Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme's lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK's domain movements on its catalytic time scale. To quantitatively measure the enzyme's entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme's conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme's rate-limiting step.conformational equilibrium ͉ rate-limiting step ͉ single-molecule FRET ͉ adenylate kinase P roteins such as enzymes are flexible with a range of motions spanning from picoseconds for localized vibrations to seconds for concerted global conformational rearrangements (1). Despite their randomly fluctuating environment, in which stochastic collisions with solvent molecules drive changes in tertiary structure, enzymes have evolved to catalyze reactions efficiently and specifically. Indeed, conformational transitions have been postulated to play a central role in enzyme functions in a wide variety of ways, including direct contribution to catalysis (2), allosteric regulation (3), and large-scale conformational changes in response to ligand binding (4). Most of our current understanding of structural motions in solution comes from NMR experiments (5) as well as from molecular dynamics simulations (6), approaches that are best suited to study dynamics in the picoto millisecond time scales. Because catalysis in enzymes frequently occurs in the submillisecond to minute time regime, our current understanding of the relationship between enzyme function and conformational dynamics comes from NMR experiments involving relatively localized motions of active site forming loops on the submillisecond time scale (7-10). However, many enzymes contain active sites located in between domains in which large-amplitude, low-frequency domain motions are required to complete their Michaelis-Menten enzyme-substrate complexes. Even simple questions regarding these transitions remain generally unanswered: What is the number and range of conformational states accessible to enzymes during their catalytic cycle? How does the enzyme's conformation respond to interac...

Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the ␣/␤-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin Acoated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.Lipid droplets, organelles that store neutral lipids, are found in nearly all types of eukaryotic cells. The most highly studied lipid droplet-associated proteins are members of the PAT 1 (for perilipin, adipophilin (also called adipose differentiation-related protein or ADRP), and TIP47) family of proteins (1) that includes perilipin, adipophilin, TIP47, and a related but structurally divergent protein, S3-12 (2, 3). Three protein isoforms of perilipins, named perilipins A, B, and C, are translated from alternatively spliced forms of mRNA. Perilipin A and S3-12 localize to lipid droplets in adipocytes (3-5), where they are the most abundant structural proteins of large mature and small nascent lipid droplets, respectively. In contrast, most other types of cells have small lipid droplets covered with adipophilin (6, 7) and TIP47 (1, 8, 9). Perilipins A and C, and adipophilin, coat the lipid droplets of steroidogenic cells (6) that store cholesterol ester precursors for steroid hormone synthesis.The members of the PAT family comprise the major structural proteins of lipid droplets. Studies in perilipin knockout mice have established an important role for perilipins in regulating lipolysis in adipocytes, and hence, controlling the mass of triacylglycerol deposited in adipose tissue (10, 11). The mechanisms by which per...

The pathogenesis of glucocorticoid-induced (GC-induced) bone loss is unclear. For example, osteoblast apoptosis is enhanced by GCs in vivo, but they stimulate bone formation in vitro. This conundrum suggests that an intermediary cell transmits a component of the bone-suppressive effects of GCs to osteoblasts in the intact animal. Bone remodeling is characterized by tethering of the activities of osteoclasts and osteoblasts. Hence, the osteoclast is a potential modulator of the effect of GCs on osteoblasts. To define the direct impact of GCs on bone-resorptive cells, we compared the effects of dexamethasone (DEX) on WT osteoclasts with those derived from mice with disruption of the GC receptor in osteoclast lineage cells (GR oc-/-mice). While the steroid prolonged longevity of osteoclasts, their bone-degrading capacity was suppressed. The inhibitory effect of DEX on bone resorption reflects failure of osteoclasts to organize their cytoskeleton in response to M-CSF. DEX specifically arrested M-CSF activation of RhoA, Rac, and Vav3, each of which regulate the osteoclast cytoskeleton. In all circumstances GR oc-/-mice were spared the impact of DEX on osteoclasts and their precursors. Consistent with osteoclasts modulating the osteoblast-suppressive effect of DEX, GR oc-/-mice are protected from the steroid's inhibition of bone formation.

The ubiquitin-proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and mis-folded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques. These structures bring some surprising insights into the functional mechanism of the proteasome and will provide invaluable guidance for genetic and biochemical studies of this key regulatory system.

To explore the role of glucocorticoids in regulation of kinase pathways during innate immune responses, we generated mice with conditional deletion of glucocorticoid receptor (GR) in macrophages (MGRKO). Activation of toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) caused greater mortality and cytokine production in MGRKO mice than in controls. Ex vivo, treatment with dexamethasone (Dex) markedly inhibited LPS-mediated induction of inflammatory genes in control but not GR-deficient macrophages. We show that Dex inhibits p38 MAPK, but not PI3K/Akt, ERK, or JNK, in control macrophages. Associated with p38 inhibition, Dex induced MAP kinase phosphatase-1 (MKP-1) in control, but not MGRKO, macrophages. Consistent with the ex vivo studies, treatment with a p38 MAPK–specific inhibitor resulted in rescue of MGRKO mice from LPS-induced lethality. Taken together, we identify p38 MAPK and its downstream targets as essential for GR-mediated immunosuppression in macrophages.

M-Au/TiO2 (M = Ag, Pd, Pt) composites were prepared through a facile one-pot photodeposition synthesis and evaluated for solar water splitting (SWS) with and without a sacrificial agent. The M-Au combination exhibits a dominant role in augmenting the H2 generation activity by forming a bi-metallic system. Degussa P25 was used as a TiO2 substrate to photodeposit Au followed by Au + M (M = Ag/Pd/Pt). The SWS activity of the M-Au/TiO2 was determined through photocatalytic H2 production in the presence of methanol as a sacrificial agent under one sun conditions with an AM1.5 filter. The highest H2 yield was observed for Pt0.5-Au1/TiO2 and was around 1.3 ± 0.07 mmol h(-1) g(-1), with an apparent quantum yield (AQY) of 6.4%. Pt0.5-Au1/TiO2 also demonstrated the same activity for 25 cycles of five hours each for 125 h. Critically, the same Pt0.5-Au1/TiO2 catalyst was active in overall SWS (OSWS) without any sacrificial agent, with an AQY = 0.8%. The amount of Au and/or Pt was varied to obtain the optimum composition and it was found that the Pt0.5-Au1/TiO2 composition exhibits the best activity. Detailed characterization by physico-chemical, spectral and microscopy measurements was carried out to obtain an in-depth understanding of the origin of the photocatalytic activity of Pt0.5-Au1/TiO2. These in-depth studies show that gold interacts predominantly with oxygen vacancies present on titania surfaces, and Pt preferentially interacts with gold for an effective electron-hole pair separation at Pt-Au interfaces and electron storage in metal particles. The Pt in Pt0.5-Au1/TiO2 is electronically and catalytically different from the Pt in Pt/TiO2 and it is predicted that the former suppresses the oxygen reduction reaction.

Cystic Fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyperexpression of IL-8 protein. However, the mechanistic link between mutations in CFTR and acquisition of the proinflammatory phenotype in the CF airway has remained elusive. We hypothesized that specific microRNAs (miRNAs) might mediate this linkage. To identify the potential link, we screened an miRNA library for differential expression in ⌬F508-CFTR and wild type CFTR lung epithelial cell lines. Of 22 differentially and significantly expressed miRNAs, we found that expression of miR-155 was more than 5-fold elevated in CF IB3-1 lung epithelial cells in culture, compared with control IB3-1/S9 cells. Clinically, miR-155 was also highly expressed in CF lung epithelial cells and circulating CF neutrophils biopsied from CF patients. We report here that high levels of miR-155 specifically reduced levels of SHIP1, thereby promoting PI3K/Akt activation. However, overexpressing SHIP1 or inhibition of PI3K in CF cells suppressed IL-8 expression. Finally, we found that phospho-Akt levels were elevated in CF lung epithelial cells and were specifically lowered by either antagomir-155 or elevated expression of SHIP1. We therefore suggest that elevated miR-155 contributes to the proinflammatory expression of IL-8 in CF lung epithelial cells by lowering SHIP1 expression and thereby activating the PI3K/Akt signaling pathway. These data suggest that miR-155 may play an important role in the activation of IL-8-dependent inflammation in CF.

The notion of spontaneous formation of an inhomogeneous superconducting state is at the heart of most theories attempting to understand the superconducting state in the presence of strong disorder. Using scanning tunneling spectroscopy and high resolution scanning transmission electron microscopy, we experimentally demonstrate that under the competing effects of strong homogeneous disorder and superconducting correlations, the superconducting state of a conventional superconductor, NbN, spontaneously segregates into domains. Tracking these domains as a function of temperature we observe that the superconducting domains persist across the bulk superconducting transition, Tc, and disappear close to the pseudogap temperature, T*, where signatures of superconducting correlations disappear from the tunneling spectrum and the superfluid response of the system.