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Principal Investigator: Prakash Shrestha
Organization: UNIVERSITY OF KENTUCKY
Fiscal Year: 2024
Award: $120,258
Funding agency: National Institute of Allergy and Infectious Diseases
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its transmission across the
world was the cause of the COVID-19 pandemic. The CoV entry is mainly initiated by the binding of furin
cleavage activated spike (S) protein with ACE2 receptor protein on the surface of host cell. Next, S2’
cleavage by transmembrane protease serine 2 (TMPRSS2) on the cell surface induce the conformational
change of S2 subunit to activate the membrane fusion and entry of virus genetic material. Therefore, S
protein has been the major target to design the vaccines and therapeutics. While a handful of
investigations have been reported about the structure of S protein:ACE2 complex, critical questions about
the detachment of S1/S2 subunits, structural changes in S2 and their dynamics to activate the membrane
fusion remain. These answers will help to improve the current understanding of the mechanism of virus
entry and should be highly significant for the development of effective preventions and therapeutics.
I propose to combine novel DNA nanoswitch calipers (DNC), single-molecule fluorescence integrated
optical tweezers, and high-throughput magnetic tweezers to study the dynamics of the molecular events
associated with the membrane fusion process of viral entry and analyze the heterogeneity of neutralizing
antibodies (nAbs). Using DNC that I developed, we showed the measurement of multiple distances within a
target biomolecule at angstrom level precision. Next, we showed the high-throughput measurements in
magnetic tweezers to analyze the heterogeneous mixture of peptides. Therefore, DNCs are useful to study
multicomponent protein-protein interactions and simultaneously monitor the structural changes associated with
the process. By executing these projects, I will have the following three important answers. First, I will measure
binding dynamics of full-length S protein and monomer/dimer ACE2 receptor proteins to understand the entire
energy landscape governing the interaction of these proteins. Second, I will measure the detachment kinetics
of S1/S2 subunits, induced by TMPRSS2 and determine the change in conformation of S2. These reactions
are critical to understand the membrane fusion process. Third, I will utilize the similar approach to measure the
binding dynamics of nAbs with S protein. By high-throughput measurements in magnetic tweezers, I will
analyze the efficacy and heterogeneity of nAbs to understand the immune response of the patients and
vaccinated individuals, and also validate DNC assay with a cell-based pseudovirus neutralization assay.
Hence, the proposed work will provide detailed insight into understanding the mechanism of SARS-
CoV-2 entry and rapid analysis of immune response of the patients. My quantitative approaches and advanced
single-molecule approaches will bring insights, into the field of virology and immunology. This study will also
give me firsthand experience in biochemistry, molecular biology, virology, and immunology, enabling me to
better apply my quantitative and physical training to more topics in biological research in the future.