA)NH

Abstract

NMR spectroscopy is by far the most versatile and information rich technique to study intrinsically disordered proteins (IDPs). While NMR is able to offer residue level information on structure and dynamics, assignment of chemical shift resonances in IDPs is not a straightforward process. Consequently, numerous pulse sequences and assignment protocols have been developed during past several years, targeted especially for the assignment of IDPs, including experiments that employ H(N), H(α) or (13)C detection combined with two to six indirectly detected dimensions. Here we propose two new HN-detection based pulse sequences, (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H, that provide correlations with (1)H(N)(i - 1), (13)C'(i - 1) and (15)N(i), and (1)H(N)(i + 1), (13)C'(i) and (15)N(i) frequencies, respectively. Most importantly, they offer sequential links across the proline bridges and enable filling the single proline gaps during the assignment. We show that the novel experiments can efficiently complement the information available from existing HNCO and intraresidual i(HCA)CO(CA)NH pulse sequences and their concomitant usage enabled >95 % assignment of backbone resonances in cytoplasmic tail of adenosine receptor A2A in comparison to 73 % complete assignment using the HNCO/i(HCA)CO(CA)NH data alone.

Quarterly intrinsic disorder digest (January-February-March, 2014)

“…Hellman et al proposed two new pulse sequences which were able to improve backbone assignments greater than 22% in the proline-rich C-terminus of adenosine receptor 2A. 72 Sahu et al took a different approach to resolving the issues involved in analyzing IDPs in the proton spectrum. The authors used carbon-detected spectra of the Pdx1 C-terminal domain to generate several pulse protocols that do not depend on 4D or greater experiments or the previously mentioned non-uniform sampling.…”

Human Adenosine A2A Receptor Binds Calmodulin with High Affinity in a Calcium-Dependent Manner

“…All A2A-ctL NMR spectra were measured in 95%/5% 50 mM HEPES, 250 mM NaCl, 1 mM TCEP-HCl, 5 mM CaCl 2 pH 7.2/D 2 O at 25 C at 800 MHz 1 H frequency using a Varian UNITY INOVA 800 NMR spectrometer (Agilent, Santa Clara, California), equipped with a cryogenically cooled 1 H, 13 C, 15 N probehead. The backbone assignment of A2A-ctL was carried out using a plethora of three-dimensional (3D) NH-detected experiments including experiments specially tailored for intrinsically disordered proteins (46,47) as well as the conventional HNCO, HNCACB, and CBCA(CO)NH (48). Proton chemical shifts were referenced with respect to residual solvent signal (4.79 ppm at 25 C and pH 7.2).…”

“…The conformational heterogeneity of intrinsically disordered proteins calls for special means for the NMR signal assignment. Indeed, to accomplish backbone assignment of A2A-ctL, we employed a panoply of novel NH-detected triple-resonance experiments that correlate 1 H N , 13C', and 15 N frequencies and simultaneously link amino acid stretches flanking single proline residues (46,47). These data were supplemented with 13 C a and 13 C b chemical shifts available from HNCACB/CBCA(CO)NH experiments (H. Tossavainen, M. Hellman, H. Piirainen, V.P.…”

HN, N, Cα, Cβ and C′ assignments of the intrinsically disordered C-terminus of human adenosine A2A receptor

“…For the assignment we employed a combination of common 3D H N -detected assignment spectra and 3D H N -detected experiments specially tailored for intrinsically disordered proteins (Mäntylahti et al 2009;Hellman et al 2014). Fast conformational averaging and chemical exchange and absence of structure-induced secondary chemical shifts result in a narrow H N chemical shift range as well as overlap of C a and C b shifts, both commonly observed for IDPs.…”