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.
Highlights
Nuclear magnetic resonance (NMR) spectroscopy is the technique of excellence to obtain structural and dynamics atomic resolution information of macromolecules, especially proteins[1]
dengue virus (DENV) C (Fig. 1a,b) was used as a model to study the relationship between the protein structure/dynamics and backbone N-H solvent accessibility
The interval used is between pH 6.0 and 7.5, a physiological range that is suitable for most proteins, including DENV C, as described ahead
Summary
Nuclear magnetic resonance (NMR) spectroscopy is the technique of excellence to obtain structural and dynamics atomic resolution information of macromolecules, especially proteins[1]. Backbone amide hydrogen exchange experiments are informative of protein N-H solvent accessibility, being related to both structure and dynamics (sensitive to the millisecond timescale)[9,10,11,12,13]. Found in the flaviviruses capsid protein structures of Dengue, West Nile and, recently, Zika viruses (with PDB IDs, respectively, 1R6R, 1SFK and 5YGH)[26,32,33,34] This approach employs a small pH variation, preserving DENV C overall structure and dynamics, which allows probing the backbone N-H groups’ solvent accessibility (using only 1H-15N HSQC peak intensities). Since the solvent accessibility of each N-H group is related to its intramolecular H-bond pattern, our approach informs on the secondary structure content This methodology is readily applicable to study other proteins structure and dynamics. The method is readily applicable to this protein as well, supporting its use
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