Fast NMR method to probe solvent accessibility and disordered regions in proteins

Abstract

Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established 1H-15N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method.

Structural and Functional Properties of the Capsid Protein of Dengue and Related Flavivirus

“…Since τ 3 values were ~6.4 ns (with a significant weight α 3 of ~0.42), this means that, at both pH values, we could measure τ c values up to a limit of ~19 ns. In both pH conditions, the τ c measured was 16.4 ± 0.5 ns at 22 °C, within the limit and higher than expected for a purely globular protein of DENV C size, as predicted [13].…”

“…The α0 domain, homologous to pep14-23, is amphipathic, with average values near 0. In the flexible fold region, which is mostly amphipathic too, there is a peak of hydrophobicity between residues 30 and 40, possibly explaining its intermediate structure/dynamics behavior [13,14]. Some peaks of hydrophobicity are observed in the α3 and α4 domains, with the most hydrophobic domain being α2, as expected from the sequence analysis (Figure 2) and from the literature [12,14,18].…”

“…Jones et al [16] measured a τ c of 13 ns at 27 °C, by NMR, which with the corrections from Equation (10) [37], corresponds to 13.4 ns at 25 °C. Given DENV C size, this implies that the protein is not globular, in line with current knowledge of DENV C structure and dynamics [12,13,14,15,16]. Fluorescence anisotropy supports an even more open and partially disordered DENV C structure, given the τ c value of 15.2 ± 0.5 ns at 25 °C Table 3, in line with in silico data (Figure 1, Figure 2, Figure 3 and Figure 4).…”

“…Eighty monomer conformations were obtained (several for each sequence) and superimposed with the DENV C homodimer partial structure deposited at the Protein Data Bank (PDB) and obtained via nuclear magnetic resonance (NMR) spectroscopy (PDB ID: 1R6R). Noteworthy, DENV [12,16], WNV [31] and ZIKV [25] C proteins form homodimers, stabilized by hydrophobic and electrostatic interactions involving their conserved fold region [12,13,14,25,31,32,33]. Since this is the most conserved region of Flavivirus C proteins sequences (Figure 2), a homodimer is thus not only a stable conformational arrangement, but also likely to occur.…”

“…DENV C contains 100 amino acid residues, which form an homodimer with an intrinsically disordered protein (IDP) region in the N-terminal followed by four α-helices, α1 to α4, per monomer [12]. Overall, the main structural/dynamics regions consist of the disordered N-terminal, a short flexible intermediate fold and, finally, a large conserved fold region, which greatly stabilizes the protein homodimer structure [12,13,14,15,16]. The C protein has an asymmetric charge distribution: one side of the dimer contains a hydrophobic pocket (α2–α2′ interface), responsible for, alongside the disordered N-terminal, the binding to host lipid droplets (LDs) [12,13,14,15,16].…”