Structural Disorder within Henipavirus Nucleoprotein and Phosphoprotein: From Predictions to Experimental Assessment

Henipaviruses are newly emerged viruses within the Paramyxoviridae family. Their negative-strand RNA genome is packaged by the nucleoprotein (N) within α-helical nucleocapsid that recruits the polymerase complex made of the L protein and the phosphoprotein (P). To date structural data on Henipaviruses are scarce, and their N and P proteins have never been characterized so far. Using both computational and experimental approaches we herein show that Henipaviruses N and P proteins possess large intrinsically disordered regions. By combining several disorder prediction methods, we show that the N-terminal domain of P (PNT) and the C-terminal domain of N (NTAIL) are both mostly disordered, although they contain short order-prone segments. We then report the cloning, the bacterial expression, purification and characterization of Henipavirus PNT and NTAIL domains. By combining gel filtration, dynamic light scattering, circular dichroism and nuclear magnetic resonance, we show that both NTAIL and PNT belong to the premolten globule sub-family within the class of intrinsically disordered proteins. This study is the first reported experimental characterization of Henipavirus P and N proteins. The evidence that their respective N-terminal and C-terminal domains are highly disordered under native conditions is expected to be invaluable for future structural studies by helping to delineate N and P protein domains amenable to crystallization. In addition, following previous hints establishing a relationship between structural disorder and protein interactivity, the present results suggest that Henipavirus PNT and NTAIL domains could be involved in manifold protein-protein interactions.


Introduction
Hendra virus (HeV), the first known member of the genus Henipavirus within the Paramyxoviridae family, emerged in 1994 as the causative agent of a sudden outbreak of acute respiratory disease in horses in Brisbane, Australia. Nipah virus (NiV), the second known member of the genus Henipavirus, came to light as the etiologic agent of an outbreak of disease in pigs and humans in Malaysia in 1998 through 1999. The initial NiV outbreak in Malaysia resulted in 265 human cases of encephalitis, including 105 deaths. The virus reemerged in Bangladesh in 2001 and outbreaks of encephalitis caused by NiV have occurred in that country almost every year since, with a case fatality rate approaching 75% (see [1] and references therein cited). Later on, fruit-eating bats were shown to be the natural reservoir of both viruses (see [2] and references therein cited).
Although the genome of HeV and NiV shares the same overall organization of members of the Paramyxovirinae subfamily, a few distinctive properties, including their much larger size, led to the creation of the Henipavirus genus to accommodate these newly emerged zoonotic viruses [3]. Currently this genus contains two virus species and a number of strains isolated from humans, bats, horses and pigs over a wide geographic area and during a period of 10 years. Noteworthy, recently Henipaviruses have also been found outside Australia and Asia, thus extending the region of potential endemicity of one of the most pathogenic virus genera known in humans [2]. The susceptibility of humans, the wide host range and interspecies transmission and the absence of therapeutics agents led to the classification of HeV and NiV as biosecurity level 4 (BSL4) pathogens [4].
Henipaviruses particles are pleomorphic and enveloped. Their negative-stranded, non-segmented RNA genome is encapsidated by the nucleoprotein (N) within a-helical nucleocapsid that has the characteristic herringbone-like structure typically observed in other Paramyxoviridae members including measles virus (MeV) [5,6,7,8] and Sendai virus (SeV) [9,10]. This helical nucleocapsid, rather than naked RNA, is the substrate used by the polymerase complex during both transcription and replication. Minigenome replicon studies showed that Henipavirus N, P and L proteins are necessary and sufficient to sustain replication of viral RNA [11]. By analogy with other Paramyxoviridae members, the polymerase complex is assumed to consist of the L protein and of the phosphoprotein (P), with this latter serving as a tethering anchor for the recruitment of L onto the nucleocapsid template.
The genome organization of Henipaviruses resembles that in the Respirovirus and Morbillivirus genera. The extra length of the Henipavirus genome mainly arises from additional unique, long untranslated sequences at the 39 end of five of the six genes. Despite their much larger genome size, the genome length is divisible by six and reverse genetics studies confirmed that NiV does obey the ''rule of six'', i.e. the genome length must be a multiple of six to replicate efficiently [11]. Overall, the proteins of Henipaviruses are typical of those of the Paramyxovirinae subfamily, with the exception of the P protein that is significantly larger than cognate proteins in the subfamily. Despite this difference in size, the organization of Henipavirus P proteins closely resembles that of other members in the subfamily. Indeed, beyond the P protein, that is translated by an mRNA co-linear with genomic RNA, the P gene of Paramyxovirinae also encodes the V and W proteins that are produced upon addition of either one or two non-templated G residues at the editing site of the P messenger (see [1] and references therein cited). The P, V and W proteins are therefore identical for the first 404 (HeV) or 406 (NiV) residues. A C protein is also encoded by the 59 end of the gene in an overlapping reading frame and is produced by an internal translational initiation mechanism, which is common to other members of Paramyxovirinae, except for Rubulaviruses. The P, V and C proteins predicted from the coding region of the P gene are indeed present in HeV-infected cells. As for NiV, although no formal proof indicates that the W protein is expressed, this latter displays anti-interferon signaling activity when expressed from cloned genes [12]. In addition, in this latter virus, the C, V and W proteins were shown to inhibit minigenome replication [13].
So far, structural and molecular information on Henipavirus proteins is scarce and limited to their surface proteins, where crystallographic studies led to the determination of the 3D structure of Henipavirus fusion (F) and attachment (G) proteins [14,15,16,17].
Previous computational and experimental studies carried out by our laboratory pointed out that MeV N and P possess large (up to 230 residues) intrinsically disordered regions (IDRs) [6,18,19,20,21,22,23,24,25]. Using computational approaches, we then extended these findings to the N and P proteins of Paramyxovirinae members [26] and showed that the presence of IDRs is a conserved feature within the replicative complex of these viruses.
IDRs are regions that lack highly populated constant secondary and tertiary structure under physiological conditions and in the absence of a partner/ligand [27] (for recent reviews on intrinsically disordered proteins -IDPs -see [28,29]). Intrinsically disordered proteins show an extremely wide diversity in their structural properties: indeed they can attain extended conformations (random coil-like) or remain globally collapsed (molten globulelike), where the latter possess regions of fluctuating secondary structure. Conformational and spectroscopic analyses showed that random coil-like proteins (RCs) can be subdivided in their turn into two major groups. While the first group consists of proteins with extended maximum dimensions typical of random coils with no (or little) secondary structure, the second group comprises the so-called premolten globules (PMGs), which are more compact (but still less compact than globular or molten globule proteins) and conserve some residual secondary structure [30,31,32,33,34,35].
As a first step towards the structural characterization of Henipavirus N and P proteins, we herein describe the results of a thorough computational analysis that shows that Henipaviruses N and P proteins possess a modular organization consisting of large (up to 400 residues) unstructured regions alternating with structured regions. We show that the N-terminal domain of P (PNT) and the C-terminal domain of N (N TAIL ) possess the peculiar sequence features that typify IDRs. We also report the cloning, bacterial expression, purification and characterization of Henipavirus N TAIL and PNT domains. Using different, complementary biochemical and biophysical methods, we confirmed the predicted disordered nature of Henipavirus N TAIL and PNT and show that they belong to the PMG subfamily within the class of IDPs.

Results
Disorder predictions and modular organization of Henipavirus N and P We first analyzed the amino acid sequences of Henipavirus N and P proteins using the MeDor metaserver for the prediction of disorder [36]. In the graphical output generated by MeDor, disordered regions, as predicted by the various predictors, are shown along with the HCA plot of the query sequence.
As shown in Fig. 1A, both nucleoproteins consist of a large (400 residues) N-terminal region (referred to as N CORE ), which is consistently predicted to be ordered by the various predictors and that is enriched in hydrophobic clusters, and of a C-terminal region (referred to as N TAIL ) that is predicted to be disordered by most predictors and that is depleted in hydrophobic clusters. Interestingly, two or three low sequence complexity regions, which are hallmarks of structural disorder [37], were found within the NiV and HeV N TAIL , respectively ( Fig. 1). Besides, analysis of a multiple sequence alignment among Henipavirus, Morbillivirus and Respirovirus N proteins (see supplementary Fig. S1) revealed a significant sequence divergence in the N TAIL region, in agreement with earlier observations pointing out a higher sequence variability in disordered regions as compared to structured ones [38]. Although the N TAIL region is mostly disordered, four short regions possessing each a hydrophobic cluster and/or a predicted (a or b) secondary structure element, are found (see Fig. 1A).
The P protein of both HeV and NiV displays a more complex organization, with regions of predicted disorder alternating with structured regions (Fig. 1B). With the only exception of the first 50 residues, which are predicted to be ordered, the P protein of both viruses possesses a spectacularly large N-terminal region of about 400 residues that is depleted in hydrophobic clusters, that is predicted to be disordered by most predictors and that possesses very few predicted secondary structure elements, a feature typifying protein regions with ''no ordered regular structure'' [39]. This region of predicted disorder encompasses the region shared by the P, V and W proteins (referred to as P N-Terminal, PNT) and further extends by approximately 65 residues towards the C-terminus (see Fig. 1B). A region predicted to be structured, and whose HCA plot is reminiscent of coiled-coil regions follows downstream (see  [26]). Notably, one or two coiled-coils are predicted within this region of the HeV (aa 546-560) or NiV (aa 482-497 and 548-562) P protein, respectively. In further support of the occurrence of a coiled-coil within this region, the majority of the best PDB hits, as provided by the 3D-PSSM server, are coiled-coils (for an example see pdb code 1sfc). By analogy with cognate Paramyxovirinae P proteins [26], this region could correspond to a putative P multimerization domain (PMD), although so far no direct information is available on the oligomeric state of the Henipavirus P protein.
The C-terminal region of both P proteins is predicted to be structured and to adopt an a-helical conformation, as judged based on the occurrence of three predicted a-helices (see Fig. 1B). Again, by analogy with the P protein of other Paramyxovirinae members, this globular region has been assumed to be the counterpart of the X domain (XD), the structure of which consists of a triple a-helical bundle [42,43,44].
PMD and XD are connected by a mixed linker region consisting of (i) a short disordered segment (aa 579-590), which also corresponds to a low complexity region in NiV P (see Fig. 1B), (ii) a region with a borderline order (aa 590-640) and finally (iii) a short disordered segment.

Sequence properties of Henipavirus N TAIL and PNT
We compared the sequence composition of Henipavirus N TAIL and PNT to that of proteins within the SWISS-PROT database (Fig. 2). Both Henipavirus N TAIL proteins ( Fig. 2A, B) have a peculiar amino acid composition, in that they are depleted in most ''order promoting'' residues (W, C, F, Y, I, V, L) and enriched in most ''disorder promoting'' residues (A, R, Q, S, P and E), as already described for the cognate N and P regions of other Paramyxovirinae members [6,19,26] and, more generally, for IDPs [30]. A similar, though less pronounced, compositional bias is observed for Henipavirus PNT (Fig. 2 C, D). Conversely, Henipavirus N CORE , PMD and XD regions do not display any significant overall relative enrichment or depletion with respect with the average amino acid composition in the SWISS-PROT database (data not shown).
Moreover, Henipavirus N TAIL and PNT are predicted to be disordered by the method based on the mean hydrophobicity/ mean net charge ratio [45], whereas PMD and XD are predicted to be ordered (Fig. 3).
In order to experimentally confirm the disorder predictions, we have expressed, purified and characterized the large regions of predicted disorder of Henipavirus N and P proteins. Taking into account the fact that the PNT region is shared by the P, V and W proteins, we reasoned that it is likely to constitute an independent, functional domain. We therefore decided to clone and characterize this latter region (residues 1-404 for HeV and 1-406 for NiV), rather than the entire disordered N-terminal P region extending up to residue 470 (see Fig. 1B). As for the N protein, we focused our efforts on the N TAIL region. , as obtained using a 14-residues window, are indicated by a green bar above the sequence. Below the sequences are shown the HCA plots and the predicted regions of disorder that are represented by bi-directional arrows. Regions highlighted in yellow and in orange correspond to either putative Molecular Recognition Elements (MoREs) or structured regions (PMD and XD) within PCT, respectively. Structured and disordered regions are represented by large and narrow boxes, respectively. The linker region connecting PMD and XD is shown by an empty, narrow box to indicate its mixed nature. The vertical line separating PNT and PCT is located at the border between the region shared by P and V and the region unique to P (see text). The hydrophobic, a-helical region at the N-terminus of P is highlighted. doi:10.1371/journal.pone.0011684.g001

Expression and purification of Henipavirus N TAIL and PNT domains
We cloned the DNA fragments of the Henipavirus N and P genes encoding N TAIL and PNT into the pDest14 expression plasmid that allows expression in E. coli of recombinant proteins under the control of the T7 promoter. Primers were designed so as to lead to constructs encoding the N TAIL and PNT domains with either an N-terminal or a C-terminal histidine tag, respectively. The E. coli Rosetta [DE3] pLysS strain (Novagen) was used for the expression of the constructs.
Both PNT and N TAIL proteins were recovered from the soluble fraction of bacterial lysates (Fig. 4, lanes SN) and were purified to homogeneity (.95%) in two steps: Immobilized Metal Affinity Chromatography (IMAC) and gel filtration (Fig. 4). The identity of all the recombinant products was confirmed by mass spectrometry analysis of the tryptic fragments obtained after digestion of the purified proteins excised from SDS-polyacrylamide gels (data not shown). Both N TAIL proteins display an abnormally slow migration in SDS-PAGE with an apparent molecular mass of 20 kDa (expected MM 15 kDa) (Fig. 4 A, B). A similar aberrant electrophoretic mobility is observed for PNT proteins (Fig. 4 C, D), where these latter migrate with an apparent molecular mass of approximately 60 kDa (expected molecular mass of 45 KDa). Noteworthy, mass spectrometry analysis confirmed that the recombinant products possess the expected molecular mass (see supplementary Figs. S2, S3, S4, S5). This abnormal behavior is therefore likely to be ascribed to a rather high content of acidic residues, as already been observed in the case of the intrinsically disordered MeV PNT [18] and N TAIL domains [6,19], and, more generally, in other IDPs [46]. Indeed, because of their biased amino acid composition, often leading to enrichment in negatively charged residues, IDPs bind less SDS than usual. As a result their apparent molecular mass is often 1.2-1.8 times higher than the real one calculated from sequence data or measured by mass spectrometry [46].
In the case of NiV PNT, a minor lower band is also observed in the final product (see Fig. 4C, lane GF). Mass spectrometry analysis of the tryptic fragments obtained after digestion of this minor band showed that it corresponds to a degradation product of PNT.

Protease sensitivity of Henipavirus N TAIL and PNT
Henipavirus PNT and N TAIL were found to be highly sensitive to proteolysis, a property that constitutes a hallmark of structural disorder (see [47] and references therein cited). Indeed, globular proteins are preferentially cleaved at exposed and flexible loops only and almost never within secondary structure elements [48,49]. The use of a protease with broad substrate specificity, such as thermolysin allows the identification of cleavage sites solely on the basis of the flexibility of the protein substrate. In order to . The relative enrichment in disorder promoting (black bars) and order-promoting (grey bars) residues is shown. Residues have been ordered on the x axis according to the TOP-IDP flexibility index as described in [138]. doi:10.1371/journal.pone.0011684.g002 assess the extent of protease sensitivity, we submitted Henipavirus PNT and N TAIL to digestion by thermolysin. As shown in Fig. 4, all the proteins are readily degraded by thermolysin after one hour incubation, a behavior that is consistent with the lack of a packed core and with an overall solvent accessibility of Henipavirus PNT and N TAIL . Conversely, lysozyme was shown to be resistant to proteolysis even after an incubation period as long as 24 hours (see supplementary Fig. S6).

Size-exclusion chromatography analyses of Henipavirus PNT and N TAIL
Since the elution volume of a protein from a gel filtration column depends on its hydrodynamic properties, we used size-exclusion chromatography (SEC) to infer the hydrodynamic properties of Henipavirus PNT and N TAIL proteins (Fig. 5). Henipavirus PNT and N TAIL are eluted form the gel filtration column as sharp peaks with an apparent molecular mass (MMapp) well above the expected one (MMtheo). These large values of apparent molecular mass indicate that these proteins are not compatible with a monomeric, globular structure (Fig. 5). Rather, such large values of apparent molecular mass can be attributed either to trimerization or to an extended conformation with low compactness of the polypeptide chain typical of IDPs [50]. Note that these very high values of molecular mass can't be ascribed to protein aggregation, since they were independent from protein concentration. In addition, note that the same elution profiles were obtained regardless of whether a sodium phosphate or Tris/ HCl buffer were used for elution and irrespective of the NaCl concentration.
The insets of Fig. 5 show the Stokes radii (R S obs ) of Henipavirus PNT and N TAIL , as deduced from the apparent molecular mass observed in gel filtration [51]. By comparing each experimentally determined R S obs with the theoretical Stokes radii expected for various conformational states (R s NF : monomeric natively folded protein; R s Trim : trimeric folded protein; R s U : fully unfolded RC state in urea; R s PMG : PMG conformation) all the protein domains were found to have Stokes radii similar to the expected values of either PMG-like IDPs or of folded trimers (see insets in Fig. 5). Indeed, as seen in Fig. 5, the R S obs of all the proteins are much larger than the corresponding R s NF values, and are very close to the values expected either for a PMG or for a folded trimer (see ratios between R S obs and R S PMG or R S Trim in insets of Fig. 5). In addition, the comparison of the hydrodynamic volume (V h obs ) of each protein, as calculated from the R S obs , with the theoretical volume values expected for the various conformational states, points out a better agreement with the expected values of PMGlike IDPs than with those of folded trimers (see ratios between V h obs and V h PMG or V h Trim in insets in Fig. 5). These studies suggest that Henipavirus PNT and N TAIL proteins either adopt a PMG conformation or are folded trimers.

NMR studies of Henipavirus PNT and N TAIL
In order to discriminate between these two latter hypotheses and to directly assess the actual conformation of Henipavirus PNT and N TAIL proteins, we studied them by 2D NMR spectroscopy. Fig. 6 shows the amide region of their NOESY spectra. The very small spread of the resonance frequencies for amide protons (between 7.8 ppm and 8.7 ppm, see frames in Fig. 6) together with the scarcity of NOEs in the amide-amide region are typical of proteins without any stable secondary structure (for examples see [6,18]), thereby supporting lack of a packed core within Henipavirus PNT and N TAIL domains.

CD studies of Henipavirus PNT and N TAIL
In further support of the absence of an ordered structure, the far-UV CD spectra of Henipavirus PNT and N TAIL at neutral pH are typical of unstructured proteins, as seen from their large negative ellipticity at 198 nm and low ellipticity at 190 nm (Fig. 7A). However, the observed ellipticity values at 200 and 222 nm of In the left part of the CH plot, a protein is predicted to be intrinsically disordered, whereas it is predicted to be structured if it falls in the right part of it (see Materials and Methods). doi:10.1371/journal.pone.0011684.g003 Henipavirus N TAIL and of NiV PNT are consistent with the existence of some residual secondary structure, as observed in IDPs adopting a PMG conformation (Fig. 7B). Indeed, Uversky noticed that IDPs can be subdivided in PMG-like and RC-like as a function of their ellipticity values at 200 and 222 nm [32]. Strikingly, except for HeV PNT that falls in the RC-like region of the plot, the other Henipavirus domains are all located in the twilight zone between RC-like and PMG-like proteins. This suggests that NiV PNT, as well as both Henipavirus N TAIL domains, do not adopt a fully extended conformation, contrary to HeV PNT that has a tendency to be more flexible and possesses less residual structure.
We also monitored the ellipticity at 222 nm of Henipavirus PNT and N TAIL proteins at increasing temperatures (Fig. 7C). In spite of the rather noisy (i.e. undulating) nature of the obtained curves, no cooperative thermal transitions were observed, as judged based on the lack of a coherent trend in the variation of the ellipticity at 222 nm as a function of the temperature (Fig. 7C). These results, once again, support lack of a rigid 3D structure [52].
To test the potential of Henipavirus PNT and N TAIL folding, we recorded their CD spectra in the presence of increasing concentrations of TFE. The solvent TFE is widely used as an empirical probe of hidden structural propensities of peptides and proteins as it mimics the hydrophobic environment experienced by proteins in proteinprotein interactions [53,54,55] (Fig. 8). All the proteins show an increasing gain of a-helicity upon addition of TFE, as indicated by the characteristic maximum at 190 nm and double minima at 208 and 222 nm (Fig. 8). The a-helical content gradually increases upon increasing the TFE concentration from 0 to 25% and then reaches a plateau, while no concomitant dose-dependent increase in the content of b structure is detected (data not shown).
Most unstructured-to-structured transitions take place in the presence of 20% TFE, a concentration at which the a-helix content is estimated to reach approximately 40% for both N TAIL and 50% for PNT proteins (Fig. 8). Note that for all the proteins, the spectra display an isodichroic point at 202 nm indicative of a two-state transition (Fig. 8).

Dynamic light scattering studies of Henipavirus PNT and N TAIL
In view of further investigating the extent of disorder within Henipavirus PNT and N TAIL proteins, we carried out dynamic light scattering (DLS) studies in the presence or absence of urea. This approach has the advantage of allowing the Stokes radius to be directly measured, as opposite to SEC analyses that only provide an estimation of the Stokes radius.
These studies showed that all Henipavirus PNT and N TAIL protein samples are highly monodisperse (99%), consisting of a single protein species. While the HeV and NiV N TAIL proteins possess a very similar R S (2862 Å or 2661 Å for NiV and HeV, respectively), the two PNT proteins differ in their R S , which was either 5063 Å or 4463 Å , depending on whether the HeV or NiV PNT protein was studied (see Table 1). For the N TAIL and NiV PNT proteins, these values are consistent (within the error bars) with the R s measured by SEC, while the R s of HeV PNT was found to be slightly larger (see Table 1).
In view of highlighting possible denaturation-induced loss of compactness, we also carried out these measurements in the presence of urea. The obtained R S values (3762 Å for both N TAIL proteins, and either 5562 Å or 5762 Å for NiV and HeV PNT, respectively) highlight a significant increase in the hydrodynamic radius in the presence of urea (see Table 1). These results argue for the presence of residual intramolecular interactions within Henipavirus PNT and N TAIL proteins under native conditions, as expected for proteins adopting a PMG conformation.

Discussion
Henipavirus PNT and N TAIL as members of the premolten globule sub-family The peculiar sequence properties of Henipavirus PNT and N TAIL suggest that these protein domains are mostly unstructured in solution.
In agreement, they show an aberrant electrophoretic migration, which is a hallmark of protein disorder [46], and display a high protease-sensitivity that argues for the lack of a packed core in these protein domains. Likewise, CD studies with increasing temperatures showed lack of any cooperative thermal unfolding, thus supporting once again the lack of a packed core. Indeed, IDPs are rather insensitive to temperature increase, with some of them having even been reported to undergo heat-induced folding (see [47] and references therein cited, and [56,57]).
The hydrodynamic properties of Henipavirus PNT and N TAIL inferred from gel filtration are consistent with these protein domains being either stable globular trimers, or extended (unstructured) monomers. Using NMR and CD we show that they are actually disordered in solution. However, with the only notable exception of HeV PNT, their far-UV spectroscopic parameters (see Fig. 7B) indicate that they are not fully unfolded, but rather they conserve some transiently populated secondary structure content typical of IDPs with a PMG conformation [32]. In addition, the mean hydrodynamic volumes and Stokes radii inferred from gel filtration are close to the values expected for native PMG conformations [32]. In further support of the occurrence of some residual structure, DLS studies pointed out a significant increase in the Stokes radius of all proteins upon addition of urea. The R S values obtained in the presence of urea are close to those expected for fully extended forms (cfr Table 1 and Fig. 5). The HeV PNT protein has a notable behavior in that  its Stokes radius, as measured by DLS, is slightly, though significantly, larger than that inferred from SEC studies. This observation nicely correlates with the above-mentioned spectroscopic properties of HeV PNT as seen by far-UV CD studies (see Fig. 7B). With the only notable exception of HeV PNT, for all the other proteins herein studied, the R S values provided by DLS were found to be very similar to those obtained by SEC. This confirms and extends previous thorough SEC analyses of several proteins revealing that the hydrodynamic radius values inferred from SEC are in very good agreement with those obtained by other hydrodynamic methods, such as viscometry, analytical ultracentrifugation and dynamic light scattering (DLS) (see [58] and references therein cited).
According to [32], Henipavirus PNT and N TAIL proteins lie in a region of the CH-plot that is consistent with the occurrence of residual intramolecular interactions typical of native PMGs. In particular, Uversky showed that intrinsic coils are more distant from the border separating structured proteins from IDPs, than intrinsic PMGs [32]. It should be pointed out however that a systematic experimental confirmation of the relationship between position in the CH-plot and content in secondary structure is still lacking, with a few discrepancies having even been experimentally observed [59]. The distance values from the border (H boundary 2H) are 0.034 and 0.007 for HeV and NiV N TAIL respectively, whereas they are 0.050 and 0.031 for HeV and NiV PNT proteins, respectively (see Materials and Methods). According to [32], these values are all consistent with the values expected for native PMGs (0.03760.033). Interestingly, although the distance value of HeV PNT is compatible with a PMG state, this protein domain has the largest distance from the border separating IDPs and structured proteins (see Fig. 3). This finding is in agreement with the spectroscopic parameters of HeV PNT that locate it in the proximity of RC-like proteins (see Fig. 7B). Notably, HeV N TAIL and NiV PNT, which fall in the same position of the ellipticity plot (see Fig. 7B), display comparable distances from the boundary of the CH-plot (see Fig. 3).
Thus, Henipavirus PNT and N TAIL can be described as nonglobular polypeptide chains, more compact than RCs, all containing some residual structure. This residual structure restrains the conformational space sampled by these proteins, thereby reducing the number of interconverting conformers in solution. In agreement, the distribution of the conformations of Henipavirus PNT and N TAIL is narrow, as seen by the relative sharpness of the elution peaks observed in gel filtration (see Fig. 5).

Residual structure and folding propensities of Henipavirus PNT and N TAIL
Analysis of the HCA plot of Henipavirus N proteins, reveals the presence within N TAIL of four short regions possessing each a hydrophobic cluster and/or a predicted (a or b) secondary structure elements , are found (see Fig. 1A). These short orderprone segments might correspond to Molecular Recognition Elements (MoREs), where these latter are short order-prone regions within IDRs with a propensity to undergo induced folding (i.e. a disorder-to-order transition) upon binding to a partner. MoREs can be divided in a-, band irregular (i.e. neither a, nor b) MoRES depending on the nature of the structural transition they undergo [60,61,62,63]. Based on the type of predicted secondary structure element, Henipavirus N TAIL domains may possess two putative a-MoREs (residues 408-422 and 473-493), a putative I- MoRE (residues 523-532) and a putative b-MoRE (residues 444-464). Thus, the residual ordered secondary structure present within Henipavirus PNT and N TAIL likely arises from transiently populated MoREs. That the conformational space sampled by these interaction-prone short segments within IDRs can be restricted even in the absence of the partner has already been reported [64] and assumed to reflect an inherent conformational preference. It has been proposed that these partly pre-configured MoREs can enable a more efficient start of the folding process induced by a binding partner through a reduction of the entropic cost of binding [46,61,62,64,65,66].
The solvent TFE mimics the hydrophobic environment experienced by proteins in protein-protein interactions and is therefore widely used as a probe to unveil the propensity of IDPs to undergo induced folding upon target binding [54]. In agreement with the presence of transiently populated a-helical segments, CD studies in the presence of TFE pointed out a clear ahelical potential in Henipavirus PNT and N TAIL . Note that the gain of a-helicity induced by TFE, although already reported for other proteins, including MeV PNT [18] and N TAIL [6,19], is not a general rule: for instance, (i) the acidic activator domain of GCN4 forms little or no a-helix in TFE concentrations as high as 30% and folds mostly as b-sheets in 50% TFE [67] and (ii) the intrinsically disordered rat seminal vesicle protein IV exclusively undergoes b transitions in the presence of TFE (P. Palladino, S. Vilasi, R. Ragone and F. Rossi, personal communication). Furthermore, in the case of MeV N TAIL , we have recently shown that TFE promotes a-helical folding of the 488-502 region only, with the downstream region only becoming slightly less mobile while retaining an extended conformation in the presence of 20% TFE [68]. The a-helical propensity of Henipavirus PNT and N TAIL is in agreement with the occurrence of two putative a-MoREs within both N TAIL proteins, as well as with the presence of a 50residues long a-helical region at the N-terminus of both PNT domains (see Fig. 1). On the other hand, secondary structure predictions point to the presence of a short b-strand within the second putative MoRE of both NiV (aa 444-452) and HeV (aa  444-449) N TAIL (see Fig. 1A), thereby suggesting that this MoRE could be a b-MoRE. However, based on the experimentally observed behavior of both N TAIL domains in the presence of TFE, this latter MoRE is probably a-helical one. This is consistent with the shape and size of hydrophobic clusters occurring in this region, as seen from the HCA plot (see Fig. 1A) [69] and is also in agreement with previous experimental observations that showed the helical nature of the cognate MeV and SeV N TAIL MoREs [19,20,42,68,70,71,72,73]. Definite answers as to the involvement of these putative MoREs in binding and in structural transitions await identification of partner(s), as well as generation of truncated N TAIL constructs and assessment of their binding abilities. By analogy with MeV and SeV, we can speculate that one such a possible binding partner could be the X domain of the P protein [19,20,25,42,68,70,71,72,73]. Future studies will address the ability of Henipavirus N TAIL domains to effectively interact with XD, and will assess possible inter-species cross-interactions, as well as possible structural transitions within N TAIL upon interaction with XD.

Structural disorder as a lessener of evolutionary constraints
Notably, PNT partially overlaps with the C protein (being encoded by the same RNA region) and the spacer region partially overlaps with the C-terminal domain of the V protein. The disordered nature of PNT and of the ''spacer'' region connecting PNT to PMD likely reflects a way of alleviating evolutionary constraints within overlapping reading frames. This observation is in agreement with previous reports pointing out a relationship between overlapping genes and structural disorder [26,74,75,76,77], as also nicely illustrated by recent findings showing that the hepatitis C virus Core +1/S protein, overlapping the Core protein gene in the +1 reading frame, is intrinsically disordered [78]. Disorder, which is encoded by a much wider portion of sequence space as compared to order, can indeed represent a strategy by which genes encoding overlapping reading frames can lessen evolutionary constraints imposed on their sequence by the overlap, allowing the encoded overlapping protein products to sample a wider sequence space without losing function.
By comparing the modular organization of the P proteins within the Paramyxovirinae subfamily, we noticed that a larger PNT domain in Henipaviruses accounts for the extra length of their P protein (cfr. Fig. 1B with Fig. 8 in [26]). This finding is consistent with the higher tolerance of disordered regions to insertions or major rearrangements as compared to ordered ones.

Functional advantage of disorder within Henipavirus N and P proteins
The results herein presented clearly show that intrinsic disorder is abundant within the replicative complex of Henipaviruses, in agreement with our previous findings for other Paramyxovirinae members (see [26] and references therein cited). One of the functional advantages of disorder is related to an increased plasticity that enables IDRs to bind to numerous structurally distinct targets [79,80,81] and allows protein interactions to occur with both high specificity and low affinity [27,30,31,34,82,83,84,85,86]. In the case of MeV, the N TAIL domain has indeed been shown to bind to numerous partners including the X domain of the P protein [6,19,20,42], the major inducible heat shock protein hsp70 [87,88,89], the interferon regulatory factor 3 [90,91], a yet unidentified protein cell receptor involved in MeV-induced immunosuppression [92,93], a nuclear export protein [94], the matrix protein [95] and, possibly, components of the cell cytoskeleton [96,97]. Likewise, both MeV and SeV PNT domains have been reported to interact with multiple partners, with the former interacting with N [98] and cellular proteins [99], and the latter interacting with the unassembled form of N (Nu) and the L protein [100,101]. By analogy with the Sendai [100,101] and human parainfluenza virus type 2 [102], the N-terminal a-helical segment within Henipavirus PNT could correspond to the Nu-binding region. Given its relative shortness and positioning upstream a disordered region, this Nterminal a-helical segment is not probably stably folded in isolation and rather only folds in cooperation with another protein. Although we can't formally rule out the possibility that Henipavirus PNT and N TAIL domains may undergo some degree of folding in the context of the full-length P and N proteins, a few studies carried out on the cognate domains of related viruses suggest that the Henipavirus PNT and N TAIL domains could display a significant amount of disorder within the entire P and N proteins either. Indeed, in SeV, the PNT region has been shown to be disordered not only in isolation but also in the context of the fulllength protein [103,104], as were the linker region between PMD and XD in the context of PCT [105] and the region upstream XD within the PX protein [106,107]. Likewise, the MeV N TAIL region is also disordered within the entire nucleoprotein, being equally accessible to monoclonal antibodies in isolation and within recombinant nucleocapsids, being highly susceptible to protease digestion and not visible in electron microscopy [6]. Definite answers as to the disordered state of PNT and N TAIL domains in the context of the full-length N and P proteins require however additional experimental work that will be the focus of future studies.
Likewise, as for the possibility that PNT and N TAIL are effectively disordered in vivo, further experimental work is required to address this question. In this regard, it is noteworthy that, so far, available data addressing the disordered state of IDPs in vivo are controversial: if in-cell NMR experiments on the natively unfolded FlgM protein indeed suggest a more folded conformation in the cellular environment of live bacteria [108], a few reports support a disordered state for other IDPs either in living cells or in the presence of crowding agents that mimic the crowded environment of cells (for examples see [6,109,110]).

Abundance of structural disorder within viruses
A recent study showed that viral proteins, and in particular RNA virus proteins, are enriched in disordered regions [111]. In that study, the authors propose that beyond affording a broad partnership, the wide occurrence of disordered regions in viral proteins could also be related to the typical high mutation rates of RNA viruses, representing a strategy for buffering the deleterious effects of mutations [111]. Taking into account these considerations, as well as the correlation between overlapping genes and disorder [74,75,76], we propose that the main advantage of the abundance of disorder within viruses would reside in pleiotropy and genetic compaction. Indeed, disorder provides a solution to reduce both genome size and molecular crowding, where a single gene would (i) encode a single (regulatory) protein product that can establish multiple interactions via its disordered regions and hence exert multiple concomitant biological effects, and/or (ii) would encode more than one product by means of overlapping reading frames. In fact, since disordered regions are less sensitive to structural constraints than ordered ones, the occurrence of disorder within one or both protein products encoded by an overlapping reading frame can represent a strategy to alleviate evolutionary constraints imposed by the overlap. As such, disorder would confer to viruses the ability to ''handle'' overlaps, thus further expanding the coding potential of viral genomes.

Conclusions
This paper represents the first report on the experimental characterization of Henipavirus N and P proteins. It also provides new perspectives in the study of disordered regions within the replicative complex of these viruses. In particular, taking into account the finding that protein-protein interactions mediated by disordered regions have been proved to be interesting drug discovery targets with the potential to increase significantly the discovery rate for new compounds [112], the present results designate the disordered regions of Henipavirus N and P proteins as promising targets for antiviral compounds. In addition, the broad molecular partnership that typifies IDRs, suggests that Henipavirus PNT and N TAIL domains could be involved in manifold proteinprotein interactions. As such, the results herein presented could orient future work towards the identification of both viral and cellular partners. In the long term, studies focused on the interactions with binding partners are expected to contribute to shed light on the molecular mechanisms of the N and P proteins of these highly pathogenic agents. On a last note, the present study is also expected to contribute to future efforts aimed at obtaining high-resolution structural data on Henipavirus N and P proteins by helping to delineate domains of these latter amenable to crystallization.
Small hydrophobic clusters occurring within mainly disordered regions, as observed in HCA plots, were assumed to correspond to putative Molecular Recognition Elements (MoREs), where these latter are short order-prone regions within IDRs that fold upon binding to a partner or ligand [60,61,62,63].

In silico amino acid composition analysis
Deviations in amino acid composition of Henipavirus PNT and N TAIL were computed as already described [30,132], using the average amino acid frequencies of the SWISS-PROT database (as obtained from http://us.expasy.org/sprot) as the reference value. The average amino acid frequencies of the SWISS-PROT database roughly corresponds to the mean composition of proteins in nature. If the average composition of an amino acid X in SWISS-PROT proteins is CW X , and CP X is the composition of X within a protein P, deviation from the composition of X of SWISS-PROT proteins was defined for P as (CP X -CW X )/CW X .
Low sequence complexity segments were searched using the SEG server (http://mendel.imp.ac.at/METHODS/seg.server. html) [133] with a trigger window length of 25, a trigger complexity of 2.2 and an extension complexity of 2.5 that work well for the identification of short non-globular domains.

Charge-Hydropathy (CH) plots
Charge-hydropathy (CH) plots were then generated as described by Uversky et al. [32]. The CH plot is divided into two regions by a line, which corresponds to the equation H = (R+1.151)/2.785, where R is the mean net charge and H is the mean hydrophobicity. In the left part of the diagram (where H,(R+1.151)/2.785), a protein is predicted as disordered, whereas it is predicted as ordered in the right part [32]. H Boundary was computed according to [32]: H Boundary = (R+1.15)/2.785. The mean net charge (R) of a protein is defined as the absolute value of the difference between the number of positively and negatively charged residues at pH 7 divided by the total number of amino acid residues. It was calculated using the program ProtParam at the EXPASY server (http://www.expasy.ch/tools). The mean hydrophobicity (H) is the sum of normalized hydrophobicities of individual residues divided by the total number of amino acid residues minus 4 residues (to take into account fringe effects in the calculation of hydrophobicity). Individual hydrophobicities were determined using the Protscale program at the EXPASY server (http://www.expasy.ch/tools), using the options ''Hphob/Kyte & Doolittle'', a window size of 5, and normalizing the scale from 0 to 1. The values computed for individual residues were then exported to a spreadsheet, summed and divided by the total number of residues minus 4 to yield (H). The net charge-hydrophobicity method is only applicable to a protein (or protein region) provided it is not composed of shorter, structurally independent modules. It might otherwise give conflicting results. It was only validated for regions .50 aa [45]. An estimation of its error rate can be drawn from Uversky [50]. In that study, no globular protein was found to have a ratio located on the left side of the line, indicating that the positive error rate for the prediction of disordered proteins must be very low. However, five unfolded proteins out of 105 -which were all borderline -were wrongly assigned as being globular, indicating a negative error rate of about 5%.

Construction of expression plasmids
The Henipavirus N TAIL and PNT constructs, encoding residues 400-532 of N and 1-406 (NiV) or 1-404 (HeV) of P, with a hexahistidine tag fused to their N-(N TAIL ) or C-termini (PNT), have been obtained by PCR using Pfx polymerase (Stratagene) and synthetic N and P genes (GenScript), optimized for the expression in E. coli, as templates. Primers (Operon) were designed to introduce a hexahistidine tag encoding sequence either at the 59 (N TAIL ) or 39 (PNT) end of the DNA fragments, as well as an AttB1 and AttB2 sites at the 59 and 39 ends of these latter, respectively. The rationale for the choice of the tag position was to reflect at best the ''natural'' organization of the proteins, where additional (natural) residues are found upstream N TAIL and downstream PNT. After purification (PCR Purification Kit, Qiagen), the PCR products were cloned into the pDest14 vector (Invitrogen) using the Gateway recombination system (Invitrogen).
Selection and amplification of DNA constructs was carried out using CaCl2-competent E. coli TAM1 cells (Active Motif). The sequence of the coding region of all expression plasmids was verified by sequencing (GenomeExpress).

Bacterial expression of Henipavirus N TAIL and PNT constructs
The E. coli Rosetta [DE3] pLysS strain (Novagen) was used for the expression of the constructs. This strain, which is optimized for the expression of recombinant proteins, also carries the lysozyme gene thus allowing a tight regulation of the expression of the recombinant gene, as well as a facilitated lysis. Cultures were grown overnight to saturation in LB medium containing 100 mg/ ml ampicilin and 34 mg/ml chloramphenicol. An aliquot of the overnight culture was diluted 1/25 in LB medium and grown at 37uC. At OD 600 of 0.7, isopropyl ß-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and the cells were grown at 37uC for 3 hours. The induced cells were harvested, washed and collected by centrifugation. The resulting pellets were frozen at 220uC.

Purification of Henipavirus N TAIL and PNT
All cellular pellets, irrespective of the recombinant protein they express, were resuspended in 5 volumes (v/w) buffer A (50 mM sodium phosphate pH 7, 300 mM NaCl, 10 mM Imidazole, 1 mM phenyl-methyl-sulphonyl-fluoride (PMSF)) supplemented with lysozyme 0.1 mg/mL, DNAse I 10 mg/mL, protease inhibitor cocktail (Roche) (one tablet for 50 mL of bacterial lysate). After a 20 min incubation with gentle agitation, the cells were disrupted by sonication (using a 750 W sonicator and 4 cycles of 30 s each at 45% power output). The lysate was clarified by centrifugation at 30,000 g for 30 min. Starting from a 1 L culture, the clarified supernatant was incubated for 1 hr with gentle shaking with 4 mL Chelating Sepharose Fast Flow Resin preloaded with Ni 2+ ions (GE, Healthcare), previously equilibrated in buffer A. The resin was washed with buffer A containing 20 mM imidazole, and the recombinant protein was eluted in buffer A containing 250 mM imidazole. Eluates were analyzed by SDS-PAGE for the presence of the desired protein product. The fractions containing the recombinant protein were combined, and then loaded onto a Superdex 200 HR 16/60 column (GE, Healthcare). N TAIL proteins were eluted in either 10 mM Tris/ HCl pH 8, 500 mM NaCl or 10 mM sodium phosphate pH 7, 150 mM NaCl depending on whether the protein was further subjected to limited proteolysis or to NMR and CD analyses, respectively. Likewise, PNT proteins were either eluted in 10 mM Tris buffer pH 8 containing 300 mM NaCl or in 10 mM sodium phosphate pH 7, 150 mM NaCl. For both HeV and NiV N TAIL proteins, which are devoid of Trp and Tyr residues, the elution was followed by monitoring the absorbance at 254 nm instead of 280 nm.
The proteins were concentrated using Centricon Plus-20 (molecular cutoff of either 5,000 Da or 10,000 Da for N TAIL or PNT proteins, respectively) (Millipore). All proteins were stored at 220uC either in the presence (PNT) or absence (N TAIL ) of 10% glycerol. HiTrap desalting columns (5 mL) (GE, Healthcare) where used to get rid of glycerol prior to NMR experiments, as well as to reduce the NaCl concentration in view of CD studies. All purification steps, except for gel filtrations, were carried out at 4uC.
Hydrodynamic characterization of Henipavirus PNT and N TAIL Apparent molecular mass (MMapp) of proteins eluted from gel filtration columns was deduced from a calibration carried out with LMW calibration kits (GE, Healthcare). The hydrodynamic radius of a protein (Stokes radius) can be deduced from its apparent molecular mass (as seen by gel filtration) [51].

Determination of protein concentration
Protein concentrations were calculated either using the theoretical absorption coefficients e (mg/ml.cm) at 280 nm as obtained using the program ProtParam at the EXPASY server (http://www.expasy.ch/tools) (PNT proteins), or the BCA protein assay reagent (Pierce) (N TAIL proteins).

Digestion of Henipavirus N TAIL and PNT by thermolysin
A thermolysin stock solution was prepared by dissolving the commercial powder (Sigma, 50-100 units/mg protein) at a concentration of 0.4 mg/mL in 10 mM Tris/HCl pH 8, 300 mM NaCl and then stored at 220uC. PNT and N TAIL samples at a concentration of 2.5 mg/mL in 10 mM Tris/HCl pH 8 supplemented with either 300 mM (PNT) or 500 mM (N TAIL ) NaCl, were used as stock solutions. Lysozyme (Euromedex) was used as a control. Digestions of proteins were performed by incubation of the protein substrate (1 mg/mL) with thermolysin in 20 mM Tris/HCl pH 8 at 26uC. Protease:protein substrate ratios were 1:100 (w/w). The extent of proteolysis was evaluated by SDS-PAGE analysis of 10 ml aliquots removed from the reaction mixture over a time course (0, 1 and 24 hours), added to 10 ml of 26 Laemli sample buffer and boiled for 5 min to inactivate the protease.

Mass Spectrometry (MALDI-TOF)
Mass analysis of Henipavirus N TAIL and PNT proteins was performed using an Autoflex II TOF/TOF. Spectra were acquired in the linear mode. Samples (0.7 mL containing 15 pmol) were mixed with an equal volume of sinapinic acid matrix solution, spotted on the target, then dried at room temperature for 10 min. The mass standard was either myoglobin or BSA depending on whether N TAIL or PNT proteins were analyzed, respectively. Proteins were analyzed in the Autoflex matrixassisted laser desorption ionization/time of flight (Bruker Daltonics, Bremen, Germany).
The identity of purified Henipavirus N TAIL and PNT proteins was confirmed by mass spectral analysis of tryptic fragments. The latter was obtained by digesting (0.25 mg trypsin) 1 mg of purified recombinant protein obtained after separation onto SDS-PAGE. The tryptic peptides were analyzed as described above and peptide fingerprints were obtained and compared with in-silico protein digest (Biotools, Bruker Daltonics, Germany). The mass standards were either autolytic tryptic peptides or peptide standards (Bruker Daltonics).

Two-dimensional Nuclear Magnetic Resonance (NMR)
PNT and N TAIL samples at a concentration of 0.1 mM in 10 mM sodium phosphate pH 7, 150 mM NaCl and 10% D 2 O were used for the acquisition of a NOESY spectrum on a 600-MHz ultra-shielded-plus Avance-III Bruker spectrometer equipped with a TCI cryo-probe. The temperature was set to 300 K and the spectra were recorded with 2048 complex points in the directly acquired dimension and 512 points in the indirectly detected dimension. Solvent suppression was achieved by using excitations sculping with gradients [134]. The data were processed using the Bruker Topspin software; they were multiplied by a sinesquared bell and zero-filled to 1 K in first dimension prior to Fourier transformation.

Circular Dichroism (CD)
CD spectra were recorded on a Jasco 810 dichrograph, equipped with a Peltier thermoregulation system, using 1-mm thick quartz cells in 10 mM sodium phosphate pH 7 at 20uC. CD spectra were measured between 190 and 260 nm, with a scanning speed of 20 nm/min and a data pitch of 0.2 nm. Spectra were averaged from three scans. Moreover, for each protein sample, at least three independent acquisitions were carried out so as to estimate the experimental error arising from sample preparation. The contribution of buffer was subtracted from experimental spectra. Spectra were smoothed using the ''means-movement'' smoothing procedure implemented in the SpectraManager package. Structural variations of both N TAIL and PNT proteins were measured as a function of changes in the initial CD spectrum upon addition of increasing concentrations of 2,2,2-trifluoroethanol (TFE) (Fluka). The final NaCl concentration in PNT and N TAIL samples was comprised between 5 and 15 mM.
Mean  [135,136]. The CDSSTR deconvolution method was used to estimate the a-helical content using the reference protein set 7. Reconstructed curves very well superimposed on the experimental ones thus attesting the reliability of the inferred ahelical percentages (data not shown).
Measurements at fixed wavelength (222 nm) were performed in the temperature range of 20uC-100uC with data pitch 20uC and temperature slope of 5uC/min with protein concentrations of 0.1 mg/mL. The buffer solutions without the proteins were used as blanks.
Dynamic light scattering studies of Henipavirus PNT and N TAIL Dynamic light scattering experiments were performed with a Zetasizer Nano-S (Malvern) at 25uC. Protein samples were diluted in 10 mM Tris/HCl pH 8 in the presence or absence of urea to a final concentration of 0.5 mg/ml. The urea concentration ranged from 6.4 M to 7.4 M for PNT and N TAIL proteins, respectively. The samples were filtered prior to the measurements (Millex syringe filters 0.22 mm, Millipore). The hydrodynamic radius was deduced from translational diffusion coefficients using the Stokes-Einstein equation. Diffusion coefficients were inferred from the analysis of the decay of the scattered intensity autocorrelation function. All calculations were performed using the software provided by the manufacturer. The relative viscosity of the samples containing 6.4 M or 7.4 M urea was assumed to be either 1.45 or 1.57, respectively, according to [137]. Figure S1 Multiple sequence alignment of Henipavirus, Morbillivirus and Respirovirus N proteins as obtained using ClustalW [113] (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and ESPript [114] (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Residues corresponding to a similarity above 60% are boxed and shown in red. Identical residues are boxed and shown in white on a red background. The front numbers correspond to the amino acid position in sequence. Dots above the alignment indicate intervals of 10 residues. Predicted secondary structure elements, as obtained using the PSIPRED server [115] (http://bioinf.cs.ucl.ac. uk/psipred/), for Hendra and Measles virus N are shown above the multiple sequence alignment. The red helix spanning residues 487-503 of Measles virus N corresponds to the helical segment observed in the crystal structure of a chimeric construct consisting of the C-terminal domain of the Measles virus P protein and of residues 486-504 of N (pdb code : 1T6O  Figure S2 Mass spectrometry (MALDI-TOF) analysis of recombinant, hexahistidine tagged NiV NTAIL purified from the soluble fraction of E. coli. Mass analysis was performed using an Autoflex II TOF/TOF. Spectra were acquired in the linear mode. The sample (0.7 mL containing 15 pmol) was mixed with an equal volume of sinapinic acid matrix solution, spotted on the target, then dried at room temperature for 10 min. The mass standard was myoglobin. Proteins were analyzed in the Autoflex matrix-assisted laser desorption ionization/time of flight (Bruker Daltonics, Bremen, Germany). A major peak with a mass slightly higher (14 953 Da) than expected (14 949 Da) was observed. The additional peak of 7 475 Da in mass, very probably corresponds to a degradation product, as the protein was shown to contain no contaminating proteins (as shown by mass spectrometry analysis of trypic fragments). Found at: doi:10.1371/journal.pone.0011684.s002 (0.05 MB DOC) Figure S3 Mass spectrometry (MALDI-TOF) analysis of recombinant, hexahistidine tagged HeV NTAIL purified from the soluble fraction of E. coli. Mass analysis was performed using an Autoflex II TOF/TOF. Spectra were acquired in the linear mode. The sample (0.7 mL containing 15 pmol) was mixed with an equal volume of sinapinic acid matrix solution, spotted on the target, then dried at room temperature for 10 min. The mass standard was myoglobin. Proteins were analyzed in the Autoflex matrix-assisted laser desorption ionization/time of flight (Bruker Daltonics, Bremen, Germany). A peak with a mass slightly higher (15 304 Da) than expected (15 241 Da) was observed. The numerous additional peaks corresponding to species of lower molecular mass likely correspond to degradation products, as the protein was found to be devoid of contaminating protein by mass spectrometry analysis of tryptic fragments. Found at: doi:10.1371/journal.pone.0011684.s003 (0.06 MB DOC) Figure S4 Mass spectrometry (MALDI-TOF) analysis of recombinant, hexahistidine tagged NiV PNT purified from the soluble fraction of E. coli. Mass analysis was performed using an Autoflex II TOF/TOF. Spectra were acquired in the linear mode. The sample (0.7 mL containing 15 pmol) was mixed with an equal volume of sinapinic acid matrix solution, spotted on the target, then dried at room temperature for 10 min. The mass standard was BSA. Proteins were analyzed in the Autoflex matrix-assisted laser desorption ionization/time of flight (Bruker Daltonics, Bremen, Germany). A major peak with a mass slightly higher (45 440 Da) than expected (45 330 Da) was observed. The additional peak (22 696 Da) very probably corresponds to a degradation product, as the protein was found to contain no contaminating proteins (as judged based by mass spectrometry analysis of tryptic fragments). Found at: doi:10.1371/journal.pone.0011684.s004 (0.06 MB DOC) Figure S5 Mass spectrometry (MALDI-TOF) analysis of recombinant, hexahistidine tagged HeV PNT purified from the soluble fraction of E. coli. Mass analysis was performed using an Autoflex II TOF/TOF. Spectra were acquired in the linear mode. The sample (0.7 mL containing 15 pmol) was mixed with an equal volume of sinapinic acid matrix solution, spotted on the target, then dried at room temperature for 10 min. The mass standard was BSA. Proteins were analyzed in the Autoflex matrix-assisted laser desorption ionization/time of flight (Bruker Daltonics, Bremen, Germany). A major peak with a mass is slightly higher (45 342 Da) than expected (45 216 Da) was observed. The additional peak (22626 Da) very probably corresponds to a degradation product, as the protein was found to contain no contaminating proteins (as judged based by mass spectrometry analysis of tryptic fragments).