Krutika Bavishi

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Krutika Bavishi’s research focuses on the dissection of the conformational dynamics of the P450 Oxidoreductase (POR). To achieve this she uses a single molecule approach which was recently also discussed in a review Krutika co-authored.

Research

In this project we are employing a single molecule fluorescence (smFRET) approach to study the conformational dynamics of the plant P450 oxidoreductase enzyme which is membrane appended and shuttles between “open” and “closed” conformations during the course of electron transfer to nature’s blow-torches- the Cytochromes P450s. The conformational dynamics are anticipated to be functionally relevant and dictating specificity towards its P450 partners. However, mechanistic details of these highly stochastic and unsynchronized structural dynamics are typically averaged out by ensemble (bulk) computational or NMR techniques. The single molecule (SM) regime harbors the potential to reveal the hidden dynamics of a system by providing a platform to directly observe and quantify the conformational fluctuations.

Inserting the fluorescently labeled enzymes on lipid patches called “nanodiscs” ensures we probe the protein structural dynamics in a system that mimics its native like environment. Besides, nanodiscs are highly monodisperse and uniform, posing as a great tool for biophysical studies.  A Total Internal Reflection Fluorescence (TIRF) microscope of high temporal resolution shall enable us to screen thousands of individual enzyme molecules in parallel to obtain high throughput SM data for statistical analysis.

On the other side, I am also involved with the Dhurrin pathway in Sorghum bicolor crop plant. I have conducting large scale expression /purification of its Cytochromes P450s enzymes (CYP79A1 and CYP71E1) using fermentation technology, which we reconstitute into nanodiscs/vesicles/amphipols and for use in a wide range of structural and functional studies. Furthermore, a detergent free technology (Styrene Maleic Acid (SMA) copolymer) has been used to isolate the Dhurrin “metabolon” from its native membranes.
(The PhD project is supported by EU-Marie Curie P4FIFTY - FP7 PEOPLE ITN 2011-289217)