Frank Gabel

Protein dynamics studied by neutron scattering

This review of protein dynamics studied by neutron scattering focuses on data collected in the last 10 years. After an introduction to thermal neutron scattering and instrumental aspects, theoretical models that have been used to interpret the data are presented and discussed. Experiments are described according to sample type, protein powders, solutions and membranes. Neutron-scattering results are compared to those obtained from other techniques. The biological relevance of the experimental results is discussed. The major conclusion of the last decade concerns the strong dependence of internal dynamics on the macromolecular environment.

Intrinsic disorder in measles virus nucleocapsids

The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. The intrinsically disordered C-terminal domain of N (NTAIL) is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. The molecular recognition element (MoRE) of NTAIL that binds P is situated 90 amino acids from the folded RNA-binding domain (NCORE) of N, raising questions about the functional role of this disordered chain. Here we report the first in situ structural characterization of NTAIL in the context of the entire N-RNA capsid. Using nuclear magnetic resonance spectroscopy, small angle scattering, and electron microscopy, we demonstrate that NTAIL is highly flexible in intact nucleocapsids and that the MoRE is in transient interaction with NCORE. We present a model in which the first 50 disordered amino acids of NTAIL are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive NCORE helical turns. The model provides a structural framework for understanding the role of NTAIL in the initiation of viral transcription and replication, placing the flexible MoRE close to the viral RNA and, thus, positioning the polymerase complex in its functional environment.

Coupling of protein and hydration-water dynamics in biological membranes

The dynamical coupling between proteins and their hydration water is important for the understanding of macromolecular function in a cellular context. In the case of membrane proteins, the environment is heterogeneous, composed of lipids and hydration water, and the dynamical coupling might be more complex than in the case of the extensively studied soluble proteins. Here, we examine the dynamical coupling between a biological membrane, the purple membrane (PM), and its hydration water by a combination of elastic incoherent neutron scattering, specific deuteration, and molecular dynamics simulations. Examining completely deuterated PM, hydrated in H2O, allowed the direct experimental exploration of water dynamics. The study of natural abundance PM in D2O focused on membrane dynamics. The temperature-dependence of atomic mean-square displacements shows inflections at 120 K and 260 K for the membrane and at 200 K and 260 K for the hydration water. Because transition temperatures are different for PM and hydration water, we conclude that ps–ns hydration water dynamics are not directly coupled to membrane motions on the same time scale at temperatures <260 K. Molecular-dynamics simulations of hydrated PM in the temperature range from 100 to 296 K revealed an onset of hydration-water translational diffusion at ≈200 K, but no transition in the PM at the same temperature. Our results suggest that, in contrast to soluble proteins, the dynamics of the membrane protein is not controlled by that of hydration water at temperatures <260 K. Lipid dynamics may have a stronger impact on membrane protein dynamics than hydration water.

Translational diffusion of hydration water correlates with functional motions in folded and intrinsically disordered proteins

Hydration water is the natural matrix of biological macromolecules and is essential for their activity in cells. The coupling between water and protein dynamics has been intensively studied, yet it remains controversial. Here we combine protein perdeuteration, neutron scattering and molecular dynamics simulations to explore the nature of hydration water motions at temperatures between 200 and 300 K, across the so-called protein dynamical transition, in the intrinsically disordered human protein tau and the globular maltose binding protein. Quasi-elastic broadening is fitted with a model of translating, rotating and immobile water molecules. In both experiment and simulation, the translational component markedly increases at the protein dynamical transition (around 240 K), regardless of whether the protein is intrinsically disordered or folded. Thus, we generalize the notion that the translational diffusion of water molecules on a protein surface promotes the large-amplitude motions of proteins that are required for their biological activity.

The structure of the box C/D enzyme reveals regulation of RNA methylation

Post-transcriptional modifications are essential to the cell life cycle, as they affect both pre-ribosomal RNA processing and ribosome assembly. The box C/D ribonucleoprotein enzyme that methylates ribosomal RNA at the 2′-O-ribose uses a multitude of guide RNAs as templates for the recognition of rRNA target sites. Two methylation guide sequences are combined on each guide RNA, the significance of which has remained unclear. Here we use a powerful combination of NMR spectroscopy and small-angle neutron scattering to solve the structure of the 390 kDa archaeal RNP enzyme bound to substrate RNA. We show that the two methylation guide sequences are located in different environments in the complex and that the methylation of physiological substrates targeted by the same guide RNA occurs sequentially. This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding.

Neutron scattering reveals extremely slow cell water in a Dead Sea organism

Intracellular water dynamics in Haloarcula marismortui, an extremely halophilic organism originally isolated from the Dead Sea, was studied by neutron scattering. The water in centrifuged cell pellets was examined by means of two spectrometers, IN6 and IN16, sensitive to motions with time scales of 10 ps and 1 ns, respectively. From IN6 data, a translational diffusion constant of 1.3 × 10−5 cm2 s−1 was determined at 285 K. This value is close to that found previously for other cells and close to that for bulk water, as well as that of the water in the 3.5 M NaCl solution bathing the cells. A very slow water component was discovered from the IN16 data. At 285 K the water-protons of this component displays a residence time of 411 ps (compared with a few ps in bulk water). At 300 K, the residence time dropped to 243 ps and was associated with a translational diffusion of 9.3 × 10−8 cm2 s−1, or 250 times lower than that of bulk water. This slow water accounts for ≈76% of cell water in H. marismortui. No such water was found in Escherichia coli measured on BSS, a neutron spectrometer with properties similar to those of IN16. It is hypothesized that the slow mobility of a large part of H. marismortui cell water indicates a specific water structure responsible for the large amounts of K+ bound within these extremophile cells.

Structural basis for the assembly of the Sxl-Unr translation regulatory complex

Genetic equality between males and females is established by chromosome-wide dosage-compensation mechanisms. In the fruitfly Drosophila melanogaster, the dosage-compensation complex promotes twofold hypertranscription of the single male X-chromosome and is silenced in females by inhibition of the translation of msl2, which codes for the limiting component of the dosage-compensation complex. The female-specific protein Sex-lethal (Sxl) recruits Upstream-of-N-ras (Unr) to the 3′ untranslated region of msl2 messenger RNA, preventing the engagement of the small ribosomal subunit. Here we report the 2.8 Å crystal structure, NMR and small-angle X-ray and neutron scattering data of the ternary Sxl–Unr–msl2 ribonucleoprotein complex featuring unprecedented intertwined interactions of two Sxl RNA recognition motifs, a Unr cold-shock domain and RNA. Cooperative complex formation is associated with a 1,000-fold increase of RNA binding affinity for the Unr cold-shock domain and involves novel ternary interactions, as well as non-canonical RNA contacts by the α1 helix of Sxl RNA recognition motif 1. Our results suggest that repression of dosage compensation, necessary for female viability, is triggered by specific, cooperative molecular interactions that lock a ribonucleoprotein switch to regulate translation. The structure serves as a paradigm for how a combination of general and widespread RNA binding domains expands the code for specific single-stranded RNA recognition in the regulation of gene expression.

NMR and small-angle scattering-based structural analysis of protein complexes in solution.

Structural analysis of multi-domain protein complexes is a key challenge in current biology and a prerequisite for understanding the molecular basis of essential cellular processes. The use of solution techniques is important for characterizing the quaternary arrangements and dynamics of domains and subunits of these complexes. In this respect solution NMR is the only technique that allows atomic- or residue-resolution structure determination and investigation of dynamic properties of multi-domain proteins and their complexes. As experimental NMR data for large protein complexes are sparse, it is advantageous to combine these data with additional information from other solution techniques. Here, the utility and computational approaches of combining solution state NMR with small-angle X-ray and Neutron scattering (SAXS/SANS) experiments for structural analysis of large protein complexes is reviewed. Recent progress in experimental and computational approaches of combining NMR and SAS are discussed and illustrated with recent examples from the literature. The complementary aspects of combining NMR and SAS data for studying multi-domain proteins, i.e. where weakly interacting domains are connected by flexible linkers, are illustrated with the structural analysis of the tandem RNA recognition motif (RRM) domains (RRM1-RRM2) of the human splicing factor U2AF65 bound to a nine-uridine (U9) RNA oligonucleotide.

From shell to cell: neutron scattering studies of biological water dynamics and coupling to activity.

An integrated picture of hydration shell dynamics and of its coupling to functional macromolecular motions is proposed from studies on a soluble protein, on a membrane protein in its natural lipid environment, and on the intracellular environment in bacteria and red blood cells. Water dynamics in multimolar salt solutions was also examined, in the context of the very slow water component previously discovered in the cytoplasm of extreme halophilic archaea. The data were obtained from neutron scattering by using deuterium labelling to focus on the dynamics of different parts of the complex systems examined.

The Low-Temperature Inflection Observed in Neutron Scattering Measurements of Proteins Is Due to Methyl Rotation: Direct Evidence Using Isotope Labeling and Molecular Dynamics Simulations

There is increasing interest in the contribution of methyl groups to the overall dynamics measured by neutron scattering experiments of proteins. In particular an inflection observed in atomic mean square displacements measured as a function of temperature on high resolution spectrometers (approximately 1 microeV) was explained by the onset of methyl group rotations. By specifically labeling a non-methyl-containing side-chain in a native protein system, the purple membrane, and performing neutron scattering measurements, we here provide direct experimental evidence that the observed inflection is indeed due to methyl group rotations. Molecular dynamics simulations reproduce the experimental data, and their analysis suggests that the apparent transition is due to methyl group rotation entering the finite instrumental resolution of the spectrometer. Methyl group correlation times measured by solid state NMR in the purple membrane, taken from previous work, support the interpretation.