Computational Investigation of Structural and Thermodynamic Properties of Beta-Barrel Membrane Proteins

Transmembrane (TM) proteins are important as they serve as gateways to permit substance transport or signaling transduction between interior and exterior of cells. Beta barrel membrane proteins (beta MPs) are one major type of TM proteins. They are solely found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplast. Beta MPs serve a multitude of essential cellular functions, including reaction catalysis, protein anchoring, metabolite transportation, and outer-membrane biogenesis. In bacteria, beta MPs are also found to be responsible for the release of virulence factors and are implicated in multidrug resistance. Dysfunctional beta MPs in mitochondria are also related to neurodegenerative diseases. The effective pore formation ability and the high stability in the membrane of beta MPs grant them huge potential in bionanotechnology. Beta MPs have drawn increasing attention in their promising application, including protein profiling, DNA sequencing, and small molecule detection. In order to investigate the roles of beta MPs in biological and pathological processes and to engineer or to design novel beta MPs for biotechnical applications, it is critical for us to understand structural and thermodynamical properties of beta MPs. Despite importance of beta MPs in biology and nanotechnology, limited availability of beta MP structures hinders understanding of their structural properties and structure-function relationship. It was estimated that there exist hundreds of beta MPs in each Gram-negative bacterium genome, while there are only around 60 non-homologous structures deposited in the Protein Data Bank when this study was conducted. This lack is due to the great difficulty of experimental determination of TM protein structures because of their amphipathic nature. It is therefore important to develop accurate and efficient computational structure prediction methods for these proteins. We have developed a method to predict the 3D structures of beta MPs. We predict strand registers and construct 3D structures of TM domains of beta MPs accurately, including proteins for which no prediction has been attempted before. Our method also accurately predicts structures from protein families with a limited number of sequences and proteins with novel folds. An average mainchain RMSD of 3.48A is achieved between predicted and experimentally resolved structures of TM domains, which is a significant improvement over a recent study. For beta MPs with NMR structures, the deviation between predictions and experimentally solved structures is similar to the difference among the NMR structures, indicating excellent prediction accuracy. Moreover, we can now accurately model the extended beta-barrels and loops in non-TM domains, increasing the overall coverage of structure prediction by 30%. In additional to structural properties, it is also important to characterize thermodynamical properties of beta MPs, which is important to understand their folding and stability, and may help in understanding the structure-function relationship. Free energy of transferring amino acid sidechains from aqueous environment into lipid bilayers, known as transfer free energy (TFE), provides important information on the thermodynamic stability of membrane proteins. However, experimental measurement of TFEs of beta MPs is challenging. A recent computational method has been developed to calculate TFEs, the results of which are in excellent agreement with experimentally measured values. However, the method does not scale up, and is limited to small beta MPs. We have improved this method and developed an approximation method, which is comparably accurate but much faster than the original method. The new method enables the calculation of TFEs of all beta MP regardless of the size of the proteins. With this method, we derived a TFE profile named General Transfer Free Energy Profile (GeTFEP) based on computation of the TFEs of 58 beta MPs. The GeTFEP agrees well with experimentally measured and computationally derived TFEs. Analysis based on the GeTFEP shows that residues in different regions of the TM segments of beta MPs have different roles during the membrane insertion process. Results further reveal the importance of the sequence pattern of TM strands in stabilizing beta MPs in the membrane environment. In addition, we show that GeTFEP can be used to predict the positioning and the orientation of beta MPs in the membrane. We also show that GeTFEP can be used to identify structurally or functionally important amino acid residue sites of beta MPs. Furthermore, the TM segments of helical membrane proteins can be accurately predicted with GeTFEP, suggesting that the GeTFEP captures fundamental thermodynamic properties of amino acid residues inside membrane, and is of general applicability in studying membrane protein. The methods reported in this thesis require only sequence information, implying their general applications to genome-wide studies. The structure prediction and the TFE characterization methods provide ways to investigate properties of novel beta MPs without conducting expensive wet lab experiments. They will also be useful in bionanotechnologies such as engineering existing beta MPs and in design novel ones

Regioisomerism in the Synthesis of a Chiral Aminotetralin Drug Compound: Unraveling Mechanistic Details and Diastereomer-Specific In-Depth NMR Investigations

During chemical process development of a novel 2-aminotetralin derivative intended for use as an antidepressant, scrutiny of the byproduct present in the drug molecule revealed a set of regioisomers. Detailed studies showed that this impurity issue originated from an early synthetic step in which a brominated tetralone motif was generated in a ring-closing protocol. It was found that this reaction was accompanied by a migration of the aromatic bromo substituent via different bromonium species along two discrete pathways. This example of the halogen dance reaction resulted in the formation of a series of tetralone impurities with a bromine distributed across all available aromatic positions of the tetralin nucleus. Subsequently, when subjected to reductive amination conditions, each of these tetralones gave rise to pairs of aminotetralins in a diastereomeric relationship. NMR investigations revealed that the alicyclic portion of the compounds thus formed displayed very complex signal patterns, which required further in-depth studies using a variety of sophisticated techniques. As a result, a deep insight into the structural features of the current 2-aminotetralin family was obtained, which is emphasized by the definition of a novel “0.2 ppm rule” allowing the absolute configuration at tetralin C-2 to be determined

Descriptive Analysis on the Impacts of Universal Zero-Markup Drug Policy on a Chinese Urban Tertiary Hospital

<div><p>Background</p><p>Universal Zero-Markup Drug Policy (UZMDP) mandates no price mark-ups on any drug dispensed by a healthcare institution, and covers the medicines not included in the China’s National Essential Medicine System. Five tertiary hospitals in Beijing, China implemented UZMDP in 2012. Its impacts on these hospitals are unknown. We described the effects of UZMDP on a participating hospital, Jishuitan Hospital, Beijing, China (JST).</p><p>Methods</p><p>This retrospective longitudinal study examined the hospital-level data of JST and city-level data of tertiary hospitals of Beijing, China (BJT) 2009–2015. Rank-sum tests and join-point regression analyses were used to assess absolute changes and differences in trends, respectively.</p><p>Results</p><p>In absolute terms, after the UZDMP implementation, there were increased annual patient-visits and decreased ratios of medicine-to-healthcare-charges (RMOH) in JST outpatient and inpatient services; however, in outpatient service, physician work-days decreased and physician-workload and inflation-adjusted per-visit healthcare charges increased, while the inpatient physician work-days increased and inpatient mortality-rate reduced. Interestingly, the decreasing trend in inpatient mortality-rate was neutralized after UZDMP implementation. Compared with BJT and under influence of UZDMP, JST outpatient and inpatient services both had increasing trends in annual patient-visits (annual percentage changes[APC] = 8.1% and 6.5%, respectively) and decreasing trends in RMOH (APC = -4.3% and -5.4%, respectively), while JST outpatient services had increasing trend in inflation-adjusted per-visit healthcare charges (APC = 3.4%) and JST inpatient service had decreasing trend in inflation-adjusted per-visit medicine-charges (APC = -5.2%).</p><p>Conclusion</p><p>Implementation of UZMDP seems to increase annual patient-visits, reduce RMOH and have different impacts on outpatient and inpatient services in a Chinese urban tertiary hospital.</p></div

Re-evaluating the geochronology of the Permian Tarim magmatic province: implications for temporal evolution of magmatism

<p>The Permian Tarim magmatic province has 118 published ages, ranging from 358 to 205 Ma, but the timing of mafic magmatism is not well constrained. We report two new secondary ion mass spectrometry U–Pb zircon dates on the Halahatang trachydacite and Wajilitag olivine clinopyroxenite, which are 287.2 ± 2.0 Ma and 283.2 ± 2.0 Ma, respectively. The trachydacite overlies the uppermost basalt and constrains the latest eruption age of basalt in northern Tarim. The latter is the first high-resolution date for the Wajilitag mafic layered intrusion. By screening all published ages, we identified 22 robust ages, ranging from 290.9 ± 4.1 to 261.7 ± 1.8 Ma. The robust ages together with our new data reveal a protracted period of mafic magmatism at <em>c</em>. 283 and <em>c</em>. 267 Ma. Silicic magmatism occurred from 291 to 272 Ma. Although the current known volume of Tarim basalt is too small to qualify as a large igneous province, the eroded and intrusive components, as well as pyroclastic deposits and silicic lavas, may increase the estimated volume. Further work is required to refine the duration of magmatism and the volume estimate of the province. </p

Trends in the inpatient mortality of Jishuitan Hospital at Xinjiekou campus, Beijing, China.

<p>Modeled Segment-1: a significant trend identified (annual percentage change[APC] = -24.31%, <i>P</i> = 0.029); Modeled Segment-2: no significant trends identified (<i>P</i> = 0.75).</p

The outpatient effects associated of the zero-markup drug policy implemented in Dec 2012 at the Jishuitan Hospital at Xinjiekou campus, Beijing, China in comparison with the Beijing tertiary hospitals.

<p>The outpatient effects associated of the zero-markup drug policy implemented in Dec 2012 at the Jishuitan Hospital at Xinjiekou campus, Beijing, China in comparison with the Beijing tertiary hospitals.</p

Summary of the differences in the trends of outpatient and inpatient services in Jishuitan hospital and the tertiary hospitals in Beijing, China.

<p>The years indicate the time segments with significant changes (<i>P</i><0.05) revealed using the join-point regression analysis. The dash-lined box indicates significant differences in the trends; JST: Jishuitan Hospital at Xinjiekou campus, Beijing, China; BJT: The tertiary hospitals in Beijing, China; ≈: No significant trends identified (<i>P</i>>0.05); → a join-point identified (join-point year in parenthesis); ↑: significantly increased annual percentage changes (<i>P</i><0.05); ↓: significantly decreased annual percentage changes (<i>P</i><0.05); * significant difference in trends between Jishuitan hospital and the tertiary hospitals in Beijing; # significant difference in trends between outpatient and inpatient services of Jishuitan hospital.</p

Theoretical framework.

<p>JST: Jishuitan Hospital at Xinjiekou campus, Beijing, China; OP: Outpatient; IP: Inpatient; ≈: No significant trends identified; ↑: significantly increased annual percentage changes (increasing trend); ↓: significantly decreased annual percentage changes (decreasing trend); * significant difference in trends between Jishuitan hospital and the tertiary hospitals in Beijing; # significant difference in trends between outpatient and inpatient services of Jishuitan hospital.</p

Comparison of the effects associated with the drug-price zero-markup policy implemented in Dec 2012 at the Jishuitan hospital at Xinjiekou campus, Beijing, China (Wilcoxon Rank-sum test).

<p>Comparison of the effects associated with the drug-price zero-markup policy implemented in Dec 2012 at the Jishuitan hospital at Xinjiekou campus, Beijing, China (Wilcoxon Rank-sum test).</p

The inpatient effects associated of the zero-markup drug policy implemented in Dec 2012 at the Jishuitan Hospital at Xinjiekou campus, Beijing, China in comparison with the Beijing tertiary hospitals.

<p>The inpatient effects associated of the zero-markup drug policy implemented in Dec 2012 at the Jishuitan Hospital at Xinjiekou campus, Beijing, China in comparison with the Beijing tertiary hospitals.</p