Model of a six immunoglobulin-like domain fragment of filamin A (16-21) built using residual dipolar couplings.

Filamins are actin-binding proteins that participate in a wide range of cell functions, including cell morphology, locomotion, membrane protein localization, and intracellular signaling. The three filamin isoforms found in humans, filamins A, B, and C, are highly homologous, and their roles are partly complementary. In addition to actin, filamins interact with dozens of other proteins that have roles as membrane receptors and channels, enzymes, signaling intermediates, and transcription factors. Filamins are composed of an N-terminal actin-binding domain and 24 filamin-type immunoglobulin-like domains (FLN) that form tail-to-tail dimers with their C-terminal FLN domain. Many of the filamin interactions including those for glycoprotein Ibα and integrins have been mapped to the region comprising FLN domains 16-21. Traditionally, FLN domains have been viewed as independent folding units, arranged in a linear chain joined with flexible linkers. Recent structural findings have shown that consecutive FLNs form more intricate superstructures. The crystal structure of filamin A domains 19-21 (FLNa19-21) revealed that domains 20 and 21 fold together and that the domain interaction can be autoregulatory. The solution structure of domains 18-19 showed a similar domain interaction, whereas domain pair 16-17 has a completely different domain packing mode. In this study, we characterize the domain organization of the FLNa domain sextet 16-21 using NMR spectroscopy. A structure model of this 60-kDa protein has been built using residual dipolar coupling restraints. RDCs and (15)N relaxation data have been used to characterize interdomain motions.

MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: application to a 557-residue IgFLNa16-21.

We describe a novel pulse sequence, MQ-HNCO-TROSY, for the measurement of scalar and residual dipolar couplings between amide proton and nitrogen in larger proteins. The experiment utilizes the whole 2T(N) polarization transfer delay for labeling of (15)N chemical shift in a constant time manner, which efficiently doubles the attainable resolution in (15)N dimension with respect to the conventional HNCO-TROSY experiment. In addition, the accordion principle is employed for measuring (J + D)(NH)s, and the multiplet components are selected with the generalized version of the TROSY scheme introduced by Nietlispach (J Biomol NMR 31:161-166, 2005). Therefore, cross peak overlap is diminished while the time period during which the (15)N spin is susceptible to fast transverse relaxation associated with the anti-TROSY transition is minimized per attainable resolution unit. The proposed MQ-HNCO-TROSY scheme was employed for measuring RDCs in high molecular weight protein IgFLNa16-21 of 557 residues, resulting in 431 experimental RDCs. Correlations between experimental and back-calculated RDCs in individual domains gave relatively low Q-factors (0.19-0.39), indicative of sufficient accuracy that can be obtained with the proposed MQ-HNCO-TROSY experiment in high molecular weight proteins.

Analgesic Plasma Concentrations of Oxycodone After Surgery for Breast Cancer-Which Factors Matter?

We investigated factors affecting analgesic oxycodone concentrations after breast cancer surgery in 1,000 women. Preoperatively, we studied heat and cold pain sensitivities and anxiety scores. Postoperatively, rest and motion pain intensities were measured and intravenous oxycodone was administered until satisfactory analgesia. At this point, the mean oxycodone concentration (variation coefficient) was 33.3 ng/mL (66%) and it was 21.7 ng/mL (69%) when the patient requested oxycodone again. At both time points, the concentrations varied >100-fold between individuals. The analgesic oxycodone concentration was increased by 21.3% per motion pain intensity score on a 0-10 scale and by 22.3% if axillary clearance was performed instead of sentinel node biopsy (P < 0.001). Forty-seven women who were older and less anxious than others (P < 0.01) required no oxycodone. Anxiety, age, chronic pain, or preoperative pain sensitivity were not independently associated with the analgesic oxycodone concentration. CYP2D6 and CYP3A genotypes did not affect analgesic concentration or duration of analgesia.

1H, 13C and 15N resonance assignments of the major extracytoplasmic domain of the cell shape-determining protein MreC from Bacillus subtilis.

MreB, MreC and MreD are essential cell shape-determining morphogenetic proteins in Gram-positive and in Gram-negative bacteria. While MreB, the bacterial homologue of the eukaryotic cytoskeletal protein actin, has been extensively studied, the roles of MreC and MreD are less well understood. They both are transmembrane proteins. MreC has a predicted single transmembrane domain and the C-terminal part outside the cell membrane. MreC probably functions as a link between the intracellular cytoskeleton and the cell wall synthesizing machinery which is located at the outer surface of the cell membrane. Also proteins involved in cell wall synthesis participate in cell morphogenesis. How these two processes are coordinated is, however, poorly understood. Bacillus subtilis (BS), a non-pathogenic Gram-positive bacterium, is widely used as a model for Gram-positive pathogens, e.g. Staphylococcus aureus (SA). Currently, the structures of MreC from BS and SA are not known. As part of our efforts to elucidate the structure-function relationships of the morphogenetic protein complexes in Gram-positive bacteria, we present the backbone and side chain resonance assignments of the extracytoplasmic domain of MreC from BS.