Previously I had worked in Prof. David Boehr’s lab as a postdoctoral associate where my main focus of research involved the use of methyl detected NMR experiments to analyze structural and dynamic characteristics of large molecular systems like RNA dependent RNA polymerase (RdRp).
My duties involved performing research full-time where I spend most of my time developing and refining NMR experiments to suit our immediate research needs. But as a part of learning experience of the graduate/undergraduate students at Drs. David Boehr’s, Scott Showalter’s and Junji Iwahara’s laboratories, I have helped and guided many students in their research endeavors. One of my skills is in my proficiency of data fitting which has helped laboratories where I have worked in the analysis of a wide spectrum of biophysical experiments (publication PDF).
Over the past academic years (2012-2015), I was working on the intrinsically disordered proteins (IDPs) project at Scott Showalter’s laboratory where I have been a vital resource in development of novel carbon detected NMR methods for the structural characterization of IDPs. My main focus was the structural characterization of intrinsically disordered proteins (IDPs). The other project in Showalter laboratory involves the study of interactions between small double-stranded RNA molecules called microRNA and proteins involved in microRNA maturation pathway. Initially this RNA project drew me to join Scott Showalter’s group in summer of 2011. The previous training during the graduate school had equipped me with understanding protein-nucleic acids interactions using NMR and other biophysical techniques. In parallel to the RNA project, I spent a considerable time developing new NMR based methods for understanding structural aspects of IDPs.
The structural elucidation of proteins and other biological macromolecules has been the central focus of the structural biology community over the last decade. Considerable effort has been put into the determination of three dimensional structure of biological macromolecules with the use of nuclear magnetic resonance (NMR), x-ray crystallography, small angle x-ray scattering (SAXS), small angle neutron scattering (SANS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM) etc. to bridge the gap between the primary and tertiary protein universe. These techniques provide a great insight into the three-dimensional arrangement of atom providing very detailed understanding of the mechanism of its biological function. This structure elucidation is only possible for proteins and other macromolecules that have a rigid three-dimensional structure, which only covers a very small portion of the complete proteome. It is also well understood that certain regions of biological molecules such as proteins remains unstructured. Sometimes these unstructured or regions with low structural complexity play a vital role in its biological function. For example, the c-terminal tail of high mobility group box 1 (HMGB1) contains a very acidic protein sequence that lacks defined 3D structure but is shown to interact with the structural regions of the protein to modulate and regulate its function. Another example of the unstructured protein is c-terminal tail of RNA polymerase that is studied for decades is known to be unstructured but plays a vital role in the regulation and modulation of transcription process. Conventional structural determination methods don’t work very well giving structural insights into these low-structurally complex protein segments. Hence there is a need for the structural understanding of these IDPs via development of new techniques and tools.
Intrinsically Disordered Proteins
IDPs are defined as proteins or regions of proteins that lack unique secondary and tertiary structure and usually can’t be crystallized by conventional methods. Some of the sequence hallmarks of IDPs are low sequence complexity and have a tendency of repeats. IDPs are usually depleted in non-polar residues and enriched in polar and charged residues with much higher prevalence of proline residue. IDPs are abundant in the proper working of biological processes acting as mediators in protein-protein interactions. These IDPs are over-represented in signaling pathways as transcription factors and “folding upon binding” has emerged as a new paradigm shift in mechanistic understanding of protein-protein interaction due to the flexible nature of these proteins. IDPs can form multiple bound conformation with multiple partners where one single segment will conform into the desired conformation for the corresponding interacting partner.
The structural complexity of IDPs pose a very challenging problem to the structural biology community where conventional methods are ill-suited. For example, structured proteins have a very well dispersed spectrum in a conventional 1H-15N hetero-nuclear single quantum coherence spectroscopy (HSQC, a very informative NMR experiment), on the other hand IDPs have very small chemical shift dispersion in the proton dimension. Due to this limitation structural biologist have not reported on IDPs over the years until the development of carbon-detect NMR methods. We were able to successfully apply 13C-15N hetero-nuclear coupled carbon detected experiments on different IDP systems that have substantial chemical shift dispersion in both dimensions for IDPs (Bruker Pulse Sequences can be found here). This has certain advantages over conventional NMR based structural methods and can be used as a great reporter of structural changes for IDPs.
13C detected NMR
I have had unique training in graduate school as well as in Showalter laboratory in the development of new NMR methods that can be used to study various biological systems. The NMR experiments are written as a pattern of radio frequency pulses at various frequencies, power and durations in a form called as pulse programming. The need for the development of 13C detected experiment is essential for the critical assessment of IDPs, one of the central focus of Showalter’s laboratory. Each carbon atom of the IDPs that is coupled with Nitrogen via a covalent bond giving rise to a NMR signal in an experiment, thus equating to one signal from each amino acid constituting the IDPs. These NMR signals are called chemical shifts that are sensitive to local chemical and electronic environment. The first step in the NMR based structural assignment is to assign the NMR chemical shifts to the corresponding covalent peptide bond connecting the amino acids. Conventional methods in NMR make use of proton, nitrogen and carbon chemical shifts in a three or more dimensional NMR experiment. Since conventional methods can’t be used in the case of IDPs as mentioned above, we were able to develop carbon detected three dimensional NMR experiments that help in the assignment of chemical shifts for IDPs. These experiments are carefully designed to provide both intra-residue as well as inter-residue information of IDPs. These NMR experiments are novel, unique and take a few days to give sequential assignment of IDPs’ chemical shifts, comparable to time scale involving conventional methods for structured proteins. A major portion of these novel NMR experiments are published here and here (Bruker Pulse Sequences can be found here). We have also developed amino-acid edited NMR experiments that show NMR signals corresponding to particular amino acids (Bruker Pulse Sequences can be found here). This helps in the chemical shift assignments of ambiguous signals.
The ultimate goal of the structural analysis of the IDPs is to gain functional knowledge providing details of its biological function. The breaking of conventional thinking of structural aspects of IDPs is one of the first steps in the understanding of IDPs functional role in biological processes. By the measurement of carbon detected NMR chemical shifts along with determination of one-, two- and three- bond scalar coupling and PRE measurements provide structural details that can be fit to hypothetical three dimensional models of IDPs. These models look like a bundle of spaghetti structures that conform to all the measured NMR experimental constraints. This is different from the conventional structural studies where one structure is enough to define the 3D protein structure and provide information on its function. This iterative process of fitting the three dimensional models into the experimental restraints is called ensemble analysis. These ensembles of IDP produced could represent their native structures that could play important role in its function. The development of these carbon detect based NMR tools will now empower us towards better understanding of IDPs’ structural characteristics and its function.
miRNA Structure mapping and RNA binding Proteins
My initial training in protein-nucleic acid interactions drew me to work in the microRNA project at Showalter laboratory. My previous experience in the molecular dynamics simulations and Scott Showalter’s aspirations to better understand mircoRNAs (miRNA) had propelled my focus on structural characterization of miRNAs. During the first year (2011-2012) at Showalter laboratory I had focused considerable time in molecular dynamics simulations on various segments of miRNAs. I had also worked along with other members in Showalter lab on various proteins involved in the miRNA maturation pathway. This had led to one publication (PDF) on RNA editing enzyme, Dicer’s interactions with double stranded RNA molecule. I have also worked with his fellow graduate student in the analysis, modelling and interpretation of the structural characterization of miRNAs using various modelling tools aided by SHAPE chemistry leading to a publication (PDF).
Kinetics of processes involving DNA binding proteins
During my time in graduate school (2006-2011) towards obtaining a doctorate degree with specialization in “Molecular Biophysics Education Track” under the supervision of Prof. Junji Iwahara in the Department of Biochemistry and Molecular Biology (BMB) and Sealy Center for Structural Biology (SCSB) at University of Texas Medical Branch at Galveston (UTMB), I focused mainly on processing involving protein-nucleic acid interactions using various biophysical methods, mainly nuclear magnetic resonance (NMR).
One of the main focuses during my doctorate research endeavor was understanding macromolecular interactions and its underlying kinetics. Macromolecular interactions do not just encompass the coming together of two or more macromolecules, but rather the speed of the interactions play a very important role in proper cellular function. In response to external stimuli, DNA binding proteins very efficiently carry out their cognate tasks by making use of different kinetic mechanisms. In my thesis I had quantitatively analyzed the kinetics of various processes involving DNA binding proteins (publication PDF, PDF, PDF, PDF, PDF, PDF).
The primary means of investigation of these macromolecular interactions in terms of structure and kinetics was done using nuclear magnetic resonance (NMR) spectroscopic methods. The theoretical description of how these NMR based methods enabling the analysis of kinetics involving macromolecules occurring at different time scales has been the inspiration of my study on macromolecular interactions (publication PDF, PDF, PDF,PDF). I was also involved in the development of TROSY based z-exchange methodology (a new NMR tool) to analyze the kinetics of translocation of a transcription factor, HoxD9 homeodomain, between two cognate DNA molecules showing the presence of different kinetic translocation mechanisms (publication PDF). One of the major mechanisms used by DNA binding protein is facilitated target search process, a kinetic shortcut, which defines the function of these DNA binding proteins and speeds up the target location. In order to understand these kinetic mechanisms, NMR based methods have been used to dissect the roles of different translocation processes and these biophysical methods are extensively analyzed using simulations to verify their validity range. Theoretical calculations were done to look into the effects of various microscopic events on the macroscopic rates measured using biophysical approaches. Another focus during my graduate study was redox regulatory systems, that continually keep the cellular environment reductive and the redox state of DNA binding proteins such as HMGB1 could play an important role in its function. The reduction/oxidation kinetics of HMGB1 along with redox regulatory proteins were extensively analyzed using quantitative NMR based methods (publication PDF). Overall my thesis provided an accurate understanding into the physiologically relevant macromolecular interactions in terms of kinetics by the development of novel biophysical methods, simulations and experiments.
I had worked with Prof. B. Jayaram at Indian Institute of Technology Delhi (IITD) on two projects directly related to my research endeavors. In 2005 I was involved in the creation of web-interface for the software used for de-novo protein folding and in the following year (2006) was involved in the creation of flexible Monte-Carlo simulation (molecular dynamics module) step in the de-novo protein folding pathway. This lead to a publication (PDF) while I was an undergraduate student at Anna University.