Protein Structure Refinement Algorithms

Protein structure prediction has remained a major challenge in structural biology for more than half a century. Accelerated and cost efficient sequencing technologies have allowed researchers to sequence new organisms and discover new protein sequences. Novel protein structure prediction technologies will allow researchers to study the structure of proteins and to determine their roles in the underlying biology processes and develop novel therapeutics. Difficulty of the problem stems from two folds: (a) describing the energy landscape that corresponds to the protein structure, commonly referred to as force field problem; and (b) sampling of the energy landscape, trying to find the lowest energy configuration that is hypothesized to be the native state of the structure in solution. The two problems are interweaved and they have to be solved simultaneously. This thesis is composed of three major contributions. In the first chapter we describe a novel high-resolution protein structure refinement algorithm called GRID. In the second chapter we present REMCGRID, an algorithm for generation of low energy decoy sets. In the third chapter, we present a machine learning approach to ranking decoys by incorporating coarse-grain features of protein structures.</p

A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNAMet. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide–alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals

Replica Exchange Monte Carlo GRID: A novel high-resolution refinement algorithm

The energy-based refinement of protein structures to atomic-level accuracy remains a major challenge in structural biology. Energy-based refinement is mainly dependent on two components: (1) sufficiently accurate force fields, and (2) efficient conformational space search algorithms. Focusing on the latter, we developed a high-resolution Replica Exchange Monte Carlo-based refinement algorithm called REMC-GRID. This method takes a three-dimensional protein structure as input and, employing an all-atom force field, attempts to improve the energy of the structure by randomly selecting residues and perturbing the backbone dihedral angles. GRID, another refinement algorithm that we had developed previously, similarly improves the energy of protein structures, but is deterministic and perturbs the backbone dihedrals and conformation of all the residues in a sequential fashion. We applied REMC-GRID and GRID to 10 high-resolution (≤ 2.8 Å) crystal structures from the Protein Data Bank and measured the energy improvements obtained and the computation times required to achieve them. REMC-GRID produced better energy improvements than GRID alone and was only moderately more expensive in the use of computational resources. In another set of experiments, we created decoy structures by randomly perturbing the backbone dihedrals, and then tested the ability of the two algorithms to return the structures back to the native conformation. REMC-GRID resulted in better recapitulation of the native conformation than GRID as measured by backbone RMSD

GRID: A high-resolution protein structure refinement algorithm

The energy-based refinement of protein structures generated by fold prediction algorithms to atomic-level accuracy remains a major challenge in structural biology. Energy-based refinement is mainly dependent on two components: (1) sufficiently accurate force fields, and (2) efficient conformational space search algorithms. Focusing on the latter, we developed a high-resolution refinement algorithm called GRID. It takes a three-dimensional protein structure as input and, using an all-atom force field, attempts to improve the energy of the structure by systematically perturbing backbone dihedrals and side-chain rotamer conformations. We compare GRID to Backrub, a stochastic algorithm that has been shown to predict a significant fraction of the conformational changes that occur with point mutations. We applied GRID and Backrub to 10 high-resolution (≤ 2.8 Å) crystal structures from the Protein Data Bank and measured the energy improvements obtained and the computation times required to achieve them. GRID resulted in energy improvements that were significantly better than those attained by Backrub while expending about the same amount of computational resources. GRID resulted in relaxed structures that had slightly higher backbone RMSDs compared to Backrub relative to the starting crystal structures. The average RMSD was 0.25 ± 0.02 Å for GRID versus 0.14 ± 0.04 Å for Backrub. These relatively minor deviations indicate that both algorithms generate structures that retain their original topologies, as expected given the nature of the algorithms

Treatment of postoperative nausea and vomiting after spinal anesthesia for cesarean delivery: A randomized, double-blinded comparison of midazolam, ondansetron, and a combination

Background: The antiemetic efficacy of midazolam and ondansetron was shown before. The aim of the present study was to compare efficacy of using intravenous midazoalm, ondansetron, and midazolam in combination with ondansetron for treatment of nausea and vomiting after cesarean delivery in parturient underwent spinal anesthesia. Materials and Methods: One hundred thirty two parturients were randomly allocated to one of three groups: group M (n = 44) that received intravenous midazoalm 30 μg/kg; group O (n = 44) that received intravenous ondansetron 8 mg; group MO (n = 44) that received intravenous midazoalm 30 μg/kg combined with intravenous ondansetron 8 mg if patients had vomiting or VAS of nausea ≥ 3 during surgery (after umbilical cord clamping) and 24 hours after that. The incidence and severity of vomiting episodes and nausea with visual analog scale (VAS) > 3 were evaluated at 2 hours, 6 hours, and 24 hours after injection of study drugs. Results: The incidence of nausea was significantly less in group MO compared with group M and group O at 6 hours postoperatively (P = 0.01). This variable was not significantly different in three groups at 2 hours and 24 hours after operation. The severity of nausea and vomiting was significantly different in three groups at 6 hours after operation (P < 0.05). Conclusion: Our study showed that using intravenous midazolam 30 μg/kg in combination with intravenous ondansetron 8 mg was superior to administering single drug in treatment of emetic symptoms after cesarean delivery under spinal anesthesia

The Prevalence of Osmophobia in Migranous and Episodic Tension Type Headaches

Background: Migraines are a neurological disease, of which the most common symptom is an intense and disabling episodic headache. Many persons experience sensory hyper excitability manifested by photophobia, phonophobia and osmophobia. This study was planned to investigate the prevalence of osmophobia in migranous and episodic tension type headache (ETTH). Materials and Methods: A semi-structured questionnaire was administered to all patients to evaluate the eventual presence of osmophobia during a headache attack and different characteristics of osmophobia were determined. Results: Osmophobia reported in 84% with migranous headache with aura, 74% of migranous patients without aura and in 43.3% of those with ETTH. In 50% of patients, osmophobia was present in all of their headache attacks, 11.7% had osmophobia in more than half of their attacks (from 10 attacks they reported osmophobia in 5-9 ones) and others had this sign in less than half of their attacks (from 10 attacks they reported osmophobia in less than 5 ones). Most frequently the offending odors were scents (88%), foods (54.2%) and cigarette smoke (62.5%). Osmophobia starts 30 min before the headache starts in 22.7% of patients. Conclusion: Osmophobia appears structurally integrated into the migraine history of the patient; however, for differential diagnosis with ETTH, other criteria are necessary

Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein

A key mechanism that Neisseria gonorrhoeae uses to achieve multidrug resistance is the expulsion of structurally different antimicrobials by the MtrD multidrug efflux protein. MtrD resembles the homologous Escherichia coli AcrB efflux protein with several common structural features, including an open cleft containing putative access and deep binding pockets proposed to interact with substrates. A highly discriminating N. gonorrhoeae strain, with the MtrD and NorM multidrug efflux pumps inactivated, was constructed and used to confirm and extend the substrate profile of MtrD to include 14 new compounds. The structural basis of substrate interactions with MtrD was interrogated by a combination of long-timescale molecular dynamics simulations and docking studies together with site-directed mutagenesis of selected residues. Of the MtrD mutants generated, only one (S611A) retained a wild-type (WT) resistance profile, while others (F136A, F176A, I605A, F610A, F612C, and F623C) showed reduced resistance to different antimicrobial compounds. Docking studies of eight MtrD substrates confirmed that many of the mutated residues play important nonspecific roles in binding to these substrates. Long-timescale molecular dynamics simulations of MtrD with its substrate progesterone showed the spontaneous binding of the substrate to the access pocket of the binding cleft and its subsequent penetration into the deep binding pocket, allowing the permeation pathway for a substrate through this important resistance mechanism to be identified. These findings provide a detailed picture of the interaction of MtrD with substrates that can be used as a basis for rational antibiotic and inhibitor design. IMPORTANCE With over 78 million new infections globally each year, gonorrhea remains a frustratingly common infection. Continuous development and spread of antimicrobial-resistant strains of Neisseria gonorrhoeae, the causative agent of gonorrhea, have posed a serious threat to public health. One of the mechanisms in N. gonorrhoeae involved in resistance to multiple drugs is performed by the MtrD multidrug resistance efflux pump. This study demonstrated that the MtrD pump has a broader substrate specificity than previously proposed and identified a cluster of residues important for drug binding and translocation. Additionally, a permeation pathway for the MtrD substrate progesterone actively moving through the protein was determined, revealing key interactions within the putative MtrD drug binding pockets. Identification of functionally important residues and substrate-protein interactions of the MtrD protein is crucial to develop future strategies for the treatment of multidrug-resistant gonorrhea.This work was supported by a Flinders Medical Research Foundation grant and was undertaken using resources from the National Computational Infrastructure (NCI), which is supported by the Australian Government. M.C. was supported by an Australian Government Research Training Program Scholarship

Coordination of Substrate Binding and Protonation in the N. gonorrhoeae MtrD Efflux Pump Controls the Functionally Rotating Transport Mechanism

Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae. The drug efflux pump primarily implicated in N. gonorrhoeae antimicrobial resistance is the inner membrane transporter MtrD, which forms part of the tripartite multiple transferable resistance (Mtr) CDE efflux system. A structure of MtrD was first solved in 2014 as a symmetrical homotrimer, and then, recently, as an asymmetrical homotrimer. Through a series of molecular dynamics simulations and mutagenesis experiments, we identify the combination of substrate binding and protonation states of the proton relay network that drives the transition from the symmetric to the asymmetric conformation of MtrD. We characterize the allosteric coupling between the functionally important local regions that control conformational changes between the access, binding, and extrusion states and allow for transition to the asymmetric MtrD conformation. We also highlight a significant rotation of the transmembrane helices caused by protonation of the proton relay network, which widens the intermonomeric gap that is a hallmark of the rotational transporter mechanism. This is the first analysis and description of the transport mechanism for the N. gonorrhoeae MtrD transporter and provides evidence that antimicrobial efflux in MtrD follows the functionally rotating transport mechanism seen in protein homologues from the same transport protein superfamily.This work was supported by a Flinders Foundation Seeding Grant, an Australian Government Research Training Program Scholarship (to M.C.), and an ANU Research School of Chemistry Honors Scholarship (to V.G.). The research was undertaken with the assistance of resources and services from the National Computational Infrastructure (NCI), which is supported by the Australian Governmen