Tailoring heme -thiolate proteins into efficient biocatalysts with high specificity and selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Tailoring Heme-Thiolate Proteins into Efficient Biocatalysts with High Specificity and Selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Controlled growth of carbon nanotubes for high performance nanoelectronics

Unique electrical and mechanical properties of single-walled carbon nanotubes (SWNTs) have made them one of the most promising candidates for next-generation nanoelectronics. Efficient utilization of the exceptional properties of SWNTs requires controlling their growth direction (e.g., vertical, horizontal) and morphologies (e.g., straight, junction, coiled). ^ In this dissertation, the catalytic effect on the branching of SWNTs, Y-shaped SWNTs (Y-SWNTs), was investigated. The formation of Y-shaped branches was found to be dependent on the composition of the catalysts. Easier carbide formers have a strong tendency to attach to the sidewall of SWNTs and thus enhance the degree of branching. Y-SWNTs based field-effect transistors (FETs) were fabricated and modulated by the metallic branch of the Y-SWNTs, exhibiting ambipolar characteristics at room temperature. A subthreshold swing of 700 mV/decade and an on/off ratio of 105 with a low off-state current of 10-13 A were obtained. The transport phenomena associated with Y- and cross-junction configurations reveals that the conduction mechanism in the SWNT junctions is governed by thermionic emission at T > 100 K and by tunneling at T < 100 K. ^ Furthermore, horizontally aligned SWNTs were synthesized by the controlled modification of external fields and forces. High performance carbon nanotube FETs and logic circuit were demonstrated utilizing the aligned SWNTs. It is found that the hysteresis in CNTFETs can be eliminated by removing absorbed water molecules on the CNT/SiO2 interface by vacuum annealing, hydrophobic surface treatment, and surface passivation. SWNT “serpentines” were synthesized by utilization of the interaction between drag force from gas flow and Van der Waals force with substrates. The curvature of bent SWNTs could be tailored by adjusting the gas flow rate, and changing the gas flow direction with respect to the step-edges on a single-crystal quartz substrate. Resistivity of bent SWNTs was observed to increase with curvature, which can be attributed to local deformations and possible chirality shift at curved part. ^ Our results show the successful synthesis of SWNTs having controllable morphologies and directionality. The capability of tailoring the electrical properties of SWNTs makes it possible to build an all-nanotube device by integrating SWNTs, having different functionalities, into complex circuits. ^

Participative Budgeting and Participant Motivation: A Review of the Literature

In their discussion - Participative Budgeting and Participant Motivation: A Review of the Literature - by Frederick J. Demicco, Assistant Professor, School of Hotel, Restaurant and Institutional Management, The Pennsylvania State University and Steven J. Dempsey, Fulton F. Galer, Martin Baker, Graduate Assistants, College of Business at Virginia Polytechnic Institute and State University, the authors initially observe: “In recent years behavioral literature has stressed the importance of participation In goal-setting by those most directly affected by those goals. The common postulate is that greater participation by employees in the various management functions, especially the planning function, will lead to improved motivation, performance, coordination, and functional behavior. The authors analyze this postulate as it relates to the budgeting process and discuss whether or not participative budgeting has a significant positive impact on the motivations of budget participants.” In defining the concept of budgeting, the authors offer: “Budgeting is usually viewed as encompassing the preparation and adoption of a detailed financial operating plan…” In furthering that statement they also furnish that budgeting’s focus is to influence, in a positive way, how managers plan and coordinate the activities of a property in a way that will enhance their own performance. In essence, framing an organization within its described boundaries, and realizing its established goals. The authors will have you know, to control budget is to control operations. What kind of parallels can be drawn between the technical methods and procedures of budgeting, and managerial behavior? “In an effort to answer this question, Ronen and Livingstone have suggested that a fourth objective of budgeting exists, that of motivation,” say the authors with attribution. “The managerial function of motivation is manipulative in nature.” Demicco, Dempsey, Galer, and Baker attempt to quantify motivation as a psychological premise using the expectancy theory, which encompasses empirical support, intuitive appeal, and ease of application to the budgetary process. They also present you with House's Path-Goal model; essentially a mathematics type formula designed to gauge motivation. You really need to see this. The views of Argyris are also explored in particular detail. Although, the Argyris study was primarily aimed at manufacturing firms, and the effects on line-supervisors of the manufacturing budgets which were used to control and evaluate their performance, its application is relevant to the hospitality industry. As the title suggests, other notables in the field of behavioral motivation theory, and participation are also referenced. “Behavioral theory has been moving away from models of purported general applicability toward contingency models that are suited for particular situations,” say the authors in closing. “It is conceivable that some time in the future, contingency models will make possible the tailoring of budget strategies to individual budget holder personalities.”

A self -testing approach for autonomic software

As researchers and practitioners move towards a vision of software systems that configure, optimize, protect, and heal themselves, they must also consider the implications of such self-management activities on software reliability. Autonomic computing (AC) describes a new generation of software systems that are characterized by dynamically adaptive self-management features. During dynamic adaptation, autonomic systems modify their own structure and/or behavior in response to environmental changes. Adaptation can result in new system configurations and capabilities, which need to be validated at runtime to prevent costly system failures. However, although the pioneers of AC recognize that validating autonomic systems is critical to the success of the paradigm, the architectural blueprint for AC does not provide a workflow or supporting design models for runtime testing. ^ This dissertation presents a novel approach for seamlessly integrating runtime testing into autonomic software. The approach introduces an implicit self-test feature into autonomic software by tailoring the existing self-management infrastructure to runtime testing. Autonomic self-testing facilitates activities such as test execution, code coverage analysis, timed test performance, and post-test evaluation. In addition, the approach is supported by automated testing tools, and a detailed design methodology. A case study that incorporates self-testing into three autonomic applications is also presented. The findings of the study reveal that autonomic self-testing provides a flexible approach for building safe, reliable autonomic software, while limiting the development and performance overhead through software reuse. ^