Valérie Belle

Mapping α‐helical induced folding within the intrinsically disordered C‐terminal domain of the measles virus nucleoprotein by site‐directed spin‐labeling EPR spectroscopy

Using site‐directed spin‐labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C‐terminal domain of measles virus nucleoprotein (NTAIL) that undergoes induced folding. In addition to four spin‐labeled NTAIL variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596‐20608), 10 new single‐site cysteine variants were designed, purified from E. coli, and spin‐labeled. These 14 spin‐labeled variants enabled us to map in detail the gain of rigidity of NTAIL in the presence of either the secondary structure stabilizer 2,2,2‐trifluoroethanol or the C‐terminal domain X (XD) of the viral phosphoprotein. Different regions of NTAIL were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488–502 and the 505–522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488–502, the NTAIL region undergoing α‐helical folding. The mobility of the 488–502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505–522 region upon binding to XD is due to the α‐helical transition occurring within the 488–502 region and not to a direct interaction with XD. Proteins 2008.

Assessing induced folding within the intrinsically disordered C-terminal domain of the Henipavirus nucleoproteins by site-directed spin labeling EPR spectroscopy

This work aims at characterizing structural transitions within the intrinsically disordered C-terminal domain of the nucleoprotein (NTAIL) from the Nipah and Hendra viruses, two recently emerged pathogens gathered within the Henipavirus genus. To this end, we used site-directed spin labeling combined with electron paramagnetic resonance spectroscopy to investigate the α-helical-induced folding that Henipavirus NTAIL domains undergo in the presence of the C-terminal X domain of the phosphoprotein (PXD). For each NTAIL protein, six positions located within four previously proposed molecular recognition elements (MoREs) were targeted for spin labeling, with three of these positions (475, 481, and 487) falling within the MoRE responsible for binding to PXD (Box3). A detailed analysis of the impact of the partner protein on the labeled NTAIL variants revealed a dramatic modification in the environment of the spin labels grafted within Box3, with the observed modifications supporting the formation of an induced α-helix within this region. In the free state, the slightly lower mobility of the spin labels grafted within Box3 as compared to the other positions suggests the existence of a transiently populated α-helix, as already reported for measles virus (MeV) NTAIL. Comparison with the well-characterized MeV NTAIL–PXD system, allowed us to validate the structural models of Henipavirus NTAIL–PXD complexes that we previously proposed. In addition, this study highlighted a few notable differences between the Nipah and Hendra viruses. In particular, the observation of composite spectra for the free form of the Nipah virus NTAIL variants spin labeled in Box3 supports conformational heterogeneity of this partly pre-configured α-helix, with the pre-existence of stable α-helical segments. Altogether these results provide insights into the molecular mechanisms of the Henipavirus NTAIL–PXD binding reaction.

Probing structural transitions in both structured and disordered proteins using site-directed spin-labeling EPR spectroscopy ‡

EPR spectroscopy is a technique that specifically detects unpaired electrons. EPR‐sensitive reporter groups (spin labels or spin probes) can be introduced into biological systems via site‐directed spin‐labeling (SDSL). The basic strategy of SDSL involves the introduction of a paramagnetic group at a selected protein site. This is usually accomplished by cysteine‐substitution mutagenesis, followed by covalent modification of the unique sulfydryl group with a selective reagent bearing a nitroxide radical. In this review we briefly describe the theoretical principles of this well‐established approach and illustrate how we successfully applied it to investigate structural transitions in both human pancreatic lipase (HPL), a protein with a well‐defined α/β hydrolase fold, and the intrinsically disordered C‐terminal domain of the measles virus nucleoprotein (NTAIL) upon addition of ligands and/or protein partners. In both cases, SDSL EPR spectroscopy allowed us to document protein conformational changes at the residue level. The studies herein summarized show that this approach is not only particularly well‐suited to study IDPs that inherently escape atomistic description by X‐ray crystallography but also provides dynamic information on structural transitions occurring within well‐characterized structured proteins for which X‐ray crystallography can only provide snapshots of the initial and final stages. Copyright

Lid opening and unfolding in human pancreatic lipase at low pH revealed by site-directed spin labeling EPR and FTIR spectroscopy.

The structural changes induced in human pancreatic lipase (HPL) by lowering the pH were investigated using a combined approach involving the use of site-directed spin labeling coupled to electron paramagnetic resonance (SDSL-EPR) and Fourier transform infrared (ATR-FTIR) spectroscopy. The secondary structure of HPL observed with ATR-FTIR spectroscopy was found to be stable in the pH range of 3.0-6.5, where HPL remained active. Using a spin-label introduced into the lid of HPL at position 249, a reversible opening of the lid controlling the access to the active site was observed by EPR spectroscopy in the pH range of 3.0-5.0. In the same pH range, some structural changes were also found to occur outside the lid in a peptide stretch located near catalytic aspartate 176, using a spin-label introduced at position 181. Below pH 3.0, ATR-FTIR measurements indicated that HPL had lost most of its secondary structure. At these pH levels, the loss of enzyme activity was irreversible and the ability of HPL to bind to lipid emulsions was abolished. The EPR spectrum of the spin-label introduced at position 181, which was typical of a spin-label having a high mobility, confirmed the drastic structural change undergone by HPL in this particular region. The EPR spectrum of the spin-label at position 249 indicated, however, that the environment of this residue within the lid was not affected at pH 3.0 in comparison with that observed in the pH range of 3.0-5.0. This finding suggests that the disulfide bridge between the hinges of the lid kept the secondary structure of the lid intact, whereas the HPL was completely unfolded.

Enlarging the Panoply of Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR): Sensitive and Selective Spin-Labeling of Tyrosine Using an Isoindoline-Based Nitroxide

Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical spin labels are functionalized to be grafted on natural or site-directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 with its partner protein, we showed that the isoindoline-based nitroxide is a good reporter to reveal changes in its local environment contrary to the previous study where the label was poorly sensitive to probe structural changes. The successful targeting of tyrosine residues with the isoindoline-based nitroxide thus offers a highly promising approach, complementary to the classical cysteine-SDSL one, which significantly enlarges the field of applications of the technique for probing protein dynamics.

Tyrosine‐Targeted Spin Labeling and EPR Spectroscopy: An Alternative Strategy for Studying Structural Transitions in Proteins

Such a difficulty was recently encountered in the study of a small and flexible chloroplast protein CP12 from the green alga Chlamydomonas reinhardtii by spin-labeling EPR spectroscopy. In this organism, CP12 contains four cysteine residues involved in two disulfide bridges in its oxidized state. Although the introduction of spin labels at the two cysteine residues of the C-terminal disulfide bridge enabled us to identify a new role of the partner protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH), it precluded the direct study of the complex formation GAPDH/CP12. [4] To overcome this difficulty, grafting of the nitroxide probe to residues other than cysteines is required. One strategy using a genetically encoded unnatural amino acid has been recently proposed. [5] The incorporation of such unnatural amino acids relies, however, on a rather complex strategy involving an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid. As such a strategy is difficult to set up, we propose an alternative method consisting of selectively targeting residues other than cysteines with a nitroxide probe. Bioconjugation of small molecules to protein residues is very challenging, and several reactions have recently been proposed to target specific residues selectively. [6] In particular, efforts have been paid to modify the aromatic amino acid side chains of tryptophan [7] and tyrosine. [8] Among them, a threecomponent Mannich-type reaction has been developed that allows the modification of tyrosine under mild, biocompatible, and metal-free conditions. [8a] Inspired by these recent studies, we present the selective grafting of a nitroxide probe to tyrosine by using the Mannich-type reaction on CP12, a protein bearing only one natural tyrosine residue. This unique tyrosine residue, located at position 78 in the sequence of a total of 80 amino acids, makes this protein an ideal candidate for demonstrating the feasibility of tyrosine-targeted spin

Dynamics of the intrinsically disordered protein CP12 in its association with GAPDH in the green alga Chlamydomonas reinhardtii: a fuzzy complex

CP12 is a widespread regulatory protein of oxygenic photosynthetic organisms that contributes to the regulation of the Calvin cycle by forming a supra-molecular complex with at least two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). CP12 shares some similarities with intrinsically disordered proteins (IDPs) depending on its redox state. In this study, site-directed spin labeling (SDSL) combined with EPR spectroscopy was used to probe the dynamic behavior of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH, the first step towards ternary complex formation. The two N-terminal cysteine residues were labeled using the classical approach while the tyrosine located at the C-terminal end of CP12 was modified following an original procedure. The results show that the label grafted at the C-terminal extremity is in the vicinity of the interaction site whereas the N-terminal region remains fully disordered upon binding to GAPDH. In conclusion, GAPDH-CP12 is a fuzzy complex, in which the N-terminal region of CP12 keeps a conformational freedom in the bound form. This fuzziness could be one of the keys to facilitate binding of PRK to CP12-GAPDH and to form the ternary supra-molecular complex.

The interaction between the measles virus nucleoprotein and the Interferon Regulator Factor 3 relies on a specific cellular environment

BackgroundThe genome of measles virus consists of a non-segmented single-stranded RNA molecule of negative polarity, which is encapsidated by the viral nucleoprotein (N) within a helical nucleocapsid. The N protein possesses an intrinsically disordered C-terminal domain (aa 401–525, NTAIL) that is exposed at the surface of the viral nucleopcapsid. Thanks to its flexible nature, NTAIL interacts with several viral and cellular partners. Among these latter, the Interferon Regulator Factor 3 (IRF-3) has been reported to interact with N, with the interaction having been mapped to the regulatory domain of IRF-3 and to NTAIL. This interaction was described to lead to the phosphorylation-dependent activation of IRF-3, and to the ensuing activation of the pro-immune cytokine RANTES gene.ResultsAfter confirming the reciprocal ability of IRF-3 and N to be co-immunoprecipitated in 293T cells, we thoroughly investigated the NTAIL-IRF-3 interaction using a recombinant, monomeric form of the regulatory domain of IRF-3. Using a large panel of spectroscopic approaches, including circular dichroism, fluorescence spectroscopy, nuclear magnetic resonance and electron paramagnetic resonance spectroscopy, we failed to detect any direct interaction between IRF-3 and either full-length N or NTAIL under conditions where these latter interact with the C-terminal X domain of the viral phosphoprotein. Furthermore, such interaction was neither detected in E. coli nor in a yeast two hybrid assay.ConclusionAltogether, these data support the requirement for a specific cellular environment, such as that provided by 293T human cells, for the NTAIL-IRF-3 interaction to occur. This dependence from a specific cellular context likely reflects the requirement for a human or mammalian cellular co-factor.

Amplitude of Pancreatic Lipase Lid Opening in Solution and Identification of Spin Label Conformational Subensembles by Combining Continuous Wave and Pulsed EPR Spectroscopy and Molecular Dynamics

The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two spin labels grafted on this enzyme. One spin label was introduced at a rigid position in HPL where an accessible cysteine residue (C181) naturally occurs. A second spin label was covalently bound to the mobile lid after introducing a cysteine residue at position 249 by site-directed mutagenesis. Double electron-electron resonance (DEER) experiments allowed the estimation of a distance of 19 +/- 2 A between the spin labels when bilabeled HPL was alone in a frozen solution, i.e., with the lid in the closed conformation. A magnetic interaction was however detected by continuous wave EPR experiments, suggesting that a fraction of bilabeled HPL contained spin labels separated by a shorter distance. These results could be interpreted by the presence of two conformational subensembles for the spin label lateral chain at position 249 when the lid was closed. The existence of these conformational subensembles was revealed by molecular dynamics experiments and confirmed by the simulation of the EPR spectrum. When the lid opening was induced by the addition of bile salts and colipase, a larger distance of 43 +/- 2 A between the two spin labels was estimated from DEER experiments. The distances measured between the spin labels grafted at positions 181 and 249 were in good agreement with those estimated from the known X-ray structures of HPL in the closed and open conformations, but for the first time, the amplitude of the lid opening was measured in solution or in a frozen solution in the presence of amphiphiles.

Conformational Selection Underlies Recognition of a Molybdoenzyme by Its Dedicated Chaperone

Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism.