Protein characterization using a computational microscope: Nanopore sequencing and biological condensates

Proteins are the workhorses of the cell, forming the basic building block of life. The structure, shape and sequence of the proteins in the human cell modulate the major biological processes in our body. Deciphering the sequence of any protein in a single-molecule measurement and understanding the molecular mechanism of structural rearrangements are the two major roadblocks in our fight against curing several deadly diseases. This thesis focuses on these two major areas of research, with the first part on single-molecule identification and sequencing of proteins and the second part on elucidating the molecular conformations of intrinsically disordered proteins in biomolecular condensates. The first part consists of six chapters describing the following areas of research in protein sequencing. Firstly, we describe a robust and inexpensive computational approach, the steric exclusion model (SEM) to calculate ionic current in nanopore systems that is orders of magnitude more efficient than all-atom molecular dynamics (MD) and yet is sensitive enough to account for the atomic structure of the nanopore and the analyte. Then in collaboration with the Oukhaled Lab at U. Cergy Pontoise France, we show the molecular mechanism underlying nanopore recognition of all twenty proteinogenic amino acids, a critical step toward realization of single-molecule protein sequencing. In collaboration with the Behrends Lab at U. Freiburg Germany, we demonstrate the use of an engineered protein nanopore to differentiate isomeric peptides, derived from human histone H4 protein of identical mass according to the positions of acetylated lysine residues. In collaboration with the Maglia Lab at U. Groningen Netherlands, we uncover the molecular mechanism of nanopore mass spectrometry using a FraC nanopore that can capture peptides of varying charge. We then combined all-atom molecular dynamics simulations and electrophysiology experiments to determine the molecular mechanism of the ionic current modulation and improve resolution of detecting biochemical reactions at single-molecule resolution using engineered nanopores. Lastly, we demonstrate a proof-of-principle proteome characterization system that relies on ionic current signatures produced by complete translocation of natively folded proteins through an array of nanopores. In the second part, we provide the first molecular-level insights into the microscopic structure of a biological condensate. Firstly, we benchmarked the efficacy of nine presently available molecular force fields in describing the structure and dynamics of a fused in sarcoma (FUS) protein. We validated and implemented the most efficient force fields in NAMD simulation package, enabling microscopic simulations of biological condensate systems containing tens of millions of atoms.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Quant Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Sarthak, Kumar

Contributors dc:contributor

• Aksimentiev, Aleksei
• Grosman, Claudio F
• Shukla, Diwakar
• Tajkhorshid, Emad

• Nanopore Sequencing
• Protein Sequencing
• Biological Condensates
• Molecular Dynamics
• Protein Conformations

Statement dc:rights

• Copyright 2023 Kumar Sarthak

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/121253

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F121253