X-ray scattering experiments with high-flux X-ray source coupled rapid mixing microchannel device and their potential for high-flux neutron scattering investigations.

Small-angle X-ray scattering provides global, shape-sensitive structural information about macromolecules in solution. Its extension to time dimension in the form of time-resolved SAXS investigations and combination with other time-resolved biophysical methods contributes immensely to the study of protein dynamics. TR-SAXS can also provide unique information about the global structures of transient intermediates during protein dynamics. An experimental set-up with low protein consumption is essential for an extensive use of TR-SAXS experiments on protein dynamics. In this direction, a newly developed 20-microchannel microfluidic continuous-flow mixer was combined with SAXS. With this set-up, we demonstrate ubiquitin unfolding dynamics after rapid mixing with the chaotropic agent Guanidinium-HCl within milliseconds using only ∼ 40 nanoliters of the protein sample per scattering image. It is suggested that, in the future, this new TR-SAXS platform will help to increase the use of time-resolved small-angle X-ray scattering, wide-angle X-ray scattering and neutron scattering experiments for studying protein dynamics in the early millisecond regime. The potential research field for this set-up includes protein folding, protein misfolding, aggregation in amyloidogenic diseases, function of intrinsically disordered proteins and various protein-ligand interactions.

Citrate sensing by the C4-dicarboxylate/citrate sensor kinase DcuS of Escherichia coli: binding site and conversion of DcuS to a C4-dicarboxylate- or citrate-specific sensor.

The histidine protein kinase DcuS of Escherichia coli senses C(4)-dicarboxylates and citrate by a periplasmic domain. The closely related sensor kinase CitA binds citrate, but no C(4)-dicarboxylates, by a homologous periplasmic domain. CitA is known to bind the three carboxylate and the hydroxyl groups of citrate by sites C1, C2, C3, and H. DcuS requires the same sites for C(4)-dicarboxylate sensing, but only C2 and C3 are highly conserved. It is shown here that sensing of citrate by DcuS required the same sites. Binding of citrate to DcuS, therefore, was similar to binding of C(4)-dicarboxylates but different from that of citrate binding in CitA. DcuS could be converted to a C(4)-dicarboxylate-specific sensor (DcuS(DC)) by mutating residues of sites C1 and C3 or of some DcuS-subtype specific residues. Mutations around site C1 aimed at increasing the size and accessibility of the site converted DcuS to a citrate-specific sensor (DcuS(Cit)). DcuS(DC) and DcuS(Cit) had complementary effector specificities and responded either to C(4)-dicarboxylates or to citrate and mesaconate. The results imply that DcuS binds citrate (similar to the C(4)-dicarboxylates) via the C(4)-dicarboxylate part of the molecule. Sites C2 and C3 are essential for binding of two carboxylic groups of citrate or of C(4)-dicarboxylates; sites C1 and H are required for other essential purposes.

NMR in the SPINE Structural Proteomics project.

This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.

Apical endocytosis in outer hair cells of the mammalian cochlea.

Outer hair cells (OHCs), the sensory-motor cells of the mammalian cochlea, contain an endocytic tubulovesicular compartment below their apical stereocilia. We have used two-photon imaging of FM1-43 in the intact epithelium to show that these cells take up membrane in a Ca(2+)-dependent manner from a distinct apical site. The uptake rate was 0.8 microm(2)/s and internalized membrane was trafficked rapidly to a compartment along the lateral wall and distinct intracellular compartments. Double labelling with FM1-43 and DiOC(6), an endoplasmic reticulum (ER) marker, showed that these compartments are part of the tubulovesicular endoplasmic reticulum of OHCs. Labelling with a lysosomal marker showed that OHC lysosomes are restricted to the apex. Using the protein marker wheat germ agglutinin (WGA-FITC) we demonstrate that apical protein internalization and trafficking is about eight times slower than membrane internalization. Using double labelling with FM1-43 and WGA-FITC, we show that membrane and protein internalization are apically colocalized but that patterns of protein and membrane traffic differ. Protein was targeted only to the most apical third of the lateral wall. In control conditions, OHCs displayed only weak WGA-FITC surface labelling at the site of endocytosis. Lowering the rate of apical endocytosis increased this surface signal. The results suggest that OHCs endocytose membrane and membrane proteins with a high turnover rate and that these cells may use apical endocytosis to sort proteins via an indirect pathway to the lateral membrane.

Measurement of 2J(H,C)- and 3J(H,C)-coupling constants by alpha/beta selective HC(C)H-TOCSY.

A new heteronuclear NMR pulse sequence for the measurement of nJ(C,H) coupling constants, the alpha/beta selective HC(C)H-TOCSY, is described. It is shown that the S3E element (Meissner et al., 1997a,b) can be used to obtain spin state selective coherence transfer in molecules, in which adjacent CH moieties are labeled with 13C. Application of the alpha/beta selective HC(C)H-TOCSY to a 10 nt RNA tetraloop 5'-CGCUUUUGCG-3', in which the four uridine residues are 13C labeled in the sugar moiety, allowed measurement of two bond and three bond J(C,H) coupling constants, which provide additional restraints to characterize the sugar ring conformation of RNA in cases of conformational averaging.

Three molecules of ubiquinone bind specifically to mitochondrial cytochrome bc1 complex.

Bifurcated electron flow to high potential "Rieske" iron-sulfur cluster and low potential heme b(L) is crucial for respiratory energy conservation by the cytochrome bc(1) complex. The chemistry of ubiquinol oxidation has to ensure the thermodynamically unfavorable electron transfer to heme b(L). To resolve a central controversy about the number of ubiquinol molecules involved in this reaction, we used high resolution magic-angle-spinning nuclear magnetic resonance experiments to show that two out of three n-decyl-ubiquinones bind at the ubiquinol oxidation center of the complex. This substantiates a proposed mechanism in which a charge transfer between a ubiquinol/ubiquinone pair explains the bifurcation of electron flow.

Determination of aliphatic side-chain conformation using cross-correlated relaxation: application to an extraordinarily stable 2'-aminoethoxy-modified oligonucleotide triplex.

The structural basis for the extraordinary stability of a triple-stranded oligonucleotide in which the third strand contains 2'-aminoethoxy-substituted riboses is investigated by NMR spectroscopy. The enhanced stability of the modified triplex in comparison to the unmodified DNA triplex of the same sequence can be attributed to strong interactions of the aminoethoxy groups of the third strand with the phosphate groups of the purine strand. In molecular dynamics calculations the aminoethoxy side chain was found to be rather flexible, allowing for the presence of hydrogen bonds between the aminoethoxy group of the third strand and two different phosphates of the backbone of the second strand. To investigate the conformational preference of the aminoethoxy side chain a new NMR method has been developed which relies on CH-CH dipolar-dipolar cross-correlated relaxation rates. The results indicate that the aminoethoxy side chains adopt mainly a gauche(+) conformation, for which only one of the two hydrogen bonds inferred by NMR and molecular dynamics simulations is possible. This demonstrates a highly specific interaction between the amino group of the third strand and one of the phosphate groups of the purine strand.