Carine van Heijenoort

Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX

The atomic structure of OmpX, the smallest member of the bacterial outer membrane protein family, has been previously established by X-ray crystallography and NMR spectroscopy. In apparent conflict with electrophysiological studies, the lumen of its transmembrane β-barrel appears too tightly packed with amino acid side chains to let any solute flow through. In the present study, high-resolution solution NMR spectra were obtained of OmpX kept water-soluble by either amphipol A8-35 or the detergent dihexanoylphosphatidylcholine. Hydrogen/deuterium exchange measurements performed after prolonged equilibration show that, whatever the surfactant used, some of the amide protons of the membrane-spanning region exchange much more readily than others, which likely reflects the dynamics of the barrel.

Inter- and intramolecular contacts in a membrane protein/surfactant complex observed by heteronuclear dipole-to-dipole cross-relaxation.

Heteronuclear dipole-to-dipole cross-relaxation has been applied to exploring intermolecular interactions and intramolecular spatial proximities in a large supramolecular structure comprised of a beta-barrel membrane protein, OmpX, in complex with a polymeric surfactant, amphipol A8-35. The experiments, performed in either the laboratory or the rotating frame, reveal the existence of intermolecular contacts between aromatic amino acids and specific groups of the polymer, in addition to intra-protein dipolar interactions, some of them involving carbonyl carbons. This study opens the perspective of collecting by NMR spectroscopy a new kind of through-space structural information involving aromatic and carbonyl (13)C atoms of large proteins.

Structural study of the membrane protein MscL using cell-free expression and solid-state NMR.

High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75kDa pentameric alpha-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.

Multifunctionality of the β‐thymosin/WH2 module: G‐actin sequestration, actin filament growth, nucleation, and severing

The β‐thymosin/WH2 actin‐binding module shows an amazing adaptation to multifunctionality. The β‐thymosins are genuine G‐actin sequesterers of moderate affinity for G‐actin, allowing an efficient regulation of the G‐actin/F‐actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first β‐thymosin domain of the protein Ciboulot makes with G‐actin a complex that supports filament growth, such as profilin–actin. We illustrate how the use of engineered chimeric proteins, actin‐binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G‐actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon‐Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G‐actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.

Structure, Function, and Evolution of the β‐Thymosin/WH2 (WASP‐Homology2) Actin‐Binding Module

Abstract:  β‐thymosins are acknowledged G‐actin sequesterers. However, in the recent years, the conserved β‐thymosins/WH2 actin‐binding module, has been identified in a large number of proteins that all interact with actin and play diverse functions in cell motility. The functional evolution of the WH2 domain has been approached by a combination of structural and biochemical methods, using thymosin β4 (Tβ4) and Ciboulot, a 3 β‐thymosin repeat protein from Drosophila as models. Ciboulot binds actin like Tβ4 but promotes actin assembly like profilin. The first repeat of Ciboulot (D1) has the profilin function of the whole protein. The crystal structure of Ciboulot‐actin shows that the major interaction with G‐actin lies in the N‐terminal amphipathic helix of D1. By point mutagenesis the sequestering activity of Tβ4 can be changed into a profilin activity. (1H, 15N)‐NMR studies show that the functional switch from inhibition to promotion of actin assembly is linked to a change in the dynamics of interaction of the central and C‐terminal regions of the WH2 domain with subdomains 1 and 2 of G‐actin. Further systematic mutagenesis studies have been performed by engineering a series of chimeras of Ciboulot and Tβ4. Proteins displaying either profilin function or enhanced sequestering activity compared to Tβ4 have been characterized. The results provide insight into the structural basis for the regulation of the multiple functions of the WH2 domain.

Robust structure-based resonance assignment for functional protein studies by NMR

High-throughput functional protein NMR studies, like protein interactions or dynamics, require an automated approach for the assignment of the protein backbone. With the availability of a growing number of protein 3D structures, a new class of automated approaches, called structure-based assignment, has been developed quite recently. Structure-based approaches use primarily NMR input data that are not based on J-coupling and for which connections between residues are not limited by through bonds magnetization transfer efficiency. We present here a robust structure-based assignment approach using mainly HN–HN NOEs networks, as well as 1H–15N residual dipolar couplings and chemical shifts. The NOEnet complete search algorithm is robust against assignment errors, even for sparse input data. Instead of a unique and partly erroneous assignment solution, an optimal assignment ensemble with an accuracy equal or near to 100% is given by NOEnet. We show that even low precision assignment ensembles give enough information for functional studies, like modeling of protein-complexes. Finally, the combination of NOEnet with a low number of ambiguous J-coupling sequential connectivities yields a high precision assignment ensemble. NOEnet will be available under: http://www.icsn.cnrs-gif.fr/download/nmr.

NOEnet–Use of NOE networks for NMR resonance assignment of proteins with known 3D structure

Motivation: A prerequisite for any protein study by NMR is the assignment of the resonances from the 15N−1H HSQC spectrum to their corresponding atoms of the protein backbone. Usually, this assignment is obtained by analyzing triple resonance NMR experiments. An alternative assignment strategy exploits the information given by an already available 3D structure of the same or a homologous protein. Up to now, the algorithms that have been developed around the structure-based assignment strategy have the important drawbacks that they cannot guarantee a high assignment accuracy near to 100%. Results: We propose here a new program, called NOEnet, implementing an efficient complete search algorithm that ensures the correctness of the assignment results. NOEnet exploits the network character of unambiguous NOE constraints to realize an exhaustive search of all matching possibilities of the NOE network onto the structural one. NOEnet has been successfully tested on EIN, a large protein of 28 kDa, using only NOE data. The complete search of NOEnet finds all possible assignments compatible with experimental data that can be defined as an assignment ensemble. We show that multiple assignment possibilities of large NOE networks are restricted to a small spatial assignment range (SAR), so that assignment ensembles, obtained from accessible experimental data, are precise enough to be used for functional proteins studies, like protein–ligand interaction or protein dynamics studies. We believe that NOEnet can become a major tool for the structure-based backbone resonance assignment strategy in NMR. Availability: The NOEnet program will be available under: http://www.icsn.cnrs-gif.fr/download/nmr Contact: carine@icsn.cnrs-gif.fr; eric.guittet@icsn.cnrs-gif.fr Supplementary Information: Supplementary data are available at Bioinformatics online.

NMR structure note: oxidized microsomal human cytochrome b5

Cytochrome b5 (cytb5) is a small membrane-bound hemoprotein present in all eukaryotic organisms. In most eukaryotic cells, cytb5 is attached to the cytosolic face of the endoplasmic reticulum and is described as the microsomal form (Mc). In vertebrates, two supplementary cytb5 can be found: soluble in the erythrocytes and membrane-bound attached to the internal face of the outer membrane in the mitochondria (OM) (Wang et al. 2007). Cytb5 can be divided into three domains: an N-terminal hydrosoluble globular domain that contains approximately 90 residues, a C-terminal hydrophobic domain about 25 residues long and a linker between the two above-mentioned domains. The hydrophilic domain contains the redox center, an iron protoporphyrin IX, ligated to the apoprotein by two histidyl residues. Mc cytb5 transfers electrons to various acceptors and is consequently involved in many different metabolic pathways. In the mixed function oxidase system, Mc cytb5 is a facultative electron donor to cytochromes P450 (Vergeres and Waskell 1995). Several mechanisms for the recognition between cytb5 and its physiological or artificial acceptors have been hypothesized. The implication of electrostatic interactions has been mostly studied with the cytochromes P450 as acceptors and is now widely accepted as a dominant factor (Schenkman et al. 1994). Nonetheless, several other factors may contribute positively to the recognition mechanism. First, the soluble domain of cytb5 lacking its C-terminal membrane domain cannot transfer electrons to membrane electron acceptors like cytochrome P450 (Dailey and Strittmatter 1978) while the full-length cytb5 can transfer electrons to both soluble and membranebound electron acceptor proteins (Vergeres and Waskell 1995). The linker is also recognized as an important factor controlling cytb5 to P450 interactions (Clarke et al. 2004). The three dimensional structures of the oxidized or reduced soluble domain of wild-type and mutant cytb5 from different species have been determined, either by X-ray or NMR. So far, no three dimensional structure of the full length protein has been reported, although some solid-state NMR data of the full-length rabbit cytb5 inserted in bicelles was Electronic supplementary material The online version of this article (doi:10.1007/s10858-010-9428-6) contains supplementary material, which is available to authorized users.

1 H, 13 C and 15 N resonance assignment of the first N-terminal RNA recognition motif (RRM) of the human heterogeneous nuclear ribonucleoprotein H (hnRNP H)

Human heterogeneous nuclear ribonucleoprotein H (hnRNP H) regulates alternative splicing of HIV-1 Tat pre-mRNA. The structure of the first N-terminal domain (residues 1–104) of hnRNP H was solved and its binding to an exonic splicing silencer (pESS2) studied. For this, all backbone and 85% of side-chain resonance frequencies were assigned.