Advances in the REDCAT Software Package

Background Residual Dipolar Couplings (RDCs) have emerged in the past two decades as an informative source of experimental restraints for the study of structure and dynamics of biological macromolecules and complexes. The REDCAT software package was previously introduced for the analysis of molecular structures using RDC data. Here we report additional features that have been included in this software package in order to expand the scope of its analyses. We first discuss the features that enhance REDCATs user-friendly nature, such as the integration of a number of analyses into one single operation and enabling convenient examination of a structural ensemble in order to identify the most suitable structure. We then describe the new features which expand the scope of RDC analyses, performing exercises that utilize both synthetic and experimental data to illustrate and evaluate different features with regard to structure refinement and structure validation. Results We establish the seamless interaction that takes place between REDCAT, VMD, and Xplor-NIH in demonstrations that utilize our newly developed REDCAT-VMD and XplorGUI interfaces. These modules enable visualization of RDC analysis results on the molecular structure displayed in VMD and refinement of structures with Xplor-NIH, respectively. We also highlight REDCAT’s Error-Analysis feature in reporting the localized fitness of a structure to RDC data, which provides a more effective means of recognizing local structural anomalies. This allows for structurally sound regions of a molecule to be identified, and for any refinement efforts to be focused solely on locally distorted regions. Conclusions The newly engineered REDCAT software package, which is available for download via the WWW from http://ifestos.cse.sc.edu webcite, has been developed in the Object Oriented C++ environment. Our most recent enhancements to REDCAT serve to provide a more complete RDC analysis suite, while also accommodating a more user-friendly experience, and will be of great interest to the community of researchers and developers since it hides the complications of software development

Concurrent Identification, Characterization, and Reconstruction Of Protein Structure and Mixed-Mode Dynamics From RDC Data Using Redcraft

A complete understanding of the structure-function relationship of proteins requires an analysis of their dynamic behaviors and the static structure. However, all current approaches to studying dynamics in proteins have their shortcomings. A conceptually attractive and alternative approach simultaneously characterizes a protein\u27s structure and its intrinsic dynamics⁠. Ideally, such an approach could solely rely on RDC data-carrying both structural and dynamical information. The major bottleneck in utilizing RDC data in recent years has been attributed to a lack of RDC analysis tools capable of extracting the pertinent information embedded within this complex data source. Here we present a comprehensive strategy for structure calculation and reconstruction of discrete state dynamics from RDC data based on the SVD method of order tensor estimation. In addition to structure determination, we provide a mechanism of producing an ensemble of conformations for the dynamical regions of a protein from RDC data. The developed methodology has been tested on simulated RDC data with ۫Hz of error from an 83 residue α protein (PDB ID 1A1Z). In nearly all instances, our method reproduced the protein structure, including the conformational ensemble, within less than 2Å. Based on our investigations, arc motions with more than 30° of rotation are recognized as internal dynamics and are reconstructed with sufficient accuracy. Furthermore, states with relative occupancies above 20% are consistently recognized and reconstructed successfully. Arc motions with a magnitude of 15° or relative occupancy of less than 10% are consistently unrecognizable as dynamical regions within the context of ± 1Hz of error. We also introduce a computational approach named REDCRAFT that allows for uncompromised and concurrent characterization of protein structure and dynamics. We have subjected DHFR (PDB-ID 1RX2), a 159-residue protein, to a fictitious but plausible, mixed-mode internal dynamics model. In this simulation, DHFR was segmented into seven regions. The two dynamical and rigid-body segments experienced an average orientational modification of 7˚ and 12˚, respectively. Observable RDC data for backbone C\u27-N, N-H, and C\u27-H were generated from 102 frames that described the molecular trajectory. The Dynamic Profile generated by REDCRAFT allowed for the recovery of individual fragments with bb-rmsd of less than 1Å and the identification of different dynamical regions of the protein. Following the recovery of fragments, structural assembly correctly assembled the four rigid fragments with respect to each other, categorized the two domains that underwent rigid-body dynamics, and identified one dynamical region for which no conserved structure can be defined. In conclusion, our approach successfully identified dynamical domains, recovery of structure where it is meaningful, and relative assembly of the domains when possible

Structural characterization of helices A and B of Kv7.2 channel bound to Calmodulin. The calcium effect

217 p.Los canales neuronales heterodiméricos Kv7.2 y Kv7.3 son el sustrato molecular de la corriente depotasio no-inactivante dominada ¿Corriente M¿. Ésta juega un papel crítico en el control de laexcitabilidad neuronal. Su mal funcionamiento desencadena consecuencias fatales causantes deenfermedades como la epilepsia neonatal benigna (BFNC) que pueden estar asociadas a encefalopatias yretraso mental. La calmodulina es una proteína auxiliar sensible al calcio que es esencial para el correctoensamblaje, tráfico y función de dichos canales. Mientras que la compresión de la regulación de estoscanales ha alcanzado niveles razonables debido a estudios bioquímicos y electrofisiológicos, poco se sabesobre la estructura y conformación. El propósito de la presente tesis es el aumentar los conocimientosestructurales de los canales Kv7.2 con la meta de mejorar el entendimiento de cómo se da la regulaciónpor calcio de estos canales, y a su vez mejorar el conocimiento de los mecanismos por los que ciertaspatologías ocurren. Para ello la tesis presenta tres fases: 1) Mejora de la expresión recombinante del Cterminaldel canal, que se ha conseguido gracias a la coexpresion de la calmodulina y de la region Cterminaldel canal. 2) Caracterización estructural del complejo CaM/Hélices_AB mediante RMN, dondese observa cómo la calmodulina abraza dos helices del C-terminal (A y B) que están orientadas de maneraantiparalela. 3) Estudio del impacto del calcio sobre el complejo donde se demuestra que no se dancambios drásticos grandes en el complejo, con lo que se puede deducir que la señalización por calcio hade ser un mecanismo más sutil, posiblemente mediante cambios en las dinámicas locales o mediantemecanismos alostéricos

From Cellular to Holistic: Development of Algorithms to Study Human Health and Diseases

The development of theoretical computational methods and their application has become widespread in the world today. In this dissertation, I present my work in the creation of models to detect and describe complex biological and health related problems. The first major part of my work centers around the creation and enhancement of methods to calculate protein structure and dynamics. To this end, substantial enhancement has been made to the software package REDCRAFT to better facilitate its usage in protein structure calculation. The enhancements have led to an overall increase in its ability to characterize proteins under difficult conditions such as high noise and low data density. Secondly, a database that allows for easy and comprehensive mining of protein structures has been created and deployed. We show preliminary results for its application to protein structure calculation. This database, among other applications, can be used to create input sets for computational models for prediction of protein structure. Lastly, I present my work on the creation of a theoretical model to describe discrete state protein dynamics. The results of this work can be used to describe many real-world dynamic systems. The second major part of my work centers around the application of machine learning techniques to create a system for the automated detection of smoking using accelerometer data from smartwatches. The first aspect of this work that will be presented is binary detection of smoking puffs. This model was then expanded to perform full cigarette session detection. Next, the model was reformulated to perform quantification of smoking (such as puff duration and the time between puffs). Lastly, a rotational matrix was derived to resolve ambiguities of smartwatches due to position of the watch on the wrist

Structural characterization of helices A and B of Kv7.2 channel bound to Calmodulin. The calcium effect

217 p.Los canales neuronales heterodiméricos Kv7.2 y Kv7.3 son el sustrato molecular de la corriente depotasio no-inactivante dominada ¿Corriente M¿. Ésta juega un papel crítico en el control de laexcitabilidad neuronal. Su mal funcionamiento desencadena consecuencias fatales causantes deenfermedades como la epilepsia neonatal benigna (BFNC) que pueden estar asociadas a encefalopatias yretraso mental. La calmodulina es una proteína auxiliar sensible al calcio que es esencial para el correctoensamblaje, tráfico y función de dichos canales. Mientras que la compresión de la regulación de estoscanales ha alcanzado niveles razonables debido a estudios bioquímicos y electrofisiológicos, poco se sabesobre la estructura y conformación. El propósito de la presente tesis es el aumentar los conocimientosestructurales de los canales Kv7.2 con la meta de mejorar el entendimiento de cómo se da la regulaciónpor calcio de estos canales, y a su vez mejorar el conocimiento de los mecanismos por los que ciertaspatologías ocurren. Para ello la tesis presenta tres fases: 1) Mejora de la expresión recombinante del Cterminaldel canal, que se ha conseguido gracias a la coexpresion de la calmodulina y de la region Cterminaldel canal. 2) Caracterización estructural del complejo CaM/Hélices_AB mediante RMN, dondese observa cómo la calmodulina abraza dos helices del C-terminal (A y B) que están orientadas de maneraantiparalela. 3) Estudio del impacto del calcio sobre el complejo donde se demuestra que no se dancambios drásticos grandes en el complejo, con lo que se puede deducir que la señalización por calcio hade ser un mecanismo más sutil, posiblemente mediante cambios en las dinámicas locales o mediantemecanismos alostéricos