The blobulator: a webtool for identification and visual exploration of hydrophobic modularity in protein sequences.

Motivation: Clusters of hydrophobic residues are known to promote structured protein stability and drive protein aggregation. Recent work has shown that identifying contiguous hydrophobic residue clusters (termed "blobs") has proven useful in both intrinsically disordered protein (IDP) simulation and human genome studies. However, a graphical interface was unavailable. Results: Here, we present the blobulator: an interactive and intuitive web interface to detect intrinsic modularity in any protein sequence based on hydrophobicity. We demonstrate three use cases of the blobulator and show how identifying blobs with biologically relevant parameters provides useful information about a globular protein, two orthologous membrane proteins, and an IDP. Other potential applications are discussed, including: predicting protein segments with critical roles in tertiary interactions, providing a definition of local order and disorder with clear edges, and aiding in predicting protein features from sequence. Availability: The blobulator GUI can be found at www.blobulator.branniganlab.org, and the source code with pip installable command line tool can be found on GitHub at www.GitHub.com/BranniganLab/blobulator.

Propofol directly binds and inhibits skeletal muscle ryanodine receptor 1 (RyR1).

As the primary Ca 2+ release channel in skeletal muscle sarcoplasmic reticulum (SR), mutations in the type 1 ryanodine receptor (RyR1) or its binding partners underlie a constellation of muscle disorders, including malignant hyperthermia (MH). In patients with MH mutations, exposure to triggering drugs such as the halogenated volatile anesthetics biases RyR1 to an open state, resulting in uncontrolled Ca 2+ release, sarcomere tension and heat production. Restoration of Ca 2+ into the SR also consumes ATP, generating a further untenable metabolic load. When anesthetizing patients with known MH mutations, the non-triggering intravenous general anesthetic propofol is commonly substituted for triggering anesthetics. Evidence of direct binding of anesthetic agents to RyR1 or its binding partners is scant, and the atomic-level interactions of propofol with RyR1 are entirely unknown. Here, we show that propofol decreases RyR1 opening in heavy SR vesicles and planar lipid bilayers, and that it inhibits activator-induced Ca 2+ release from SR in human skeletal muscle. In addition to confirming direct binding, photoaffinity labeling using m- azipropofol (AziP m ) revealed several putative propofol binding sites on RyR1. Prediction of binding affinity by molecular dynamics simulation suggests that propofol binds at least one of these sites at clinical concentrations. These findings invite the hypothesis that in addition to propofol not triggering MH, it may also be protective against MH by inhibiting induced Ca 2+ flux through RyR1.

Probing the Conformational Space of the Cannabinoid Receptor 2 and a Systematic Investigation of DNP-Enhanced MAS NMR Spectroscopy of Proteins in Detergent Micelles.

Tremendous progress has been made in determining the structures of G-protein coupled receptors (GPCR) and their complexes in recent years. However, understanding activation and signaling in GPCRs is still challenging due to the role of protein dynamics in these processes. Here, we show how dynamic nuclear polarization (DNP)-enhanced magic angle spinning nuclear magnetic resonance in combination with a unique pair labeling approach can be used to study the conformational ensemble at specific sites of the cannabinoid receptor 2. To improve the signal-to-noise, we carefully optimized the DNP sample conditions and utilized the recently introduced AsymPol-POK as a polarizing agent. We could show qualitatively that the conformational space available to the protein backbone is different in different parts of the receptor and that a site in TM7 is sensitive to the nature of the ligand, whereas a site in ICL3 always showed large conformational freedom.

Open-channel structure of a pentameric ligand-gated ion channel reveals a mechanism of leaflet-specific phospholipid modulation.

Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the activated, open-channel state of a pLGIC by specific, state-dependent binding to this site.

Best practices for resilient automation of biomolecular simulations.

As molecular dynamics simulation projects require ever-increasing amounts of simulation time, they become more complex to implement and more susceptible to unintended interruptions. This makes the task of the investigator in managing such projects more difficult and time-consuming. Here, we present a few potential operational problems that commonly arise in such simulation projects, possibly unanticipated considerations for solutions to these problems, and discussions of potential solutions that provide fault tolerance and automation. Example scripts are provided, along with the rationale for their design.

Ketamine Metabolite (2R,6R)-Hydroxynorketamine Interacts with µ and κ Opioid Receptors.

Ketamine is an anesthetic, analgesic, and antidepressant whose secondary metabolite (2R,6R)-hydroxynorketamine (HNK) has N-methyl-d-aspartate-receptor-independent antidepressant activity in a rodent model. In humans, naltrexone attenuates its antidepressant effect, consistent with opioid pathway involvement. No detailed biophysical description is available of opioid receptor binding of ketamine or its metabolites. Using molecular dynamics simulations with free energy perturbation, we characterize the binding site and affinities of ketamine and metabolites in µ and κ opioid receptors, finding a profound effect of the protonation state. G-protein recruitment assays show that HNK is an inverse agonist, attenuated by naltrexone, in these receptors with IC50 values congruous with our simulations. Overall, our findings are consistent with opioid pathway involvement in ketamine function.

Cholesterol as a modulator of cannabinoid receptor CB2 signaling.

Signaling through integral membrane G protein-coupled receptors (GPCRs) is influenced by lipid composition of cell membranes. By using novel high affinity ligands of human cannabinoid receptor CB2, we demonstrate that cholesterol increases basal activation levels of the receptor and alters the pharmacological categorization of these ligands. Our results revealed that (2-(6-chloro-2-((2,2,3,3-tetramethylcyclopropane-1-carbonyl)imino)benzo[d]thiazol-3(2H)-yl)ethyl acetate ligand (MRI-2646) acts as a partial agonist of CB2 in membranes devoid of cholesterol and as a neutral antagonist or a partial inverse agonist in cholesterol-containing membranes. The differential effects of a specific ligand on activation of CB2 in different types of membranes may have implications for screening of drug candidates in a search of modulators of GPCR activity. MD simulation suggests that cholesterol exerts an allosteric effect on the intracellular regions of the receptor that interact with the G-protein complex thereby altering the recruitment of G protein.

Mechanistic insights into volatile anesthetic modulation of K2P channels.

K2P potassium channels are known to be modulated by volatile anesthetic (VA) drugs and play important roles in clinically relevant effects that accompany general anesthesia. Here, we utilize a photoaffinity analog of the VA isoflurane to identify a VA-binding site in the TREK1 K2P channel. The functional importance of the identified site was validated by mutagenesis and biochemical modification. Molecular dynamics simulations of TREK1 in the presence of VA found multiple neighboring residues on TREK1 TM2, TM3, and TM4 that contribute to anesthetic binding. The identified VA-binding region contains residues that play roles in the mechanisms by which heat, mechanical stretch, and pharmacological modulators alter TREK1 channel activity and overlaps with positions found to modulate TASK K2P channel VA sensitivity. Our findings define molecular contacts that mediate VA binding to TREK1 channels and suggest a mechanistic basis to explain how K2P channels are modulated by VAs.

Azi-medetomidine: Synthesis and Characterization of a Novel α2 Adrenergic Photoaffinity Ligand.

Agonists at the α2 adrenergic receptor produce sedation, increase focus, provide analgesia, and induce centrally mediated hypotension and bradycardia, yet neither their dynamic interactions with adrenergic receptors nor their modulation of neuronal circuit activity is completely understood. Photoaffinity ligands of α2 adrenergic agonists have the potential both to capture discrete moments of ligand-receptor interactions and to prolong naturalistic drug effects in discrete regions of tissue in vivo. We present here the synthesis and characterization of a novel α2 adrenergic agonist photolabel based on the imidazole medetomidine called azi-medetomidine. Azi-medetomidine shares protein association characteristics with its parent compound in experimental model systems and by molecular dynamics simulation of interactions with the α2A adrenergic receptor. Azi-medetomidine acts as an agonist at α2A adrenergic receptors, and produces hypnosis in Xenopus laevis tadpoles. Azi-medetomidine competes with the α2 agonist clonidine at α2A adrenergic receptors, which is potentiated by photolabeling, and azi-medetomidine labels moieties on the α2A adrenergic receptor as determined by mass spectrometry in a manner consistent with a simulated model. This novel α2 adrenergic agonist photolabel can serve as a powerful tool for in vitro and in vivo investigations of adrenergic signaling.

Behavioural patterns of electrolyte repletion in intensive care units: lessons from a large electronic dataset.

Repletion of electrolytes often depends on provider-specific behavior and hospital policy. We examined the pattern of electrolyte repletion across several intensive care units (ICU) in a large healthcare system from 2010-2015. This included 109 723 potassium repletions, 51 833 magnesium repletions, 2 306 calcium repletions, 8 770 phosphate repletions, and 3 128 249 visit-days over 332 018 visits. Potassium, magnesium, and calcium were usually repleted within the institutional reference range. In contrast, the bulk of phosphate repletion was done with pre-repletion serum level below the reference range. The impact of repletion on post-repletion levels was significant but uniformly small. The pre-repletion serum level had a significant inverse correlation with the post-repletion level of each electrolyte. Potassium, magnesium and phosphate follow-up labs were scheduled in 9-10 hours after their repletion. In contrast, calcium was rechecked in less than 20 minutes. Routine repletion of potassium, magnesium and calcium had no effect on the incidence of tachyarrhythmias. We estimated the expense from electrolyte repletion within the reference range was approximately $1.25 million. Absent a specific clinical indication, repleting electrolytes when the serum concentration are within normative values may represent an avenue for cost savings, staff burden unload and potential reduction in frequency of complications in the ICUs.

Molecular Mechanics Parameterization of Anesthetic Molecules.

Anesthetic drug molecules are being increasingly studied through the use of computational methods such as molecular dynamics (MD). Molecular mechanics force fields require the investigator to supply parameters for the force field equation, which are not available for novel molecules. Careful selection of these parameters is critical for simulations to produce meaningful results. Therefore, this chapter presents a state-of-the-art method for determining these parameters by comparison to quantum mechanics calculations and experimental quantities. Ketamine is used as an example to demonstrate the process.

An Automated Critical Event Screening and Notification System to Facilitate Preanesthesia Record Review.

BACKGROUND: Anesthesia information management systems make prior anesthesia records readily available for review when patients return for a subsequent procedure but may create a problem of too much documentation to review in a limited amount of time. We implemented a screening tool to facilitate the identification of critical documentation for review. METHODS: An algorithm was developed to electronically search prior anesthesia records for predefined critical events and flag records containing these events. Our web-based daily case schedule was modified to contain a warning message for any patient on the schedule who has a prior record flagged by the system, in addition to a preexisting hyperlink to view the relevant record. A retrospective analysis was performed to determine the impact of the warning messages on the frequency with which the care team reviewed these records before providing anesthesia care. RESULTS: The screening algorithm flagged 13% of archived cases as critical. There were 3329 and 3369 cases in the 6 months before and after system implementation, respectively, that had prior critical records available for review at that time. One or more of these critical records were viewed before the subsequent case start in 39% vs 59% (P < .01) of cases in the pre- versus postimplementation periods. Subgroup analysis revealed that the increase was greatest for attending anesthesiologists working alone. CONCLUSIONS: We created a system to automatically detect critical events in prior anesthesia records for the purpose of forewarning the anesthesia care team when the same patient returns for another procedure. Inclusion of these warnings on the daily case schedule was associated with an increased frequency of preanesthesia review of old records.

iGAS: A framework for using electronic intraoperative medical records for genomic discovery.

OBJECTIVE: Design and implement a HIPAA and Integrating the Healthcare Enterprise (IHE) profile compliant automated pipeline, the integrated Genomics Anesthesia System (iGAS), linking genomic data from the Mount Sinai Health System (MSHS) BioMe biobank to electronic anesthesia records, including physiological data collected during the perioperative period. The resulting repository of multi-dimensional data can be used for precision medicine analysis of physiological readouts, acute medical conditions, and adverse events that can occur during surgery. MATERIALS AND METHODS: A structured pipeline was developed atop our existing anesthesia data warehouse using open-source tools. The pipeline is automated using scheduled tasks. The pipeline runs weekly, and finds and identifies all new and existing anesthetic records for BioMe participants. RESULTS: The pipeline went live in June 2015 with 49.2% (n=15,673) of BioMe participants linked to 40,947 anesthetics. The pipeline runs weekly in minimal time. After eighteen months, an additional 3671 participants were enrolled in BioMe and the number of matched anesthetic records grew 21% to 49,545. Overall percentage of BioMe patients with anesthetics remained similar at 51.1% (n=18,128). Seven patients opted out during this time. The median number of anesthetics per participant was 2 (range 1-144). Collectively, there were over 35 million physiologic data points and 480,000 medication administrations linked to genomic data. To date, two projects are using the pipeline at MSHS. CONCLUSION: Automated integration of biobank and anesthetic data sources is feasible and practical. This integration enables large-scale genomic analyses that might inform variable physiological response to anesthetic and surgical stress, and examine genetic factors underlying adverse outcomes during and after surgery.

Common Internal Allosteric Network Links Anesthetic Binding Sites in a Pentameric Ligand-Gated Ion Channel.

General anesthetics bind reversibly to ion channels, modifying their global conformational distributions, but the underlying atomic mechanisms are not completely known. We examine this issue by way of the model protein Gloeobacter violaceous ligand-gated ion channel (GLIC) using computational molecular dynamics, with a coarse-grained model to enhance sampling. We find that in flooding simulations, both propofol and a generic particle localize to the crystallographic transmembrane anesthetic binding region, and that propofol also localizes to an extracellular region shared with the crystallographic ketamine binding site. Subsequent simulations to probe these binding modes in greater detail demonstrate that ligand binding induces structural asymmetry in GLIC. Consequently, we employ residue interaction correlation analysis to describe the internal allosteric network underlying the coupling of ligand and distant effector sites necessary for conformational change. Overall, the results suggest that the same allosteric network may underlie the actions of various anesthetics, regardless of binding site.

A Retrospective Study of Success, Failure, and Time Needed to Perform Awake Intubation.

BACKGROUND: Awake intubation is the standard of care for management of the anticipated difficult airway. The performance of awake intubation may be perceived as complex and time-consuming, potentially leading clinicians to avoid this technique of airway management. This retrospective review of awake intubations at a large academic medical center was performed to determine the average time taken to perform awake intubation, its effects on hemodynamics, and the incidence and characteristics of complications and failure. METHODS: Anesthetic records from 2007 to 2014 were queried for the performance of an awake intubation. Of the 1,085 awake intubations included for analysis, 1,055 involved the use of a flexible bronchoscope. Each awake intubation case was propensity matched with two controls (1:2 ratio), with similar comorbidities and intubations performed after the induction of anesthesia (n = 2,170). The time from entry into the operating room until intubation was compared between groups. The anesthetic records of all patients undergoing awake intubation were also reviewed for failure and complications. RESULTS: The median time to intubation for patients intubated post induction was 16.0 min (interquartile range: 13 to 22) from entrance into the operating room. The median time to intubation for awake patients was 24.0 min (interquartile range: 19 to 31). The complication rate was 1.6% (17 of 1,085 cases). The most frequent complications observed were mucous plug, endotracheal tube cuff leak, and inadvertent extubation. The failure rate for attempted awake intubation was 1% (n = 10). CONCLUSIONS: Awake intubations have a high rate of success and low rate of serious complications and failure. Awake intubations can be performed safely and rapidly.

Convergent transmission of RNAi guide-target mismatch information across Argonaute internal allosteric network.

In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand "seed region" have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of a large number of internal allosteric pathways.

Thermodynamic basis of selectivity in guide-target-mismatched RNA interference.

Silencing in RNAi is strongly affected by guide-strand/target-mRNA mismatches. Target nucleation is thought to occur at positions 2-8 of the guide ("seed region"); successful hybridization in this region is the primary determinant of target-binding affinity and hence target cleavage. To define a molecular basis for the target sequence selectivity in RNAi, we studied all possible distinct single mismatches in seven positions of the seed region-a total of 21 substitutions. We report results from soft-core thermodynamic integration simulations to determine changes in targeting binding-free energies to Argonaute due to single mismatches in the guide strand, which arise during binding of an imperfectly matched target mRNA. In agreement with experiment, most mismatches impair target binding, consistent with a prominent role for binding affinity changes in RNAi sequence selectivity. Individual Argonaute residues located near the mismatched base pair are found to contribute significantly to binding affinity changes. We also use this methodology to analyze the mismatch-dependent free energy changes for dissociation of a DNA•RNA hybrid from Argonaute, as a model for the escape of miRNAs from the silencing pathway. Several mismatched sequences of the miRNA have increased affinity to Argonaute, implying that some mismatches may reduce the probability for escape. Furthermore, calculations of base-substitution-dependent free energy changes for binding ssDNA reveal mild sequence sensitivity as expected for guide strand binding to Argonaute. Our findings give a thermodynamic basis for RNAi target sequence selectivity and suggest that miRNA mismatches may increase silencing effectiveness and thus could be evolutionarily advantageous.