Efficient Sampling of Protein Loop Regions Using Conformational Hashing Complemented with Random Coordinate Descent.

De novo construction of loop regions is an important problem in computational structural biology. Compared to regions with well-defined secondary structure, loops tend to exhibit significant conformational heterogeneity. As a result, their structures are often ambiguous when determined using experimental data obtained by crystallography, cryo-EM, or NMR. Although structurally diverse models could provide a more relevant representation of proteins in their native states, obtaining large numbers of biophysically realistic and physiologically relevant loop conformations is a resource-consuming task. To address this need, we developed a novel loop construction algorithm, Hash/RCD, that combines knowledge-based conformational hashing with random coordinate descent (RCD). This hybrid approach achieved a closure rate of 100% on a benchmark set of 195 loops in 29 proteins that range from 3 to 31 residues. More importantly, the use of templates allows Hash/RCD to maintain the accuracy of state-of-the-art coordinate descent methods while reducing sampling time from over 400 to 141 ms. These results highlight how the integration of coordinate descent with knowledge-based sampling overcomes barriers inherent to either approach in isolation. This method may facilitate the identification of native-like loop conformations using experimental data or full-atom scoring functions by allowing rapid sampling of large numbers of loops. In this manuscript, we investigate and discuss the advantages, bottlenecks, and limitations of combining conformational hashing with RCD. By providing a detailed technical description of the Hash/RCD algorithm, we hope to facilitate its implementation by other researchers.

Specificity of the chromophore-binding site in human cone opsins.

The variable composition of the chromophore-binding pocket in visual receptors is essential for vision. The visual phototransduction starts with the cis-trans isomerization of the retinal chromophore upon absorption of photons. Despite sharing the common 11-cis-retinal chromophore, rod and cone photoreceptors possess distinct photochemical properties. Thus, a detailed molecular characterization of the chromophore-binding pocket of these receptors is critical to understanding the differences in the photochemistry of vision between rods and cones. Unlike for rhodopsin (Rh), the crystal structures of cone opsins remain to be determined. To obtain insights into the specific chromophore-protein interactions that govern spectral tuning in human visual pigments, here we harnessed the unique binding properties of 11-cis-6-membered-ring-retinal (11-cis-6mr-retinal) with human blue, green, and red cone opsins. To unravel the specificity of the chromophore-binding pocket of cone opsins, we applied 11-cis-6mr-retinal analog-binding analyses to human blue, green, and red cone opsins. Our results revealed that among the three cone opsins, only blue cone opsin can accommodate the 11-cis-6mr-retinal in its chromophore-binding pocket, resulting in the formation of a synthetic blue pigment (B6mr) that absorbs visible light. A combination of primary sequence alignment, molecular modeling, and mutagenesis experiments revealed the specific amino acid residue 6.48 (Tyr-262 in blue cone opsins and Trp-281 in green and red cone opsins) as a selectivity filter in human cone opsins. Altogether, the results of our study uncover the molecular basis underlying the binding selectivity of 11-cis-6mr-retinal to the cone opsins.

Crowd sourcing difficult problems in protein science.

Dedicated computing resources are expensive to develop, maintain, and administrate. Frequently, research groups require bursts of computing power, during which progress is still limited by available computing resources. One way to alleviate this bottleneck would be to use additional computing resources. Today, many computing devices remain idle most of the time. Passive volunteer computing exploits this unemployed reserve of computing power by allowing device-owners to donate computing time on their own devices. Another complementary way to alleviate bottlenecks in computing resources is to use more efficient algorithms. Engaging volunteer computing employs human intuition to help solve challenging problems for which efficient algorithms are difficult to develop or unavailable. Designing engaging volunteer computing projects is challenging but can result in high-quality solutions. Here, we highlight four examples.

Complex binding pathways determine the regeneration of mammalian green cone opsin with a locked retinal analogue.

Phototransduction is initiated when the absorption of light converts the 11-cis-retinal chromophore to its all-trans configuration in both rod and cone vertebrate photoreceptors. To sustain vision, 11-cis-retinal is continuously regenerated from its all-trans conformation through a series of enzymatic steps comprising the "visual or retinoid" cycle. Abnormalities in this cycle can compromise vision because of the diminished supply of 11-cis-retinal and the accumulation of toxic, constitutively active opsin. As shown previously for rod cells, attenuation of constitutively active opsin can be achieved with the unbleachable analogue, 11-cis-6-membered ring (11-cis-6mr)-retinal, which has therapeutic effects against certain degenerative retinal diseases. However, to discern the molecular mechanisms responsible for this action, pigment regeneration with this locked retinal analogue requires delineation also in cone cells. Here, we compared the regenerative properties of rod and green cone opsins with 11-cis-6mr-retinal and demonstrated that this retinal analogue could regenerate rod pigment but not green cone pigment. Based on structural modeling suggesting that Pro-205 in green cone opsin could prevent entry and binding of 11-cis-6mr-retinal, we initially mutated this residue to Ile, the corresponding residue in rhodopsin. However, this substitution did not enable green cone opsin to regenerate with 11-cis-6mr-retinal. Interestingly, deletion of 16 N-terminal amino acids in green cone opsin partially restored the binding of 11-cis-6mr-retinal. These results and our structural modeling indicate that a more complex binding pathway determines the regeneration of mammalian green cone opsin with chromophore analogues such as 11-cis-6mr-retinal.

Hydrogen/Deuterium Exchange Mass Spectrometry of Human Green Opsin Reveals a Conserved Pro-Pro Motif in Extracellular Loop 2 of Monostable Visual G Protein-Coupled Receptors.

Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled with mass spectrometry to map conformational changes in green cone opsin upon light activation. We then compared these findings with those reported for rhodopsin. The extent of H/D exchange in green cone opsin was greater than in rhodopsin in the dark and bleached states, suggesting a higher structural heterogeneity for green cone opsin. Further analysis revealed that green cone opsin exists as a dimer in both dark (inactive) and bleached (active) states, and that the predicted glycosylation sites at N32 and N34 are indeed glycosylated. Comparison of deuterium uptake between inactive and active states of green cone opsin also disclosed a reduced solvent accessibility of the extracellular N-terminal region and an increased accessibility of the chromophore binding site. Increased H/D exchange at the extracellular side of transmembrane helix four (TM4) combined with an analysis of sequence alignments revealed a conserved Pro-Pro motif in extracellular loop 2 (EL2) of monostable visual GPCRs. These data present new insights into the locus of chromophore release at the extracellular side of TM4 and TM5 and provide a foundation for future functional evaluation.

Structural Insights into the Drosophila melanogaster Retinol Dehydrogenase, a Member of the Short-Chain Dehydrogenase/Reductase Family.

The 11-cis-retinylidene chromophore of visual pigments isomerizes upon interaction with a photon, initiating a downstream cascade of signaling events that ultimately lead to visual perception. 11-cis-Retinylidene is regenerated through enzymatic transformations collectively called the visual cycle. The first and rate-limiting enzymatic reaction within this cycle, i.e., the reduction of all-trans-retinal to all-trans-retinol, is catalyzed by retinol dehydrogenases. Here, we determined the structure of Drosophila melanogaster photoreceptor retinol dehydrogenase (PDH) isoform C that belongs to the short-chain dehydrogenase/reductase (SDR) family. This is the first reported structure of a SDR that possesses this biologically important activity. Two crystal structures of the same enzyme grown under different conditions revealed a novel conformational change of the NAD+ cofactor, likely representing a change during catalysis. Amide hydrogen-deuterium exchange of PDH demonstrated changes in the structure of the enzyme upon dinucleotide binding. In D. melanogaster, loss of PDH activity leads to photoreceptor degeneration that can be partially rescued by transgenic expression of human RDH12. Based on the structure of PDH, we analyzed mutations causing Leber congenital amaurosis 13 in a homology model of human RDH12 to obtain insights into the molecular basis of RDH12 disease-causing mutations.

Image registration and averaging of low laser power two-photon fluorescence images of mouse retina.

Two-photon fluorescence microscopy (TPM) is now being used routinely to image live cells for extended periods deep within tissues, including the retina and other structures within the eye . However, very low laser power is a requirement to obtain TPM images of the retina safely. Unfortunately, a reduction in laser power also reduces the signal-to-noise ratio of collected images, making it difficult to visualize structural details. Here, image registration and averaging methods applied to TPM images of the eye in living animals (without the need for auxiliary hardware) demonstrate the structural information obtained with laser power down to 1 mW. Image registration provided between 1.4% and 13.0% improvement in image quality compared to averaging images without registrations when using a high-fluorescence template, and between 0.2% and 12.0% when employing the average of collected images as the template. Also, a diminishing return on image quality when more images were used to obtain the averaged image is shown. This work provides a foundation for obtaining informative TPM images with laser powers of 1 mW, compared to previous levels for imaging mice ranging between 6.3 mW [Palczewska G., Nat Med.20, 785 (2014) Sharma R., Biomed. Opt. Express4, 1285 (2013)].

Characterization of the Domain Orientations of E. coli 5'-Nucleotidase by Fitting an Ensemble of Conformers to DEER Distance Distributions.

Escherichia coli 5'-nucleotidase is a two-domain enzyme exhibiting a unique 96° domain motion that is required for catalysis. Here we present an integrated structural biology study that combines DEER distance distributions with structural information from X-ray crystallography and computational biology to describe the population of presumably almost isoenergetic open and closed states in solution. Ensembles of models that best represent the experimental distance distributions are determined by a Monte Carlo search algorithm. As a result, predominantly open conformations are observed in the unliganded state indicating that the majority of enzyme molecules await substrate binding for the catalytic cycle. The addition of a substrate analog yields ensembles with an almost equal mixture of open and closed states. Thus, in the presence of substrate, efficient catalysis is provided by the simultaneous appearance of open conformers (binding substrate or releasing product) and closed conformers (enabling the turnover of the substrate).

Semi-automated discrimination of retinal pigmented epithelial cells in two-photon fluorescence images of mouse retinas.

Automated image segmentation is a critical step toward achieving a quantitative evaluation of disease states with imaging techniques. Two-photon fluorescence microscopy (TPM) has been employed to visualize the retinal pigmented epithelium (RPE) and provide images indicating the health of the retina. However, segmentation of RPE cells within TPM images is difficult due to small differences in fluorescence intensity between cell borders and cell bodies. Here we present a semi-automated method for segmenting RPE cells that relies upon multiple weak features that differentiate cell borders from the remaining image. These features were scored by a search optimization procedure that built up the cell border in segments around a nucleus of interest. With six images used as a test, our method correctly identified cell borders for 69% of nuclei on average. Performance was strongly dependent upon increasing retinosome content in the RPE. TPM image analysis has the potential of providing improved early quantitative assessments of diseases affecting the RPE.

BCL::MP-fold: Membrane protein structure prediction guided by EPR restraints.

For many membrane proteins, the determination of their topology remains a challenge for methods like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Electron paramagnetic resonance (EPR) spectroscopy has evolved as an alternative technique to study structure and dynamics of membrane proteins. The present study demonstrates the feasibility of membrane protein topology determination using limited EPR distance and accessibility measurements. The BCL::MP-Fold (BioChemical Library membrane protein fold) algorithm assembles secondary structure elements (SSEs) in the membrane using a Monte Carlo Metropolis (MCM) approach. Sampled models are evaluated using knowledge-based potential functions and agreement with the EPR data and a knowledge-based energy function. Twenty-nine membrane proteins of up to 696 residues are used to test the algorithm. The RMSD100 value of the most accurate model is better than 8 Å for 27, better than 6 Å for 22, and better than 4 Å for 15 of the 29 proteins, demonstrating the algorithms' ability to sample the native topology. The average enrichment could be improved from 1.3 to 2.5, showing the improved discrimination power by using EPR data.

Postoperative observation of children after endoscopic type 1 posterior laryngeal cleft repair.

OBJECTIVES: To report the perioperative management and surgical outcomes in a large series of pediatric patients with endoscopically repaired type 1 posterior laryngeal cleft (PLC). STUDY DESIGN: Case series with chart review. SETTING: Urban, tertiary care, free-standing pediatric hospital. SUBJECTS AND METHODS: Patients who underwent endoscopic carbon dioxide laser-assisted repair of type 1 posterior laryngeal clefts between January 2006 and December 2012. Medical records were reviewed. RESULTS: Fifty-four patients (34 male) underwent repair of type 1 PLC. Median age was 25.5 months (range, 2-120 months). Indications for repair included aspiration (n = 39; 72%), chronic bronchitis (n = 13; 24%), and stridor with feeds (n = 2; 4%). No children remained intubated postoperatively. Thirty-three patients (61%) stayed in overnight observation ("Obs PLC") and 21 patients (39%) stayed in the pediatric intensive care unit ("PICU PLC") postoperatively. Between Obs PLC and PICU PLC groups, there was no significant difference in age (mean 22 vs 30 months, respectively; P = .28). Comorbidities were similar between the groups. Symptoms improved in 41 of the 54 patients (76%). No postoperative complications were noted. Two patients required revision PLC repair. The cost of admitting a patient to a lower acuity location was estimated to be 60% less per day than cost of a PICU admission. CONCLUSIONS: The endoscopic surgical repair of a type 1 PLC is successful and has a low morbidity and complication rate. Patients may be safely managed in an observation unit and without postoperative intubation. This approach achieved a marked cost reduction in postoperative care.

Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye.

Two-photon excitation microscopy can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in subcellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all-trans-retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here, we report repetitive, dynamic imaging of these compounds in live mice through the pupil of the eye. By leveraging advanced adaptive optics, we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium by their characteristic localization, spectral properties and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light- and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions.

Energetic analysis of the rhodopsin-G-protein complex links the α5 helix to GDP release.

We present a model of interaction of Gi protein with the activated receptor (R*) rhodopsin, which pinpoints energetic contributions to activation and reconciles the ß2 adrenergic receptor-Gs crystal structure with new and previously published experimental data. In silico analysis demonstrated energetic changes when the Gα C-terminal helix (α5) interacts with the R* cytoplasmic pocket, thus leading to displacement of the helical domain and GDP release. The model features a less dramatic domain opening compared with the crystal structure. The α5 helix undergoes a 63° rotation, accompanied by a 5.7-Å translation, that reorganizes interfaces between α5 and α1 helices and between α5 and ß6-α5. Changes in the ß6-α5 loop displace αG. All of these movements lead to opening of the GDP-binding pocket. The model creates a roadmap for experimental studies of receptor-mediated G-protein activation.

RosettaEPR: rotamer library for spin label structure and dynamics.

An increasingly used parameter in structural biology is the measurement of distances between spin labels bound to a protein. One limitation to these measurements is the unknown position of the spin label relative to the protein backbone. To overcome this drawback, we introduce a rotamer library of the methanethiosulfonate spin label (MTSSL) into the protein modeling program Rosetta. Spin label rotamers were derived from conformations observed in crystal structures of spin labeled T4 lysozyme and previously published molecular dynamics simulations. Rosetta's ability to accurately recover spin label conformations and EPR measured distance distributions was evaluated against 19 experimentally determined MTSSL labeled structures of T4 lysozyme and the membrane protein LeuT and 73 distance distributions from T4 lysozyme and the membrane protein MsbA. For a site in the core of T4 lysozyme, the correct spin label conformation (Χ1 and Χ2) is recovered in 99.8% of trials. In surface positions 53% of the trajectories agree with crystallized conformations in Χ1 and Χ2. This level of recovery is on par with Rosetta performance for the 20 natural amino acids. In addition, Rosetta predicts the distance between two spin labels with a mean error of 4.4 Å. The width of the experimental distance distribution, which reflects the flexibility of the two spin labels, is predicted with a mean error of 1.3 Å. RosettaEPR makes full-atom spin label modeling available to a wide scientific community in conjunction with the powerful suite of modeling methods within Rosetta.

Pediatric obstructive sleep apnea syndrome.

Pediatric obstructive sleep apnea syndrome (OSAS) is a common health problem diagnosed and managed by various medical specialists, including family practice physicians, pediatricians, pulmonologists, and general and pediatric otolaryngologists. If left untreated, the sequelae can be severe. Over the last decade, significant advancements have been made in the evidence-based management of pediatric OSAS. This article focuses on the current understanding of this disease, its management, and related clinical practice guidelines.

Conformation of receptor-bound visual arrestin.

Arrestin-1 (visual arrestin) binds to light-activated phosphorylated rhodopsin (P-Rh*) to terminate G-protein signaling. To map conformational changes upon binding to the receptor, pairs of spin labels were introduced in arrestin-1 and double electron-electron resonance was used to monitor interspin distance changes upon P-Rh* binding. The results indicate that the relative position of the N and C domains remains largely unchanged, contrary to expectations of a "clam-shell" model. A loop implicated in P-Rh* binding that connects ß-strands V and VI (the "finger loop," residues 67-79) moves toward the expected location of P-Rh* in the complex, but does not assume a fully extended conformation. A striking and unexpected movement of a loop containing residue 139 away from the adjacent finger loop is observed, which appears to facilitate P-Rh* binding. This change is accompanied by smaller movements of distal loops containing residues 157 and 344 at the tips of the N and C domains, which correspond to "plastic" regions of arrestin-1 that have distinct conformations in monomers of the crystal tetramer. Remarkably, the loops containing residues 139, 157, and 344 appear to have high flexibility in both free arrestin-1 and the P-Rh*complex.

Treatment strategies for lateral sphenoid sinus recess cerebrospinal fluid leaks.

OBJECTIVE: To highlight concepts critical to achieving successful repair and avoiding intracranial complications in the treatment of cerebrospinal fluid (CSF) leaks from the lateral recess of the sphenoid sinus (LRS). DESIGN: Outcomes study. SETTING: Tertiary referral university hospital. PATIENTS: Eleven patients with LRS CSF leaks from June 2008 to June 2010. INTERVENTIONS: Endoscopic transpterygoid approach and multilayer repair of skull base defect in the LRS. MAIN OUTCOME MEASURES: Recurrence, graft techniques, postoperative intracranial pressure (ICP), and use of ventriculoperitoneal (VP) shunt. RESULTS: Thirteen CSF leaks originating in the LRS were surgically repaired in 11 patients; 2 patients required bilateral leak repair. The endoscopic transpterygoid approach was used in 12 of 13 repairs. Eight patients had failed attempts at repair prior to presentation (4 endoscopic sphenoidotomies and 4 middle cranial fossa [MCF] approaches). One patient presented with a temporal lobe abscess following hydroxyapatite "obliteration" to seal off the LRS. This required a combined MCF/transpterygoid approach to drain the abscess, remove the encephalocele and hydroxyapatite, and seal the skull base defect. In 2 cases, the LRS was left patent owing to concerns of inadequate mucosal extirpation. The median duration of follow-up was 10.8 months (range, 2-29 months). One patient experienced a failure (2 months after repair), which was successfully sealed on the second attempt. Postoperatively, 5 patients required VP shunts, and 5 were maintained on acetazolamide for elevated ICP (average, 26.7 cm H2O in 8 patients; presumed elevated in 2 patients). CONCLUSIONS: The current study demonstrated a 92% success rate using the endoscopic transpterygoid approach for LRS skull base defects providing support for routine use in the treatment algorithm. Poor outcomes were observed with previous surgical attempts to obstruct the LRS without repairing the skull base defect.

Cystic fibrosis transmembrane conductance regulator modulation by the tobacco smoke toxin acrolein.

OBJECTIVES/HYPOTHESIS: Evidence indicates that decreased mucociliary clearance (MCC) is a major contributing feature to chronic rhinosinusitis. Tobacco-smoke exposure is thought to inhibit transepithelial Cl(-) secretion, a major determinant of airway surface liquid hydration and MCC. The objective of the current study was to evaluate the effects of acrolein exposure (a prominent tobacco smoke toxin) on vectorial Cl(-) transport through the major apical anion channel cystic fibrosis transmembrane conductance regulator (CFTR) in sinonasal epithelium. STUDY DESIGN: In vitro investigation. METHODS: Primary murine nasal septal epithelia (MNSE; wild-type and transgenic CFTR(-/-)) cultures were exposed to acrolein in Ussing chambers and the effects on Cl(-) secretion investigated using pharmacologic manipulation. Cellular cyclic adenosine monophosphate (cAMP) signaling and cytotoxicity were also investigated. RESULTS: Acrolein stimulated Cl(-) secretion (ΔI(SC) - change in short-circuit current in µA/cm(2)) at concentrations similar to smoker's airways (100 µM, 15.8 ± 2.2 vs. 2.4 ± 0.8 [control]; P < .0001), suppressed forskolin-stimulated C- transport at 300 µM (13.3 ± 1.2 vs. 19.9 ± 1.0; P < .01), and completely abolished all transport at 500 µM (-1.1 ± 1.6). Stimulated Cl(-) secretion was solely reliant upon the presence of CFTR (confirmed in transgenic CFTR(-/-) MNSE), but independent of cAMP signaling. Inhibition at higher concentrations was not secondary to cellular cytotoxicity. CONCLUSIONS: The present study demonstrates that acrolein has complex but pronounced interaction with the major apical Cl(-) transport mechanism that uses CFTR. Further investigations are required to determine acrolein's impact as a tobacco smoke constituent on mucociliary transport.

MRSA and non-MRSA otorrhea in children: a comparative study of clinical course.

OBJECTIVE: To test the perception that post-tympanostomy tube otorrhea caused by methicillin-resistant Staphylococcus aureus (MRSA) is a more virulent disease than otorrhea caused by other pathogens by analyzing the clinical differences and disease courses in children diagnosed with otorrhea caused by MRSA bacteria vs non-MRSA bacteria. DESIGN: Retrospective review. SETTING: Tertiary children's hospital. PATIENTS: We retrospectively examined the medical records of children who presented to a tertiary children's hospital from January 1, 2003, to December 31, 2008, with otorrhea that occurred after tympanostomy tube insertion. MAIN OUTCOME MEASURES: Otorrhea culture records were used to group the 1079 patients into those whose otitis media was due to MRSA (n = 170) and those with non-MRSA otitis media (n = 909). From the non-MRSA group, we randomly selected an age-matched group of 170 and examined the differences between the MRSA and age-matched non-MRSA groups in organisms isolated by culture, demographic factors (including type of medical insurance), medical history, treatments, surgical procedures performed, audiometric data, and other admissions for infection-related illnesses. RESULTS: The overall incidence of MRSA in this series was about 16% (170 of 1079 patients). Of the 170 eligible children in each age-matched group, 135 with MRSA otorrhea and 141 with non-MRSA otorrhea had data in every category selected for statistical analysis. The groups did not differ significantly in type of insurance; history of tympanostomy tube placement, cholesteatoma, or prematurity; number or type (minor/major) of surgical procedures performed; or risk of subsequent infection-related diagnoses. More patients in the MRSA group received intravenous antibiotic therapy (11% vs 3.6%; P < .001). CONCLUSION: In this study, a diagnosis of otorrhea due to MRSA did not carry an increased risk for surgical procedures or infection-associated sequelae compared with a diagnosis of non-MRSA otorrhea.