Heather L. Gordon

The Leaf Epidermome of Catharanthus roseus Reveals Its Biochemical Specialization

Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis–enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-long-chain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed.

Evidence that histidine forms a coordination bond to the A0A and A0B chlorophylls and a second H-bond to the A1A and A1B phylloquinones in M688HPsaA and M668HPsaB variants of Synechocystis sp. PCC 6803

The axial ligands of the acceptor chlorophylls, A(0A) and A(0B), in Photosystem I are the Met sulfur atoms of M688(PsaA) and M668(PsaB). To determine the role of the Met, His variants were generated in Synechocystis sp. PCC 6803. Molecular dynamics simulations on M688H(PsaA) show that there exist low energy conformations with the His coordinated to A(0A) and possibly H-bonded to A(1A). Transient EPR studies on M688H(PsaA) indicate a more symmetrical electron spin distribution in the A(1A) phyllosemiquinone ring consistent with the presence of an H-bond to the C1 carbonyl. Ultrafast optical studies on the variants show that the 150fs charge separation between P₇₀₀ and A(0) remains unaffected. Studies on the ns timescale show that 57% of the electrons are transferred from A(0A)(-) to A(1A) in M688H(PsaA) and 48% from A(0B)(-) to A(1B) in M668H(PsaB); the remainder recombine with P₇₀₀(+) with 1/e times of 25ns and 37ns, respectively. Those electrons that reach A(1A) and A(1B) in the branch carrying the mutation are not transferred to FX, but recombine with P₇₀₀(+) with 1/e times of ~15μs and ~5μs, respectively. Hence, the His is coordinated to A0 in all populations, but in a second population, the His may be additionally H-bonded to A(1). Electron transfer from A(0) to A(1) occurs only in the latter, but the higher redox potentials of A(0) and A(1) as a result of the stronger coordination bond to A(0) and the proposed second H-bond to A(1) preclude electron transfer to the Fe/S clusters.

A ‘Partially Clothed’ Pivot Algorithm for Model Polyelectrolyte Solutions

Abstract For the Monte Carlo study of polymer configurations, the “pivot algorithm” has proved very effective for both lattice and continuum models. Its efficiency deteriorates for a polyion immersed in a bath of free ions, however, since to pivot a substantial segment of the polyion without any of its sheath of counterions carries a large energy expense. This article reports an attempt to relieve that problem by including, in pivot moves, a portion of the ion atmosphere. It is shown that this leads to substantial gains, at least for moderately low ionic concentrations, and allows useful estimation of difficult quantities, such as persistence length.

Efficient generation of low-energy folded states of a model protein. II. Automated histogram filtering

A number of short, Monte Carlo simulated annealing runs are performed on a highly frustrated 69-mer off-lattice model protein, consisting of a chain of 69 beads that are either hydrophobic, hydrophilic, or neutral in nature, and which demonstrably folds into a six-stranded β-barrel structure. We employ an iterative, consensus-based scheme to cluster the 725 nonbonded distances between the hydrophobic beads using, in tandem, Ward’s method for hierarchical clustering and k-means partitional clustering. We also independently analyze the same data using computer-automated histogram filtering, a technology designed to cluster high-dimensional data, without the tedium and subjectivity required by our iterative implementation of the two classical clustering methods. The memberships of low-energy clusters obtained from both classical clustering and automated histogram filtering approaches are remarkably similar. Nonbonded distance constraints are derived from these clusters and from small sets of the original unc...

Efficient generation of low-energy folded states of a model protein

A number of short simulated annealing runs are performed on a highly-frustrated 46-“residue” off-lattice model protein. We perform, in an iterative fashion, a principal component analysis of the 946 nonbonded interbead distances, followed by two varieties of cluster analyses: hierarchical and k-means clustering. We identify several distinct sets of conformations with reasonably consistent cluster membership. Nonbonded distance constraints are derived for each cluster and are employed within a distance geometry approach to generate many new conformations, previously unidentified by the simulated annealing experiments. Subsequent analyses suggest that these new conformations are members of the parent clusters from which they were generated. Furthermore, several novel, previously unobserved structures with low energy were uncovered, augmenting the ensemble of simulated annealing results, and providing a complete distribution of low-energy states. The computational cost of this approach to generating low-energy conformations is small when compared to the expense of further Monte Carlo simulated annealing runs.A number of short simulated annealing runs are performed on a highly-frustrated 46-“residue” off-lattice model protein. We perform, in an iterative fashion, a principal component analysis of the 946 nonbonded interbead distances, followed by two varieties of cluster analyses: hierarchical and k-means clustering. We identify several distinct sets of conformations with reasonably consistent cluster membership. Nonbonded distance constraints are derived for each cluster and are employed within a distance geometry approach to generate many new conformations, previously unidentified by the simulated annealing experiments. Subsequent analyses suggest that these new conformations are members of the parent clusters from which they were generated. Furthermore, several novel, previously unobserved structures with low energy were uncovered, augmenting the ensemble of simulated annealing results, and providing a complete distribution of low-energy states. The computational cost of this approach to generating low-ener...

Introduction of a hydrogen bond between phylloquinone PhQA and a threonine side-chain OH group in photosystem i

The phylloquinone acceptor PhQ(A) in photosystem I binds to the protein through a single H-bond to the backbone nitrogen of PsaA-L722. Here, we investigate the effect of this H-bond on the electron transfer (ET) kinetics by substituting threonine for PsaA-L722. Room temperature spin-polarized transient EPR measurements show that in the PsaA-L722T mutant, the rate of PhQ(A)(-) to F(X) ET increases and the hyperfine coupling to the 2-methyl group of PhQ(A) is much larger than in the wild type. Molecular dynamics simulations and ONIOM type electronic structure calculations indicate that it is possible for the OH group of the Thr side chain to form an H-bond to the carbonyl oxygen atom, O(4) of the phylloquinone, and that this results in an increase in the 2-methyl hyperfine couplings as observed in the transient EPR data. The Arrhenius plot of the PhQ(A)(-) to F(X) ET in the PsaA-L722T mutant suggests that the increased rate is probably the result of a slight change in the electronic coupling between PhQ(A)(-) and F(X). The strong deviation from Arrhenius behavior observed at ∼200 K can be reproduced using a semiclassical model, which takes the zero-point energy of the mode coupled to the ET into account. However, since the change in slope of the Arrhenius plot occurs at the protein glass transition temperature, it is argued that it could be the result of a change in the protein relaxation dynamics at this temperature rather than quantum mechanical effects.

Structure propensities in mutated polyglutamine peptides

Polyglutamine is a naturally occurring peptide found within several proteins in neuronal cells of the brain, and its aggregation has been implicated in several neurodegenerative diseases, including Huntington’s disease. The resulting aggregates have been demonstrated to possess β-sheet structure, and experimental evidence has demonstrated that aggregation begins with a nucleus composed of a single peptide. In this paper, we computationally examined the structural tendencies of mutant polyglutamine peptides that were studied experimentally, and found to aggregate with varying efficiencies. Low-energy structures were generated for each peptide by simulated annealing molecular dynamics, and were analyzed quantitatively by various geometry-based methods. In all simulations, the carboxy-terminal end of each peptide was constrained to a β-turn-β-strand structure to simulate a situation in which β-structure formation has initiated due to interaction with a seed or a growing oligomer/aggregate. Our results suggest the experimentally-observed inhibition of aggregation to be due to localized conformational restraint on the peptide backbone, which in turn confines the peptide to native coil structure, discouraging transition towards the β-sheet structure required for aggregation.

PRIORITY SETTING IN AN ACUTE CARE HOSPITAL IN ARGENTINA: A QUALITATIVE CASE STUDY

Purpose: To describe and evaluate priority setting in an Acute Care hospital in Argentina, using Accountability for Reasonableness, an ethical framework for fair priority setting. 
Methods: Case Study involving key informant interviews and document review. Thirty respondents were identified using a snowball sampling strategy. A modified thematic approach was used in analyzing the data. 
Results: Priorities are primarily determined at the Department of Health. The committee which is supposed to set priorities within the hospital was thought not to have much influence. Decisions were based on government policies and objectives, personal relationships, economic, political, historical and arbitrary reasons. Decisions at the DOH were publicized through internet; however, apart from the tenders and a general budget, details of hospital decisions were not publicized. CATA provided an accessible but ineffective forum for appeals. There were no clear mechanisms for appeals and leadership to ensure adherence to a fair process.
Conclusions: In spite of their efforts to ensure fairness, Priority setting in the study hospital did not meet all the four conditions of a fair process. Policy discussions on improving legitimacy and fairness provided an opportunity for improving fairness in the hospital and Accountability for Reasonableness might be a useful framework for analysis and for identifying and improving strategies.

Protein structure generation and elucidation: applications of automated histogram filtering cluster analysis

Abstract This chapter reviews automated histogram filtering (AHF), a recently developed approach to cluster analysis, and describes two applications of this methodology, illustrating its power as a tool for analysis of biomolecular simulation data. The first is to cluster low-energy conformations of a model protein, generated by Monte Carlo-simulated annealing. From ensuing cluster memberships, we arrive at distance constraints to serve as input to distance-geometry software. Additional low-energy structures are subsequently generated by the latter software with remarkable efficiency. The second application is to cluster molecular dynamics snapshots of an all-atom model of the response regulator protein Spo0F of Bacillus subtilis . Two clusters are uncovered, reflecting our having sampled two distinct basins on the potential energy surface. We subject snapshots for each cluster to AHF cluster analysis, and perform an orthogonal rotation of the dominant so-called ‘principal components’. We monitor the loadings of each interatomic distance variable along the rotated principal components. To visualize our results, we construct squared-loadings plots, revealing secondary structural elements that undergo concerted motions along so-called ‘collective coordinates’. Dominant collective coordinates are found to be associated with contiguous stretches of amino acid residues implicated in the biological functioning of Spo0F.

Water-mediated interactions in the CRP-cAMP-DNA complex: Does water mediate sequence-specific binding at the DNA primary-kink site?

The cyclic AMP receptor protein (CRP) of Escherichia coli binds preferentially to DNA sequences possessing a T:A base pair at position 6 (at which the DNA becomes kinked), but with which it does not form any direct interactions. It has been proposed that indirect readout is involved in CRP-DNA binding, in which specificity for this base pair is primarily related to sequence effects on the energetic susceptibility of the DNA to kink formation. In the current study, the possibility of contributions to indirect readout by water-mediated hydrogen bonding of CRP with the T:A base pair was investigated. A 1.0 ns molecular dynamics simulation of the CRP-cAMP-DNA complex in explicit solvent was performed, and assessed for water-mediated CRP-DNA hydrogen bonds; results were compared to several X-ray crystal structures of comparable complexes. While several water-mediated CRP-DNA hydrogen bonds were identified, none of these involved the T:A base pair at position 6. Therefore, the sequence specificity for this base pair is not likely enhanced by water-mediated hydrogen bonding with the CRP.