Mixed Ligand Passivation as the Origin of Near-Unity Emission Quantum Yields in CsPbBr3 Nanocrystals.

Key features of syntheses, involving the quaternary ammonium passivation of CsPbBr3 nanocrystals (NCs), include stable, reproducible, and large (often near-unity) emission quantum yields (QYs). The archetypical example involves didodecyl dimethyl ammonium (DDDMA+)-passivated CsPbBr3 NCs where robust QYs stem from interactions between DDDMA+ and NC surfaces. Despite widespread adoption of this synthesis, specific ligand-NC surface interactions responsible for large DDDMA+-passivated NC QYs have not been fully established. Multidimensional nuclear magnetic resonance experiments now reveal a new DDDMA+-NC surface interaction, beyond established "tightly bound" DDDMA+ interactions, which strongly affects observed emission QYs. Depending upon the existence of this new DDDMA+ coordination, NC QYs vary broadly between 60 and 85%. More importantly, these measurements reveal surface passivation through unexpected didodecyl ammonium (DDA+) that works in concert with DDDMA+ to produce near-unity (i.e., >90%) QYs.

Coupled intra- and interdomain dynamics support domain cross-talk in Pin1.

The functional mechanisms of multidomain proteins often exploit interdomain interactions, or "cross-talk." An example is human Pin1, an essential mitotic regulator consisting of a Trp-Trp (WW) domain flexibly tethered to a peptidyl-prolyl isomerase (PPIase) domain, resulting in interdomain interactions important for Pin1 function. Substrate binding to the WW domain alters its transient contacts with the PPIase domain via means that are only partially understood. Accordingly, we have investigated Pin1 interdomain interactions using NMR paramagnetic relaxation enhancement (PRE) and molecular dynamics (MD) simulations. The PREs show that apo-Pin1 samples interdomain contacts beyond the range suggested by previous structural studies. They further show that substrate binding to the WW domain simultaneously alters interdomain separation and the internal conformation of the WW domain. A 4.5-µs all-atom MD simulation of apo-Pin1 suggests that the fluctuations of interdomain distances are correlated with fluctuations of WW domain interresidue contacts involved in substrate binding. Thus, the interdomain/WW domain conformations sampled by apo-Pin1 may already include a range of conformations appropriate for binding Pin1's numerous substrates. The proposed coupling between intra-/interdomain conformational fluctuations is a consequence of the dynamic modular architecture of Pin1. Such modular architecture is common among cell-cycle proteins; thus, the WW-PPIase domain cross-talk mechanisms of Pin1 may be relevant for their mechanisms as well.

NMR Relaxation Dispersion Reveals Macrocycle Breathing Dynamics in a Cyclodextrin-based Rotaxane.

A distinctive feature of mechanically interlocked molecules (MIMs) is the relative motion between the mechanically bonded components, and often it is the functional basis for artificial molecular machines and new functional materials. Optimization of machine or materials performance requires knowledge of the underlying atomic-level mechanisms that control the motion. The field of biomolecular NMR spectroscopy has developed a diverse set of pulse schemes that can characterize molecular dynamics over a broad time scale, but these techniques have not yet been used to characterize the motion within MIMs. This study reports the first observation of NMR relaxation dispersion related to MIM motion. The rotary (pirouette) motion of α-cyclodextrin (αCD) wheels was characterized in a complementary pair of rotaxanes with pirouetting switched ON or OFF. 13C and 1H NMR relaxation dispersion measurements reveal previously unknown exchange dynamics for the αCD wheels in the pirouette-ON rotaxane with a rate constant of 2200 s-1 at 298 K and an activation barrier of ΔF‡ = 43 ± 3 kJ/mol. The exchange dynamics disappear in the pirouette-OFF rotaxane, demonstrating their switchable nature. The 13C and 1H sites exhibiting relaxation dispersion suggest that the exchange involves "macrocycle breathing", in which the αCD wheel fluctuates between a contracted or expanded state, the latter enabling diffusive rotary motion about the axle. The substantial insight from these NMR relaxation dispersion methods suggests similar dynamic NMR methods can illuminate the fast time scale (microsecond to millisecond) mechanisms of intercomponent motion in a wide range of MIMs.

Reveglucosidase alfa (BMN 701), an IGF2-Tagged rhAcid α-Glucosidase, Improves Respiratory Functional Parameters in a Murine Model of Pompe Disease.

Pompe disease is a rare neuromuscular disorder caused by an acid α-glucosidase (GAA) deficiency resulting in glycogen accumulation in muscle, leading to myopathy and respiratory weakness. Reveglucosidase alfa (BMN 701) is an insulin-like growth factor 2-tagged recombinant human acid GAA (rhGAA) that enhances rhGAA cellular uptake via a glycosylation-independent insulin-like growth factor 2 binding region of the cation-independent mannose-6-phosphate receptor (CI-MPR). The studies presented here evaluated the effects of Reveglucosidase alfa treatment on glycogen clearance in muscle relative to rhGAA, as well as changes in respiratory function and glycogen clearance in respiratory-related tissue in a Pompe mouse model (GAAtm1Rabn/J). In a comparison of glycogen clearance in muscle with Reveglucosidase alfa and rhGAA, Reveglucosidase alfa was more effective than rhGAA with 2.8-4.7 lower EC50 values, probably owing to increased cellular uptake. The effect of weekly intravenous administration of Reveglucosidase alfa on respiratory function was monitored in Pompe and wild-type mice using whole body plethysmography. Over 12 weeks of 20-mg/kg Reveglucosidase alfa treatment in Pompe mice, peak inspiratory flow (PIF) and peak expiratory flow (PEF) stabilized with no compensation in respiratory rate and inspiratory time during hypercapnic and recovery conditions compared with vehicle-treated Pompe mice. Dose-related decreases in glycogen levels in both ambulatory and respiratory muscles generally correlated to changes in respiratory function. Improvement of murine PIF and PEF were similar in magnitude to increases in maximal inspiratory and expiratory pressure observed clinically in late onset Pompe patients treated with Reveglucosidase alfa (Byrne et al., manuscript in preparation).

Expanded Substrate Activity of OXA-24/40 in Carbapenem-Resistant Acinetobacter baumannii Involves Enhanced Binding Loop Flexibility.

Gram-negative bacteria resist ß-lactam antibiotics primarily by deploying ß-lactamase proteins that hydrolytically destroy the antibiotics. In clinical settings, these bacteria are producing variant ß-lactamases with "gain-of-activity" mutations that inactivate a broader range of ß-lactams. Learning how these mutations broaden substrate activity is important for coping with ß-lactam resistance. Here, we investigate a gain of activity mutation in OXA-24/40, a carbapenem-hydrolyzing class D ß-lactamase (CHDL) in Acinetobacter baumannii. OXA-24/40 was originally active against penicillin and carbapenem classes of ß-lactams, but a clinical variant of OXA-24/40, the single-site substitution mutant P227S, has emerged with expanded activity that now includes advanced cephalosporins and the monobactam aztreonam. Using solution-state nuclear magnetic resonance (NMR) spectroscopy, we have compared the site-specific backbone dynamics of wild-type OXA-24/40 and the P227S variant. P227S changes local backbone flexibility in segments that are important for both binding and hydrolysis of carbapenem and cephalosporin substrates. Our results suggest that mutation-induced changes in sequence-specific dynamics can expand substrate activity and thus highlight the role of protein conformational dynamics in antibiotic resistance. To the best of our knowledge, this is the first NMR study of CHDL conformational dynamics and its impact on the expansion of ß-lactam antibiotic resistance.

Investigation of signal transduction routes within the sensor/transducer protein BlaR1 of Staphylococcus aureus.

The transmembrane antibiotic sensor/signal transducer protein BlaR1 is part of a cohort of proteins that confer ß-lactam antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) [Fisher, J. F., Meroueh, S. O., and Mobashery, S. (2005) Chem. Rev. 105, 395-424; Llarrull, L. I., Fisher, J. F., and Mobashery, S. (2009) Antimicrob. Agents Chemother. 53, 4051-4063; Llarrull, L. I., Toth, M., Champion, M. M., and Mobashery, S. (2011) J. Biol. Chem. 286, 38148-38158]. Specifically, BlaR1 regulates the inducible expression of ß-lactamases that hydrolytically destroy ß-lactam antibiotics. The resistance phenotype starts with ß-lactam antibiotic acylation of the BlaR1 extracellular domain (BlaRS). The acylation activates the cytoplasmic protease domain through an obscure signal transduction mechanism. Here, we compare protein dynamics of apo versus antibiotic-acylated BlaRS using nuclear magnetic resonance. Our analyses reveal inter-residue interactions that relay acylation-induced perturbations within the antibiotic-binding site to the transmembrane helix regions near the membrane surface. These are the first insights into the process of signal transduction by BlaR1.

Revealing cell-surface intramolecular interactions in the BlaR1 protein of methicillin-resistant Staphylococcus aureus by NMR spectroscopy.

In methicillin-resistant Staphylococcus aureus, ß-lactam antibiotic resistance is mediated by the transmembrane protein BlaR1. The antibiotic sensor domain BlaR(S) and the L2 loop of BlaR1 are on the membrane surface. We used NMR to investigate interactions between BlaR(S) and a water-soluble peptide from L2. This peptide binds BlaR(S) proximal to the antibiotic acylation site as an amphipathic helix. Acylation of BlaR(S) by penicillin G does not disrupt binding. These results suggest a signal transduction mechanism whereby the L2 helix, partially embedded in the membrane, propagates conformational changes caused by BlaR(S) acylation through the membrane via transmembrane segments, leading to antibiotic resistance.

Stereospecific gating of functional motions in Pin1.

Pin1 is a modular enzyme that accelerates the cis-trans isomerization of phosphorylated-Ser/Thr-Pro (pS/T-P) motifs found in numerous signaling proteins regulating cell growth and neuronal survival. We have used NMR to investigate the interaction of Pin1 with three related ligands that include a pS-P substrate peptide, and two pS-P substrate analogue inhibitors locked in the cis and trans conformations. Specifically, we compared the ligand binding modes and binding-induced changes in Pin1 side-chain flexibility. The cis and trans binding modes differ, and produce different mobility in Pin1. The cis-locked inhibitor and substrate produced a loss of side-chain flexibility along an internal conduit of conserved hydrophobic residues, connecting the domain interface with the isomerase active site. The trans-locked inhibitor produces a weaker conduit response. Thus, the conduit response is stereoselective. We further show interactions between the peptidyl-prolyl isomerase and Trp-Trp (WW) domains amplify the conduit response, and alter binding properties at the remote peptidyl-prolyl isomerase active site. These results suggest that specific input conformations can gate dynamic changes that support intraprotein communication. Such gating may help control the propagation of chemical signals by Pin1, and other modular signaling proteins.

Interdomain interactions support interdomain communication in human Pin1.

Pin1 is an essential mitotic regulator consisting of a peptidyl-prolyl isomerase (PPIase) domain flexibly tethered to a smaller Trp-Trp (WW) binding domain. Communication between these domains is important for Pin1 in vivo activity; however, the atomic basis for this communication has remained elusive. Our previous nuclear magnetic resonance (NMR) studies of Pin1 functional dynamics suggested that weak interdomain contacts within Pin1 enable allosteric communication between the domain interface and the distal active site of the PPIase domain.1,2 A necessary condition for this hypothesis is that the intrinsic properties of the PPIase domain should be sensitive to interdomain contact. Here, we test this sensitivity by generating a Pin1 mutant, I28A, which weakens the wild-type interdomain contact while maintaining the overall folds of the two domains. Using NMR, we show that I28A leads to altered substrate binding affinity and isomerase activity. Moreover, I28A causes long-range perturbations to conformational flexibility in both domains, for both the apo and substrate-complexed states of the protein. These results show that the distribution of conformations sampled by the PPIase domain is sensitive to interdomain contact and strengthen the hypothesis that such contact supports interdomain allosteric communication in Pin1. Other modular systems may exploit interdomain interactions in a similar manner.

Kinetic isotope effects support the twisted amide mechanism of Pin1 peptidyl-prolyl isomerase.

The Pin1 peptidyl-prolyl isomerase catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac-Phe-Phe-phosphoSer-Pro-Arg-p-nitroaniline, were synthesized in unlabeled form, and with deuterium-labeled Ser-d3 and Pro-d7 amino acids. Kinetic data were collected as a function of Pin1 concentration to measure kinetic isotope effects (KIEs) on catalytic efficiency (kcat/Km). The normal secondary (2°) KIE value measured for the Ser-d3 substrate (kH/kD = 1.6 ± 0.2) indicates that the serine carbonyl does not rehybridize from sp(2) to sp(3) in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2° KIE can be explained by hyperconjugation between Ser α-C-H/D and C═O and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2° KIE value (kH/kD = 0.86 ± 0.08) measured for the Pro-d7 substrate indicates rehybridization of the prolyl nitrogen from sp(2) to sp(3) during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (kH2O/kD2O = 0.92 ± 0.12), indicating little or no involvement of exchangeable protons in the mechanism. These results support the formation of a simple twisted amide transition state as the mechanism for peptidyl prolyl isomerization catalyzed by Pin1. A model of the reaction mechanism is presented using crystal structures of Pin1 with ground state analogues and an inhibitor that resembles a twisted amide transition state.

Airway epithelial miRNA expression is altered in asthma.

RATIONALE: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. OBJECTIVES: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13-regulated miRNAs. METHODS: We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. MEASUREMENTS AND MAIN RESULTS: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. CONCLUSIONS: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway epithelial cell differentiation. Clinical trial registered with www.clinicaltrials.gov (NCT 00595153).

Angiogenesis and Tissue Repair Depend on Platelet Dosing and Bioformulation Strategies Following Orthobiological Platelet-Rich Plasma Procedures: A Narrative Review.

Angiogenesis is the formation of new blood vessel from existing vessels and is a critical first step in tissue repair following chronic disturbances in healing and degenerative tissues. Chronic pathoanatomic tissues are characterized by a high number of inflammatory cells; an overexpression of inflammatory mediators; such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1); the presence of mast cells, T cells, reactive oxygen species, and matrix metalloproteinases; and a decreased angiogenic capacity. Multiple studies have demonstrated that autologous orthobiological cellular preparations (e.g., platelet-rich plasma (PRP)) improve tissue repair and regenerate tissues. There are many PRP devices on the market. Unfortunately, they differ greatly in platelet numbers, cellular composition, and bioformulation. PRP is a platelet concentrate consisting of a high concentration of platelets, with or without certain leukocytes, platelet-derived growth factors (PGFs), cytokines, molecules, and signaling cells. Several PRP products have immunomodulatory capacities that can influence resident cells in a diseased microenvironment, inducing tissue repair or regeneration. Generally, PRP is a blood-derived product, regardless of its platelet number and bioformulation, and the literature indicates both positive and negative patient treatment outcomes. Strangely, the literature does not designate specific PRP preparation qualifications that can potentially contribute to tissue repair. Moreover, the literature scarcely addresses the impact of platelets and leukocytes in PRP on (neo)angiogenesis, other than a general one-size-fits-all statement that "PRP has angiogenic capabilities". Here, we review the cellular composition of all PRP constituents, including leukocytes, and describe the importance of platelet dosing and bioformulation strategies in orthobiological applications to initiate angiogenic pathways that re-establish microvasculature networks, facilitating the supply of oxygen and nutrients to impaired tissues.

Lysine Nzeta-decarboxylation switch and activation of the beta-lactam sensor domain of BlaR1 protein of methicillin-resistant Staphylococcus aureus.

The integral membrane protein BlaR1 of methicillin-resistant Staphylococcus aureus senses the presence of ß-lactam antibiotics in the milieu and transduces the information to the cytoplasm, where the biochemical events that unleash induction of antibiotic resistance mechanisms take place. We report herein by two-dimensional and three-dimensional NMR experiments of the sensor domain of BlaR1 in solution and by determination of an x-ray structure for the apo protein that Lys-392 of the antibiotic-binding site is posttranslationally modified by N(ζ)-carboxylation. Additional crystallographic and NMR data reveal that on acylation of Ser-389 by antibiotics, Lys-392 experiences N(ζ)-decarboxylation. This unique process, termed the lysine N(ζ)-decarboxylation switch, arrests the sensor domain in the activated ("on") state, necessary for signal transduction and all the subsequent biochemical processes. We present structural information on how this receptor activation process takes place, imparting longevity to the antibiotic-receptor complex that is needed for the induction of the antibiotic-resistant phenotype in methicillin-resistant S. aureus.

Sequence-specific dynamics modulate recognition specificity in WW domains.

The current canon attributes the binding specificity of protein-recognition motifs to distinctive chemical moieties in their constituent amino acid sequences. However, we show for a WW domain that the sequence crucial for specificity is an intrinsically flexible loop that partially rigidifies upon ligand docking. A single-residue deletion in this loop simultaneously reduces loop flexibility and ligand binding affinity. These results suggest that sequences of recognition motifs may reflect natural selection of not only chemical properties but also dynamic modes that augment specificity.

Sarcoidosis blood transcriptome reflects lung inflammation and overlaps with tuberculosis.

RATIONALE: Sarcoidosis is a granulomatous disease of unknown etiology, although M. tuberculosis may play a role in the pathogenesis. The traditional view holds that inflammation in sarcoidosis is compartmentalized to involved organs. OBJECTIVES: To determine whether whole blood gene expression signatures reflect inflammatory pathways in the lung in sarcoidosis and whether these signatures overlap with tuberculosis. METHODS: We analyzed transcriptomic data from blood and lung biopsies in sarcoidosis and compared these profiles with blood transcriptomic data from tuberculosis and other diseases. MEASUREMENTS AND MAIN RESULTS: Applying machine learning algorithms to blood gene expression data, we built a classifier that distinguished sarcoidosis from health in derivation and validation cohorts (92% sensitivity, 92% specificity). The most discriminative genes were confirmed by quantitative PCR and correlated with disease severity. Transcript profiles significantly induced in blood overlapped with those in lung biopsies and identified shared dominant inflammatory pathways (e.g., Type-I/II interferons). Sarcoidosis and tuberculosis shared more overlap in blood gene expression compared with other diseases using the 86-gene signature reported to be specific for tuberculosis and the sarcoidosis signature presented herein, although reapplication of machine learning algorithms could identify genes specific for sarcoidosis. CONCLUSIONS: These data indicate that blood transcriptome analysis provides a noninvasive method for identifying inflammatory pathways in sarcoidosis, that these pathways may be leveraged to complement more invasive procedures for diagnosis or assessment of disease severity, and that sarcoidosis and tuberculosis share overlap in gene regulation of specific inflammatory pathways.

Electromyographic Characteristics of a Single Motion Shoulder Exercise: A Pilot Study Investigating a Novel Shoulder Exercise.

BACKGROUND: Shoulder exercises focused on strengthening the rotator cuff and scapular stabilizing muscles as well as addressing scapular dyskinesis and motor control have been shown to improve rotator cuff function and decrease shoulder pain. A single motion shoulder exercise that effectively activates the rotator cuff and scapular stabilizing muscles, engages the scapulohumeral rhythm, and includes eccentric contractions may be more effective and easier for patients to consistently perform as compared to multiple standard shoulder exercises. PURPOSE: To compare the electromyographic muscle activation of key shoulder complex muscles during a single motion exercise and individual exercises (standard exercises) typically included in shoulder rehabilitation protocols. STUDY DESIGN: Case-controlled, cohort study. METHODS: Nineteen healthy men and women without shoulder pain or dysfunction were studied. Muscle activity of the rotator cuff and scapular stabilizing muscles (supraspinatus, infraspinatus, teres minor, trapezius [upper, middle and lower], serratus anterior, middle deltoid) was measured using surface EMG while subjects performed, in a standing position, several standard shoulder exercises typically included in shoulder rehabilitation protocols (resisted shoulder flexion, abduction in the scapular plane/scaption, external rotation, extension) and a single motion shoulder exercise consisting of a continuous movement creating the shape of "Figure of 8" in the transverse plane. The subjects used a weight between 5-15 pounds that produced muscle activation at 40-60% maximum voluntary isometric contraction (MVIC) for shoulder external rotation. That weight was then used for all of the exercises performed by the subject. The single highest EMG reading for each of the eight muscles studied, expressed as a percentage of MVIC, at any point during the second, third and fourth repetitions in a five repetition set was used to compare the single motion shoulder exercise and each exercise in the standard exercises set. RESULTS: Ten men and nine women between 18-65 years of age were tested. No significant difference (p=.05) between the exercises was noted for the supraspinatus, infraspinatus, teres minor, serratus anterior, middle deltoid or upper trapezius. There was a significant difference favoring the standard exercises in the middle and lower trapezius. (p= 0.0109 and 0.0002 respectively). CONCLUSION: In this pilot study, muscle activation during the single motion, Figure of 8 pattern exercise was not significantly different from the standard shoulder exercises in six of eight key muscles that are usually included in shoulder rehabilitation protocols. The exceptions were the middle and lower trapezius which were activated to a significantly higher degree with the standard exercises. Further evaluation of the clinical effectiveness of the single motion shoulder exercise is needed. LEVEL OF EVIDENCE: Level 3b.

Modeling conformational ensembles of slow functional motions in Pin1-WW.

Protein-protein interactions are often mediated by flexible loops that experience conformational dynamics on the microsecond to millisecond time scales. NMR relaxation studies can map these dynamics. However, defining the network of inter-converting conformers that underlie the relaxation data remains generally challenging. Here, we combine NMR relaxation experiments with simulation to visualize networks of inter-converting conformers. We demonstrate our approach with the apo Pin1-WW domain, for which NMR has revealed conformational dynamics of a flexible loop in the millisecond range. We sample and cluster the free energy landscape using Markov State Models (MSM) with major and minor exchange states with high correlation with the NMR relaxation data and low NOE violations. These MSM are hierarchical ensembles of slowly interconverting, metastable macrostates and rapidly interconverting microstates. We found a low population state that consists primarily of holo-like conformations and is a "hub" visited by most pathways between macrostates. These results suggest that conformational equilibria between holo-like and alternative conformers pre-exist in the intrinsic dynamics of apo Pin1-WW. Analysis using MutInf, a mutual information method for quantifying correlated motions, reveals that WW dynamics not only play a role in substrate recognition, but also may help couple the substrate binding site on the WW domain to the one on the catalytic domain. Our work represents an important step towards building networks of inter-converting conformational states and is generally applicable.

Osteoblast expression of an engineered Gs-coupled receptor dramatically increases bone mass.

Osteoblasts are essential for maintaining bone mass, avoiding osteoporosis, and repairing injured bone. Activation of osteoblast G protein-coupled receptors (GPCRs), such as the parathyroid hormone receptor, can increase bone mass; however, the anabolic mechanisms are poorly understood. Here we use "Rs1," an engineered GPCR with constitutive G(s) signaling, to evaluate the temporal and skeletal effects of G(s) signaling in murine osteoblasts. In vivo, Rs1 expression induces a dramatic anabolic skeletal response, with midfemur girth increasing 1,200% and femur mass increasing 380% in 9-week-old mice. Bone volume, cellularity, areal bone mineral density, osteoblast gene markers, and serum bone turnover markers were also elevated. No such phenotype developed when Rs1 was expressed after the first 4 weeks of postnatal life, indicating an exquisite temporal sensitivity of osteoblasts to Rs1 expression. This pathway may represent an important determinant of bone mass and may open future avenues for enhancing bone repair and treating metabolic bone diseases.

Communication breakdown: protein dynamics and drug design.

Mauldin et al. (2009) use NMR to show that drug binding can break up collective protein motions necessary for function. We discuss their findings in the context of drug discovery in pharmaceutical research.

Arginine Modulates Carbapenem Deactivation by OXA-24/40 in Acinetobacter baumannii.

The resistance of Gram-negative bacteria to ß-lactam antibiotics stems mainly from ß-lactamase proteins that hydrolytically deactivate the ß-lactams. Of particular concern are the ß-lactamases that can deactivate a class of ß-lactams known as carbapenems. Carbapenems are among the few anti-infectives that can treat multi-drug resistant bacterial infections. Revealing the mechanisms of their deactivation by ß-lactamases is a necessary step for preserving their therapeutic value. Here, we present NMR investigations of OXA-24/40, a carbapenem-hydrolyzing Class D ß-lactamase (CHDL) expressed in the gram-negative pathogen, Acinetobacter baumannii. Using rapid data acquisition methods, we were able to study the "real-time" deactivation of the carbapenem known as doripenem by OXA-24/40. Our results indicate that OXA-24/40 has two deactivation mechanisms: canonical hydrolytic cleavage, and a distinct mechanism that produces a ß-lactone product that has weak affinity for the OXA-24/40 active site. The mechanisms issue from distinct active site environments poised either for hydrolysis or ß-lactone formation. Mutagenesis reveals that R261, a conserved active site arginine, stabilizes the active site environment enabling ß-lactone formation. Our results have implications not only for OXA-24/40, but the larger family of CHDLs now challenging clinical settings on a global scale.