Laurence Blanchard

Quantitative conformational analysis of partially folded proteins from residual dipolar couplings: application to the molecular recognition element of Sendai virus nucleoprotein.

A significant fraction of proteins coded in the human proteome do not fold into stable three-dimensional structures but are either partially or completely unfolded. A key feature of this family of proteins is their proposed capacity to undergo a disorder-to-order transition upon interaction with a physiological partner. The mechanisms governing protein folding upon interaction, in particular the extent to which recognition elements are preconfigured prior to formation of molecular complexes, can prove difficult to resolve in highly flexible systems. Here, we develop a conformational model of this type of protein, using an explicit description of the unfolded state, specifically modified to allow for the presence of transient secondary structure, and combining this with extensive measurement of residual dipolar couplings throughout the chain. This combination of techniques allows us to quantitatively analyze the level and nature of helical sampling present in the interaction site of the partially folded C-terminal domain of Sendai virus nucleoprotein (N(TAIL)). Rather than fraying randomly, the molecular recognition element of N(TAIL) preferentially populates three specific overlapping helical conformers, each stabilized by an N-capping interaction. The unfolded strands adjacent to the helix are thereby projected in the direction of the partner protein, identifying a mechanism by which they could achieve nonspecific encounter interactions prior to binding. This study provides experimental evidence for the molecular basis of helix formation in partially folded peptide chains, carrying clear implications for understanding early steps of protein folding.

The protein moiety modulates the redox potential in cytochromes c

Cytochrome c is one of the most thoroughly documented oxidoreduction proteins. Its electron transfer activity, which involves an association between the heme group and the polypeptidic chain, is correlated with the redox potential value of the heme group. The redox potential covers a wide range up to 0.8 V, an extreme case being observed in the low-potential cytochromes c from sulfate reducing bacteria. On of the main roles of the polypeptidic moiety consists of modulating the redox potential value of the heme group. In this paper, some structural factors that seem likely to be involved in maintaining the redox potential value are described.

Structural Disorder within Sendai Virus Nucleoprotein and Phosphoprotein: Insight into the Structural Basis of Molecular Recognition

Intrinsically disordered regions of significant length are present throughout eukaryotic genomes, and are particularly prevalent in viral proteins. Due to their inherent flexibility, these proteins inhabit a conformational landscape that is too complex to be described by classical structural biology. The elucidation of the role that conformational flexibility plays in molecular function will redefine our understanding of the molecular basis of biological function, and the development of appropriate technology to achieve this aim remains one of the major challenges for the future of structural biology. NMR is the technique of choice for studying intrinsically disordered proteins, providing information about structure, flexibility and interactions at atomic resolution even in completely disordered proteins. In particular residual dipolar couplings (RDCs) are sensitive and powerful tools for determining local and long-range structural behaviour in flexible proteins. Here we describe recent applications of the use of RDCs to quantitatively describe the level of local structure in intrinsically disordered proteins involved in replication and transcription in Sendai virus.

Letter to the Editor: Assignment of the 1H, 15N and 13C resonances of the nucleocapsid-binding domain of the Sendai virus Phosphoprotein

Dominique Mariona,∗, Nicolas Tarbouriechb, Rob W.H. Ruigrokc,d, Wilhelm P. Burmeisterb,d & Laurence Blancharda aInstitut de Biologie Structurale ‘Jean-Pierre Ebel’ (UMR 5075 CEA-CNRS-UJF), 41 rue Jules Horowitz, 38027 Grenoble cedex 1, France; bESRF, BP 220, 38043 Grenoble cedex 9, France; cEMBL, Grenoble Outstation, B.P. 181, 38042 Grenoble cedex 9, France; dLaboratoire de Virologie Moleculaire et Structurale, EA 2939, Faculte de Medecine de Grenoble, 38700 La Tronche, France

Intramolecular electron transfer in ferredoxin II from Desulfovibrio desulfuricans Norway.

In order to elucidate the role of the two (4Fe-4S) clusters in ferredoxins and to determine whether an electron-transfer mechanism may occur between the clusters, the in vitro reduction of cytochrome c3 and cytochrome c553 by Desulfovibrio desulfuricans Norway ferredoxin II was studied using spectrophotometric techniques. Ferredoxin II, covalently cross-linked with either cytochrome c3 or c553, is an obligate intermediate in cytochrome reduction by pyruvate dehydrogenase. Both titration of the complex formation under 1H-NMR spectroscopy and cross-linking experiments between ferredoxin II and either cytochrome c3 or cytochrome c553 gave a stoichiometric ratio of 1:1. Modelling the protein yielded differences between the charge distributions around the two (Fe-S) clusters. The fact that Cluster 2 is blocked in the electron-transfer domain facing the cytochrome interacting heme, indicates Cluster 1 receives electron from pyruvate dehydrogenase. Consecutively, cytochrome reduction occurs owing to an intramolecular electron exchange between the two clusters of the ferredoxin. The properties of two (Fe-S) cluster ferredoxins are compared to those of monocluster ferredoxins and discussed in evolutionary terms.

Investigation of oxidation state-dependent conformational changes in Desulfovibrio vulgaris Hildenborough cytochrome C553 by two-dimensional 1H-NMR spectra

Two‐dimensional nuclear magnetic resonance spectroscopy (2D‐NMR) was used to assign the proton resonances of ferricytochrome C 553 from Desulfovibrio vulgaris Hildenborough. The spin systems of 76 out of 79 amino acids were identified by J‐correlation spectroscopy (COSY and HOHAHA) in H2O and D2O and correlated by nuclear Overhauser effect spectroscopy (NOESY). The proton chemical shifts are compared in both oxidized and reduced states of the protein at 23°C and pH 5.9. Chemical shift variations between reduced and oxidized states are due to the paramagnetic contribution. Medium and longrange nOe demonstrate the lack of major changes between the two redox states. NMR data provide evidence that in this low oxidoreduction potential cytochrome, the oxidized state is more rigid than the reduced state.

Parallel screening and optimization of protein constructs for structural studies.

A major challenge in structural biology remains the identification of protein constructs amenable to structural characterization. Here, we present a simple method for parallel expression, labeling, and purification of protein constructs (up to 80 kDa) combined with rapid evaluation by NMR spectroscopy. Our approach, which is equally applicable for manual or automated implementation, offers an efficient way to identify and optimize protein constructs for NMR or X‐ray crystallographic investigations.

New conformational properties induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough ferricytochrome c553 using isotopic exchanges monitored by mass spectrometry

In order to study the conformational stability induced by the replacement of Tyr‐64 in Desulfovibrio vulgaris Hildenborough (DvH) cytochrome c 553, fast peptic digestion of deuterated protein followed by separation and measurement of related peptides using liquid chromatography coupled to electrospray ionization mass spectrometry was performed. We show that the H‐bonding and/or solvent accessibility properties were modified by the single‐site mutation. The mutant proteins can be classified into two groups: the Y64F and Y64L mutants with nearly unchanged deuterium incorporation compared to the wild‐type protein and the Y64S, Y64V and Y64A mutants with increased deuterium incorporation. The 70–74 peptide was the most affected by mutation of Tyr‐64, the phenylalanine mutant inducing slight stabilization whereas the serine mutant was significantly destabilized. In addition, from the analysis of the overlapping 37–57 and 38–57 peptides we can conclude that the amide proton of Tyr‐38 has been replaced by deuterium in all proteins.