A distinct mechanism for the ABC transporter BtuCD–BtuF revealed by the dynamics of complex formation

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a diverse array of substrates across cell membranes. We present here the dynamics of complex formation of three structurally characterized ABC transporters—the BtuCD vitamin B_(12) importer and MetNI d/l-methionine importer from Escherichia coli and the Hi1470/1 metal-chelate importer from Haemophilus influenzae—in complex with their cognate binding proteins. Similarly to other ABC importers, MetNI interacts with its binding protein with low affinity (K_d ~10^(−4) M). In contrast, BtuCD–BtuF and Hi1470/1–Hi1472 form stable, high-affinity complexes (K_d ~10^(−13) and 10^(−9) M, respectively). In BtuCD–BtuF, vitamin B_(12) accelerates the complex dissociation rate ~10^7-fold, with ATP having an additional destabilizing effect. The findings presented here highlight substantial mechanistic differences between BtuCD–BtuF, and likely Hi1470/1–Hi1472, and the better-characterized maltose and related ABC transport systems, indicating that there is considerable mechanistic diversity within this large protein super-family

Noncanonical role for the binding protein in substrate uptake by the MetNI methionine ATP Binding Cassette (ABC) transporter

The Escherichia coli methionine ABC transporter MetNI exhibits both high-affinity transport toward L-methionine and broad specificity toward methionine derivatives, including D-methionine. In this work, we characterize the transport of D-methionine derivatives by the MetNI transporter. Unexpectedly, the N229A substrate-binding deficient variant of the cognate binding protein MetQ was found to support high MetNI transport activity toward D-selenomethionine. We determined the crystal structure at 2.95 Å resolution of the ATPγS-bound MetNIQ complex in the outward-facing conformation with the N229A apo MetQ variant. This structure revealed conformational changes in MetQ providing substrate access through the binding protein to the transmembrane translocation pathway. MetQ likely mediates uptake of methionine derivatives through two mechanisms: in the methionine-bound form delivering substrate from the periplasm to the transporter (the canonical mechanism) and in the apo form by facilitating ligand binding when complexed to the transporter (the noncanonical mechanism). This dual role for substrate-binding proteins is proposed to provide a kinetic strategy for ABC transporters to transport both high- and low-affinity substrates present in a physiological concentration range

The High-Affinity E. coli Methionine ABC Transporter: Structure and Allosteric Regulation

The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)–binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell

Non-Fermi liquid behavior and scaling of low frequency suppression in optical conductivity spectra of CaRuO3_3

Optical conductivity spectra $\sigma_1(\omega)$ of paramagnetic CaRuO$_3$ are investigated at various temperatures. At T=10 K, it shows a non-Fermi liquid behavior of $\sigma_1(\omega)\sim 1/{\omega}^{\frac 12}$, similar to the case of a ferromagnet SrRuO$_3$. As the temperature ($T$) is increased, on the other hand, $\sigma_1(\omega)$ in the low frequency region is progressively suppressed, deviating from the 1/{\omega}^{\frac 12%}-dependence. Interestingly, the suppression of $\sigma_1(\omega)$ is found to scale with $\omega /T$ at all temperatures. The origin of the $$ scaling behavior coupled with the non-Fermi liquid behavior is discussed.Comment: 4 pages, 3 figure

The Funnel Approach to the Precrystallization Production of Membrane Proteins

Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a “funnel approach” to overcoming these difficulties and demonstrate its efficacy in a case study of 36 prokaryotic P-type transporters. A diverse ensemble of modified constructs is generated and tested for expression in Escherichia coli, membrane localization, detergent extraction, and homogeneity. High-throughput methodologies are implemented throughout the process to facilitate identification of promising targets. We find that the choice of promoter, the choice of source organism providing the cloned gene, and, most importantly, the position of the affinity tag have a great effect on successful production. The latter had pronounced effects at all tested levels, from expression levels observed in whole cells to the extent of membrane insertion, and even on protein function. Following the initial streamlined screening, we were able to fine-tune and produce 9 of the 36 targets as materials suitable for crystallization or other structural studies

Phonon `notches' in a-b -plane optical conductivity of high-Tc superconductors

It is shown that a correlation between the positions of the $c$-axis longitudinal optic ($LO_c$) phonons and ``notch''-like structures in the $a$-$b$ plane conductivity of high-$T_c$ superconductors results from phonon-mediated interaction between electrons in different layers. It is found that the relative size of the notches depends on $\lambda_{ph}(\Omega_{ph}/\gamma_{ph})$, where $\lambda_{ph}$, $\Omega_{ph}$ and $\gamma_{ph}$ are the effective coupling strength, the frequency and the width of the optical phonon which is responsible for the notch. Even for $\lambda_{ph}\approx 0.01$ the effect can be large if the phonon is very sharp.Comment: 5 pages, REVTeX, 4 uuencoded figure

Infrared Excess in the Be Star Delta Scorpii

We present infrared photometric observations of the Be binary system delta Scorpii obtained in 2006. The J,H and K magnitudes are the same within the errors compared to observations taken 10 months earlier. We derive the infrared excess from the observation and compare this to the color excess predicted by a radiative equilibrium model of the primary star and its circumstellar disk. We use a non-LTE computational code to model the gaseous envelope concentrated in the star's equatorial plane and calculate the expected spectral energy distribution and Halpha emission profile of the star with its circumstellar disk. Using the observed infrared excess of delta Sco, as well as Halpha spectroscopy bracketing the IR observations in time, we place constraints on the radial density distribution in the circumstellar disk. Because the disk exhibits variability in its density distribution, this work will be helpful in understanding its dynamics.Comment: 12 pages, 14 figures, to be published in PASP May 200

Local symmetries of the non-Abelian two-form

It is proposed that a non-Abelian adjoint two-form in BF type theories transform inhomogeneously under the gauge group. The resulting restrictions on invariant actions are discussed. The auxiliary one-form which is required for maintaining vector gauge symmetry transforms like a second gauge field, and hence cannot be fully absorbed in the two-form. But it can be replaced, via a vector gauge transformation, by the usual gauge field, leading to gauge equivalences between different types of theories. A new type of symmetry also appears, one which depends on local functions but cannot be generated by constraints. It is connected to the identity in the limit of a vanishing global parameter, so it should be called a semiglobal symmetry. The corresponding conserved currents and BRST charges are parametrized by the space of flat connections.Comment: RevTeX4, 11 pages, minor correction