BS-MS Research Projects

Linker molecules for AFM measurement (Chemistry)
In recent years single molecule force spectroscopy has become a work-horse in understanding how these macromolecules function under controlled environments. The initial measurements in this field were mainly focused on measuring forces required to unfold a protein molecule using a sharp tip which is able to pull on a "single" molecule of interest. In last two decades force spectroscopy has become an important tool to understand how protein function is affected by its interaction with other small molecules. In order to take research in this area further, it is important that a protein is pulled selectively from either C terminus or N-terminus. It is also important to avoid effects of tip and sample surfaces as the molecule unfolds. We plan to develop some imaginative strategies to synthesize linker molecules, which will bind selectively to these protein ends, and also to tip or sample surfaces. See figure. Such molecular level handle allows pulling on single proteins in AFM experiments.
After construction of these handles and required bio-conjugation on tip and substrate, it will be used on a commercial AFM (JPK) in our lab.

Construction of poly-proteins for single molecule AFM measurements. (Biochemistry)
In a typical AFM pulling experiment, proteins are sparsely distributed over a surface and sharp tip is brought in contact with these proteins, and pulled away. This sequence, referred to as force curve measurement, is then repeated over an area of few square mircrometers. Such force measurements are spatially separated from each other by few nanometers. Although the surface is carefully cleaned and chemically treated, there is a possibility of stray organic impurities. A tip can pick up such an organic molecule and un-fold it as much as it can pick up a protein of interest deposited on the surface. It is difficult to correctly distinguish a protein un-folding event out of thousandsof such force curves. In order to avoid such spurious unfolding events entering into final analysis, researchers prepare poly-proteins, a repeated chain having 4 to 8 proteins connected to each other. A force curve showing repeated un-folding events,matching the number of protein units in a poly-protein chain is treated as data, whereas other force curves are ignored. See figure. In order to undertake protein pulling experiments, it is crucial to construct a poly-protein chain having repeats of the protein of interest. Using a polymerase chain reaction (PCR)-based strategy, one can synthesize genes that encode for multiple copies of the same protein connected to each other via covalent bonds. Such constructs can subsequently be expressed and purified from bacterial sources and used for AFM measurements. After construction of poly-proteins, it will be used for experiments with a JPK commercial AFM in our lab. The project will involve both construction of polyprotein and measurement using AFM and student will get trained on using AFM for pulling proteins.

Instrumentation: Development of scanned probe techniques

Development of small-amplitude AFM using fiber-based interferometer (Physics, Mechanical and Electronics engineering)
Currently, a fifth year BS-MS student is working on this project (Prashant Sharma). The instrument is ready. We plan to start protein pulling experiments with this new AFM. It has around 500 times more sensitivity in sensing cantilever displacements compared to commercial AFMs. We will be able to measure changes in cantilever amplitude of the order of fraction of an Å. Interested student will take this instrument further to perform actual measurements in order to see how this new methodology works in the area of protein folding.

Development of NSOM probes (Physics)
A fourth year student is already working on this project. We are in process of making first batch of tips ready. Interested student will take this further to use it in actual NSOM set-up partly developed.