Adenosine triphosphate (ATP) hydrolysis in the nitrogenase complex controls the cycle of association and dissociation between the electron donor adenosine triphosphatase (ATPase) (Fe-protein) and its target catalytic protein (MoFe-protein), driving the reduction of dinitrogen into ammonia. Crystal structures in different nucleotide states have been determined that identify conformational changes in the nitrogenase complex during ATP turnover. These structures reveal distinct and mutually exclusive interaction sites on the MoFe-protein surface that are selectively populated, depending on the Fe-protein nucleotide state. A consequence of these different docking geometries is that the distance between redox cofactors, a critical determinant of the intermolecular electron transfer rate, is coupled to the nucleotide state. More generally, stabilization of distinct docking geometries by different nucleotide states, as seen for nitrogenase, could enable nucleotide hydrolysis to drive the relative motion of protein partners in molecular motors and other systems.

High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a ''hybrid'' droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as Ϸ140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in Ϸ10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 l of protein solution, Ϸ1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.droplets ͉ plugs ͉ protein structure ͉ high-throughput ͉ miniaturization T his work reports a ''hybrid'' microfluidic approach that uses nanoliter plugs to perform screening and optimization simultaneously in the same experiment. To validate this method using a challenging problem, we demonstrate its compatibility with crystallization of membrane proteins. Small-scale screening and optimization experiments are important for biological assays, chemical screening, and protein crystallization (1-3). Screening and optimization are usually carried out sequentially. In the case of protein crystallization, random sparse matrix screening initially identifies the precipitants that may lead to crystallization. Subsequent gradient optimization establishes concentrations of these precipitants that lead to diffractionquality crystals (4). Combining screening and optimization steps into a single hybrid experiment would eliminate the need to wait for the outcome of the initial screen before carrying out subsequent optimizations. Furthermore, a hybrid experiment would reduce the false negatives (5) associated with screens performed at a single concentration. The hybrid experiment could also be more conclusive, because a single batch of the s...

Although outnumbered more than 20:1 by rod photoreceptors, cone cells in the human eye mediate daylight vision and are critical for visual acuity and color discrimination. A variety of human retinal diseases, e.g. age-related macular degeneration (AMD), are characterized by a progressive loss of cone photoreceptors, but the low abundance of cones and the absence of a macula in non-primate mammalian retinas have made it difficult to investigate them directly owing to lack of suitable experimental models. Conventional rodents (laboratory mice and rats) are nocturnal rod dominated species with few cones in the retina, while investigating other animals with cone-rich retinas presents various logistic and technical difficulties. Past work, originating in the early 1900s, has begun to provide insights into cone ultrastructure, but has yet to afford a unified model of cone cell organization. This review summarizes this past work and focuses on the recent introduction of special mammalian models (transgenic mice and diurnal rats rich in cones) that promise to reveal a more unified model of cone photoreceptor organization and its role in retinal diseases. These new mammalian models should allow new investigative techniques such as atomic force microscopy and cryo-electron tomography to advance our understanding of cone photoreceptors, much as has been done with rod photoreceptors.

Enhanced S-cone syndrome (ESCS), featuring an excess number of S cones, manifests as a progressive retinal degeneration that leads to blindness. Here, through optical imaging, we identified an abnormal interface between photoreceptors and the retinal pigment epithelium (RPE) in 9 patients with ESCS. The neural retina leucine zipper transcription factor-knockout (Nrl(-/-)) mouse model demonstrates many phenotypic features of human ESCS, including unstable S-cone-positive photoreceptors. Using massively parallel RNA sequencing, we identified 6203 differentially expressed transcripts between wild-type (Wt) and Nrl(-/-) mouse retinas, with 6 highly significant differentially expressed genes of the Pax, Notch, and Wnt canonical pathways. Changes were also obvious in expression of 30 genes involved in the visual cycle and 3 key genes in photoreceptor phagocytosis. Novel high-resolution (100 nm) imaging and reconstruction of Nrl(-/-) retinas revealed an abnormal packing of photoreceptors that contributed to buildup of photoreceptor deposits. Furthermore, lack of phagosomes in the RPE layer of Nrl(-/-) retina revealed impairment in phagocytosis. Cultured RPE cells from Wt and Nrl(-/-) mice illustrated that the phagocytotic defect was attributable to the aberrant interface between ESCS photoreceptors and the RPE. Overcoming the retinal phagocytosis defect could arrest the progressive degenerative component of this disease.

This paper reports a method for the production of arrays of nanolitre plugs with distinct chemical compositions. One of the primary constraints on the use of plug-based microfluidics for large scale biological screening is the difficulty of fabricating arrays of chemically distinct plugs on the nanolitre scale. Here, using microfluidic devices with several T-junctions linked in series, a single input array of large (approximately 320 nL) plugs was split to produce 16 output arrays of smaller (approximately 20 nL) plugs; the composition and configuration of these arrays were identical to that of the input. This paper shows how the passive break-up of plugs in T-junction microchannel geometries can be used to produce a set of smaller-volume output arrays useful for chemical screening from a single large-volume array. A simple theoretical description is presented to describe splitting as a function of the Capillary number, the capillary pressure, the total pressure difference across the channel, and the geometric fluidic resistance. By accounting for these considerations, plug coalescence and plug-plug contamination can be eliminated from the splitting process and the symmetry of splitting can be preserved. Furthermore, single-outlet splitting devices were implemented with both valve- and volume-based methods for coordinating the release of output arrays. Arrays of plugs containing commercial sparse matrix screens were obtained from the presented splitting method and these arrays were used in protein crystallization trials. The techniques presented in this paper may facilitate the implementation of high-throughput chemical and biological screening.

A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.

Biological membranes are densely packed with membrane proteins that occupy approximately half of their volume. In almost all cases, membrane proteins in the native state lack the higherorder symmetry required for their direct study by diffraction methods. Despite many technical difficulties, numerous crystal structures of detergent solubilized membrane proteins have been determined that illustrate their internal organization. Among such proteins, class A G protein-coupled receptors have become amenable to crystallization and high resolution X-ray diffraction analyses. The derived structures of native and engineered receptors not only provide insights into their molecular arrangements but also furnish a framework for designing and testing potential models of transformation from inactive to active receptor signaling states and for initiating rational drug design.

Photoreceptor cell death is the proximal cause of blindness in many retinal degenerative disorders; hence, understanding the gene regulatory networks that promote photoreceptor survival is at the forefront of efforts to combat blindness. Down-regulation of the microRNA (miRNA)-processing enzyme DICER1 in the retinal pigmented epithelium has been implicated in geographic atrophy, an advanced form of age-related macular degeneration (AMD). However, little is known about the function of DICER1 in mature rod photoreceptor cells, another retinal cell type that is severely affected in AMD. Using a conditional-knockout (cKO) mouse model, we report that loss of DICER1 in mature postmitotic rods leads to robust retinal degeneration accompanied by loss of visual function. At 14 wk of age, cKO mice exhibit a 90% reduction in photoreceptor nuclei and a 97% reduction in visual chromophore compared with those in control littermates. Before degeneration, cKO mice do not exhibit significant defects in either phototransduction or the visual cycle, suggesting that miRNAs play a primary role in rod photoreceptor survival. Using comparative small RNA sequencing analysis, we identified rod photoreceptor miRNAs of the miR-22, miR-26, miR-30, miR-92, miR-124, and let-7 families as potential factors involved in regulating the survival of rods.