Visible spectroscopy in the DIII-D divertor

Spectroscopy measurements in the DIII-D divertor have been carried out with a survey spectrometer which provides simultaneous registration of the visible spectrum over the region 400--900 nm with a resolution of 0.2 nm. Broad spectral coverage is achieved through use of a fiberoptic transformer assembly to map the curved focal plane of a fast (f/3) Rowland-circle spectrograph into a rastered format on the rectangular sensor area of a two-dimensional CCD camera. Vertical grouping of pixels during CCD readout integrates the signal intensity over the height of each spectral segment in the rastered image, minimizing readout time. For the full visible spectrum, readout time is 50 ms. Faster response time (< 10 ms) may be obtained by selecting for readout just a small number of the twenty spectral segments in the image on the CCD. Simultaneous recording of low charge states of carbon, oxygen and injected impurities has yielded information about gas recycling and impurity behavior at the divertor strike points. Transport of lithium to the divertor region during lithium pellet injection has been studied, as well as cumulative deposition of lithium on the divertor targets from pellet injection over many successive discharges

Peptide exchange on MHC-I by TAPBPR is driven by a negative allostery release cycle.

Chaperones TAPBPR and tapasin associate with class I major histocompatibility complexes (MHC-I) to promote optimization (editing) of peptide cargo. Here, we use solution NMR to investigate the mechanism of peptide exchange. We identify TAPBPR-induced conformational changes on conserved MHC-I molecular surfaces, consistent with our independently determined X-ray structure of the complex. Dynamics present in the empty MHC-I are stabilized by TAPBPR and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized according to the global stability of the final pMHC-I product and anneal in a native-like conformation to be edited by TAPBPR. Our results demonstrate an inverse relationship between MHC-I peptide occupancy and TAPBPR binding affinity, wherein the lifetime and structural features of transiently bound peptides control the regulation of a conformational switch located near the TAPBPR binding site, which triggers TAPBPR release. These results suggest a similar mechanism for the function of tapasin in the peptide-loading complex

Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome–nascent chain complexes

The translational diffusion of macromolecules can be examined non-invasively by stimulated echo (STE) NMR experiments to accurately determine their molecular sizes. These measurements can be important probes of intermolecular interactions and protein folding and unfolding, and are crucial in monitoring the integrity of large macromolecular assemblies such as ribosome–nascent chain complexes (RNCs). However, NMR studies of these complexes can be severely constrained by their slow tumbling, low solubility (with maximum concentrations of up to 10 μM), and short lifetimes resulting in weak signal, and therefore continuing improvements in experimental sensitivity are essential. Here we explore the use of the paramagnetic longitudinal relaxation enhancement (PLRE) agent NiDO2A on the sensitivity of 15N XSTE and SORDID heteronuclear STE experiments, which can be used to monitor the integrity of these unstable complexes. We exploit the dependence of the PLRE effect on the gyromagnetic ratio and electronic relaxation time to accelerate recovery of 1H magnetization without adversely affecting storage on N z during diffusion delays or introducing significant transverse relaxation line broadening. By applying the longitudinal relaxation-optimized SORDID pulse sequence together with NiDO2A to 70S Escherichia coli ribosomes and RNCs, NMR diffusion sensitivity enhancements of up to 4.5-fold relative to XSTE are achieved, alongside ~1.9-fold improvements in two-dimensional NMR sensitivity, without compromising the sample integrity. We anticipate these results will significantly advance the use of NMR to probe dynamic regions of ribosomes and other large, unstable macromolecular assemblies

Probing Microsecond Time Scale Dynamics in Proteins by Methyl 1H Carr−Purcell−Meiboom−Gill Relaxation Dispersion NMR Measurements. Application to Activation of the Signaling Protein NtrCr

To study microsecond processes by relaxation dispersion NMR spectroscopy, low power deposition and short pulses are crucial and encourage the development of experiments that employ H-1 Carr-Purcell-Meiboom-Gill (CPMG) pulse trains. Herein, a method is described for the comprehensive study of microsecond to millisecond time scale dynamics of methyl groups in proteins, exploiting their high abundance and favorable relaxation properties. In our approach, protein samples are produced using [H-1, C-13]-D-glucose in similar to 100% D2O, which yields CHD2 methyl groups for alanine, valine, threonine, isoleucine, leucine, and methionine residues with high abundance, in an otherwise largely deuterated background. Methyl groups in such samples can be sequence-specifically assigned to near completion, using C-13 TOCSY NMR spectroscopy, as was recently demonstrated (Often, R.; et al. J. Am. Chem. Soc. 2010, 132, 2952-2960). In this Article, NMR pulse schemes are presented to measure H-1 CPMG relaxation dispersion profiles for CHD2 methyl groups, in a vein similar to that of backbone relaxation experiments. Because of the high deuteration level of methyl-bearing side chains, artifacts arising from proton scalar coupling during the CPMG pulse train are negligible, with the exception of Ile-delta 1 and Thr-gamma 2 methyl groups, and a pulse scheme is described to remove the artifacts for those residues. Strong C-13 scalar coupling effects, observed for several leucine residues, are removed by alternative biochemical and NMR approaches. The methodology is applied to the transcriptional activator NtrC(r), for which an inactive/active state transition was previously measured and the motions in the microsecond time range were estimated through a combination of backbone N-15 CPMG dispersion NMR spectroscopy and a collection of experiments to determine the exchange-free component to the transverse relaxation rate. Exchange contributions to the H-1 line width were detected for 21 methyl groups, and these probes were found to collectively report on a local structural rearrangement around the phosphorylation site, with a rate constant of (15.5 +/- 0.5) x 10(3) per second (i.e., tau(ex) = 64.7 +/- 1.9 mu s). The affected methyl groups indicate that, already before phosphorylation, a substantial, transient rearrangement takes place between helices 3 and 4 and strands 4 and 5. This conformational equilibrium allows the protein to gain access to the active, signaling state in the absence of covalent modification through a shift in a pre-existing dynamic equilibrium. Moreover, the conformational switching maps exactly to the regions that differ between the solution NMR structures of the fully inactive and active states. These results demonstrate that a cost-effective and quantitative study of protein methyl group dynamics by H-1 CPMG relaxation dispersion NMR spectroscopy is possible and can be applied to study functional motions on the microsecond time scale that cannot be accessed by backbone N-15 relaxation dispersion NMR. The use of methyl groups as dynamics probes extends such applications also to larger proteins

Methyl group assignment using pseudocontact shifts with PARAssign

Macromolecular Biochemistr

Minimum Free Energy Path of Ligand-Induced Transition in Adenylate Kinase

Large-scale conformational changes in proteins involve barrier-crossing transitions on the complex free energy surfaces of high-dimensional space. Such rare events cannot be efficiently captured by conventional molecular dynamics simulations. Here we show that, by combining the on-the-fly string method and the multi-state Bennett acceptance ratio (MBAR) method, the free energy profile of a conformational transition pathway in Escherichia coli adenylate kinase can be characterized in a high-dimensional space. The minimum free energy paths of the conformational transitions in adenylate kinase were explored by the on-the-fly string method in 20-dimensional space spanned by the 20 largest-amplitude principal modes, and the free energy and various kinds of average physical quantities along the pathways were successfully evaluated by the MBAR method. The influence of ligand binding on the pathways was characterized in terms of rigid-body motions of the lid-shaped ATP-binding domain (LID) and the AMP-binding (AMPbd) domains. It was found that the LID domain was able to partially close without the ligand, while the closure of the AMPbd domain required the ligand binding. The transition state ensemble of the ligand bound form was identified as those structures characterized by highly specific binding of the ligand to the AMPbd domain, and was validated by unrestrained MD simulations. It was also found that complete closure of the LID domain required the dehydration of solvents around the P-loop. These findings suggest that the interplay of the two different types of domain motion is an essential feature in the conformational transition of the enzyme

Modular protein-RNA interactions regulating mRNA metabolism: a role for NMR

Here we review the role played by transient interactions between multi-functional proteins and their RNA targets in the regulation of mRNA metabolism, and we describe the important function of NMR spectroscopy in the study of these systems. We place emphasis on a general approach for the study of different features of modular multi-domain recognition that uses well-established NMR techniques and that has provided important advances in the general understanding of post-transcriptional regulation

Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion

We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental 13C/15N/2H isotope-labeled single-stranded DNA in amounts sufficient for NMR, based on the highly efficient self-primed PCR. To achieve this, new approaches are introduced and combined. (i) Asymmetric endonuclease double digestion of tandem-repeated PCR product. (ii) T4 DNA ligase mediated ligation of two ssDNA segments. (iii) In vitro dNTP synthesis, consisting of in vitro rNTP synthesis followed by enzymatic stereo-selective reduction of the C2′ of the rNTP, and a one-pot add-up synthesis of dTTP from dUTP. The method is demonstrated on two ssDNAs: (i) a 36-nt three-way junction, selectively 13C9/15N3/2H(1′,2″,3′,4′,5′,5″)-dC labeled and (ii) a 39-nt triple-repeat three-way junction, selectively 13C9/15N3/2H(1′,2″,3′,4′,5′,5″)-dC and 13C9/15N2/2H(1′,2″,3′,4′,5′,5″)-dT labeled in segment C20-C39. Their NMR spectra show the spectral simplification, while the stereo-selective 2H-labeling in the deoxyribose of the dC-residues, straightforwardly provided assignment of their C1′–H2′ and C2′–H2′ resonances. The labeling protocols can be extended to larger ssDNA molecules and to more than two segments

Miocene waterfowl and other birds from central Otago, New Zealand

Copyright © The Natural History Museum 2007Abundant fossil bird bones from the lower Bannockburn Formation, Manuherikia Group, an Early-Middle Miocene lacustrine deposit, 16–19 Ma, from Otago in New Zealand, reveal the “St Bathans Fauna” (new name), a first Tertiary avifauna of land and freshwater birds from New Zealand. At least 23 species of birds are represented by bones, and probable moa, Aves: Dinornithiformes, by eggshell. Anatids dominate the fauna with four genera and five species described as new: a sixth and largest anatid species is represented by just one bone. This is the most diverse Early-Middle Miocene duck fauna known worldwide. Among ducks, two species of dendrochenines are most numerous in the fauna, but a tadornine is common as well. A diving petrel (Pelecanoididae: Pelecanoides) is described, so extending the geological range of this genus worldwide from the Pliocene to the Middle Miocene, at least. The remaining 16 taxa are left undescribed but include: a large species of gull (Laridae); two small waders (Charadriiformes, genus indet.), the size of Charadrius bicinctus and Calidris ruficollis, respectively; a gruiform represented by one specimen similar to Aptornis; abundant rail (Rallidae) bones, including a common flightless rail and a rarer slightly larger taxon, about the size of Gallirallus philippensis; an ?eagle (Accipitridae); a pigeon (Columbidae); three parrots (Psittacidae); an owlet nightjar (Aegothelidae: Aegotheles sp.); a swiftlet (Apodidae: Collocalia sp.); and three passerine taxa, of which the largest is a member of the Cracticidae. The absence of some waterbirds, such as anserines (including swans), grebes (Podicipedidae) and shags (Phalacrocoracidae), among the abundant bones, indicates their probable absence from New Zealand in the Early-Middle Miocene.T. H. Worthy, A. J. D. Tennyson, C. Jones, J. A. McNamara and B. J. Dougla