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Abstract:

our present work focuses on biophysical studies of Rab2B in cell-mimicking environments. Two of the mostly commonly used macromolecular crowders, Ficoll 70 and PEG 8000, have been used and their influence on the conformational properties of Rab2B (recombinantly expressed and purified) studied. The data obtained thus far show significant modulations of the structure and conformation of Rab2B in the crowded milieu.

Introduction:

Materials and Methods:

PAGE (Gel electrophoresis)
Dialysis
Column chromatography (Ni-NTA )
UV-Vis spectrophotometry
CD and steady state fluorimeter
Time resolved fluorescence spectroscopy (TCSPC)

References:

H. R. Bourne, D. A. Sanders and F. McCormick, The GTPase superfamily: conserved structure and molecular mechanism, Proc. Natl. Acad. Sci. USA, 1987, 84, 1871-1875.
Protein–protein interactions in a crowded environment Apratim Bhattachaya ,¹ Young C. Kim,² and Jeetain Mittal¹
Influence of crowded cellular environments on protein folding, binding, and oligomerization: Biological consequences and potentials of atomistic modeling : Edited by Wilhelm , by Huan-XiangZhou.
Irina M. Kuznetsova,^1,2 Konstantin K. Turoverov,^1,2 and Vladimir N. Uversky^1,3,4,5,*,What Macromolecular Crowding Can Do to a Protein, 2014 , 15(12): 23090–23140
Saikat Biswas,† Jayanta Kundu,† Sanjib K. Mukherjee, and Pramit Kumar Chowdhury*, Mixed Macromolecular Crowding: A Protein and Solvent Perspective, 2018 , 3(4), 4316–4330.

Acknowledgement:

I would like to express my deepest gratitude to DST-INSPIRE, IIT Delhi, my Supervisor and my fellow labmates. Thank You.

Conclusions:

Till now we have used two synthetic crowders PEG-8000 and F-70 to mimick the intracellular environment which shows formation of intermediate state in between native and denatured state of the protein. We have also studied the chemical and thermal denaturation of the protein which reveals information regarding the stability of the protein.

Department of Chemistry

Industrial Significance:

This protein controls vesicular traffic and has a close relationship with colon tumors. Hence, biophysical characterization of this protein may help to diagnose disease like colon cancer and will help to discover drug compatible to the disease.

Technology Readiness Level:

Rab2B in Cell Mimicking Environment: A Biophysical Perspective

Jyoti Rani Nath¹, Z.A. Parray¹, Pramit Kumar Chowdhury^1*

Email ID: cyz198125@iitd.ac.in

Result:

:: Biophysical Studies of Rab2B ::

:: UV-Visible Spectrophotometry ::

Industry Day Theme # (Others)

Rab proteins control vesicular traffic in Eukaryotic cells. Small GTPase Rab2 is a resident of pre-golgi intermediates and is required for protein transport from the Endoplasmic Reticulum to the Golgi complex. It has been proposed that Rab2B has a close relationship with colon tumors. These proteins act as molecular switches between a GDP-bound inactive form and GTP-bound active form in which they recruit downstream effectors onto membranes. On the other hand the intracellular environment is a rather crowded one with the concentration reaching values as high as 400 g/L and that too in an organelle dependent fashion, with the ER and Golgi typically having a very high local density of macromolecules.

TRL : 4

Technology Validated in Lab