Inherent Structural Disorder and Dimerisation of Murine Norovirus NS1-2 Protein

Human noroviruses are highly infectious viruses that cause the majority of acute, non-bacterial epidemic gastroenteritis cases worldwide. The first open reading frame of the norovirus RNA genome encodes for a polyprotein that is cleaved by the viral protease into six non-structural proteins. The first non-structural protein, NS1-2, lacks any significant sequence similarity to other viral or cellular proteins and limited information is available about the function and biophysical characteristics of this protein. Bioinformatic analyses identified an inherently disordered region (residues 1–142) in the highly divergent N-terminal region of the norovirus NS1-2 protein. Expression and purification of the NS1-2 protein of Murine norovirus confirmed these predictions by identifying several features typical of an inherently disordered protein. These were a biased amino acid composition with enrichment in the disorder promoting residues serine and proline, a lack of predicted secondary structure, a hydrophilic nature, an aberrant electrophoretic migration, an increased Stokes radius similar to that predicted for a protein from the pre-molten globule family, a high sensitivity to thermolysin proteolysis and a circular dichroism spectrum typical of an inherently disordered protein. The purification of the NS1-2 protein also identified the presence of an NS1-2 dimer in Escherichia coli and transfected HEK293T cells. Inherent disorder provides significant advantages including structural flexibility and the ability to bind to numerous targets allowing a single protein to have multiple functions. These advantages combined with the potential functional advantages of multimerisation suggest a multi-functional role for the NS1-2 protein

Common Features at the Start of the Neurodegeneration Cascade

A single-molecule study reveals that neurotoxic proteins share common structural features that may trigger neurodegeneration, thus identifying new targets for therapy and diagnosis

Unequivocal single-molecule force spectroscopy of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) are predicted to represent about one third of the eukaryotic proteome. The dynamic ensemble of conformations of this steadily growing class of proteins has remained hardly accessible for bulk biophysical techniques. However, single-molecule techniques provide a useful means of studying these proteins. Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is one of such techniques, which has certain peculiarities that make it an important methodology to analyze the biophysical properties of IDPs. However, several drawbacks inherent to this technique can complicate such analysis. We have developed a protein engineering strategy to overcome these drawbacks such that an unambiguous mechanical analysis of proteins, including IDPs, can be readily performed. Using this approach, we have recently characterized the rich conformational polymorphism of several IDPs. Here, we describe a simple protocol to perform the nanomechanical analysis of IDPs using this new strategy, a procedure that in principle can also be followed for the nanomechanical analysis of any protein. © 2012 Springer Science+Business Media New York.Peer Reviewe