This Broadening Participation Research Initiation Grants in Engineering (BRIGE) grant provides funding to understand the mechanism of DNA motors dictating their moving polarity. DNA motors are molecular machines that move DNA in living cells to accomplish numerous essential tasks. Despite their enormous significance, our understanding of their mechanics that drives the motion of DNA is limited. Specifically, it remains largely elusive how a DNA motor maintains specific polarity with high fidelity when moving DNA strands, either from the 3' to the 5' end of a DNA strand for a 3'-5' motor, or the opposite for a 5'-3' motor. Surprisingly, the structures of a 3'-5' motor and a 5'-3' motor are similar, further challenging the mechanical studies of these motors to explain their distinct polarities. This project aims to study the mechanics of both 3'-5' and 5'-3' DNA motors to achieve the following two research goals: (1) Determine the force transduction of 3'-5' and 5'-3' DNA motors, and (2) Determine structural effects on the deformation of 3'-5' and 5'-3' DNA motors.
The mechanical studies of DNA motors will enable us to better understand the design principles of DNA motors, facilitating the future design of DNA motors with specified polarity. The results of the study will open doors to new applications such as motor-guided DNA sequencing, motor-delivered gene drugs, and motor-folded 3D DNA scaffolds for nanobiotechnology applications. In terms of education goals, this research will be highly interdisciplinary that requires knowledge in multiple areas, so it will advance the next-generation engineers in integrating knowledge learned from engineering and biological disciplines. This project will specifically emphasize the broadening of participation of underrepresented groups by providing research and lecture opportunities for minority high-school students.
Myosin VI possesses multiple unique features and is the only myosin known to move toward the (-)-end of actin filaments. This study used sequence and structure analyses to identify residues that are specific for myosin VI. Comparing the amino acids within myosin VI as well as between myosin VI and other myosins, we identified a series of myosin VI residues in the head region that are not only highly conserved in myosin VI, but whose amino acids are also distinct from those of other myosins. The majority of these myosin VI-specific residues have never been examined or speculated for their functional roles in myosin VI. Many of these residues locate in or adjacent to the converter domain. Among these residues, M701, P444, and F763 were revealed as unique for myosin VI. To further identify the mechanistic roles of these residues, we computationally mutated these three residues and examined their effects on conformational equilibrium using molecular dynamics simulation. We found that within a short 50-ns simulation, the conformational equilibria of mutated M701 and F763 were significantly deviated from the original states, indicating their important roles in linking the converter domain and the motor domain in the prestroke structure, potentially critical for the special rotation angle of myosin VI. Hepatitis C virus (HCV) NS3 helicase couples ATP binding and hydrolysis to polynucleotide unwinding. Understanding the regulation mechanism of ATP binding will facilitate targeting of the ATP binding site for potential therapeutic development for hepatitis C. T324, an amino acid residue connecting domains 1 and 2 of NS3 helicase, has been suggested as part of a flexible hinge for opening of the ATP binding cleft, although the detailed mechanism remains largely unclear. We used computational simulation to examine the mutational effect of T324 on the dynamics of the ATP binding site. A mutant model, T324A, of the NS3 helicase apo structure was created and energy minimized. Molecular dynamics simulation was conducted for both wild-type and the T324A mutant apo structures to compare their differences. For the mutant structure, histogram analysis of pairwise distances between residues in domains 1 and 2 (E291-Q460, K210-R464 and R467-T212) showed that separation between the two domains was reduced by ~10% and the standard deviation by ~33%. Root mean square fluctuation (RMSF) analysis demonstrated that residues in close proximity to residue 324 have at least 30% RMSF value reductions in the mutant structure. Solvent RMSF analysis showed that more water molecules are trapped near D290 and H293 in domain 1 to form an extensive interaction network constraining cleft opening. We also demonstrated that the T324A mutation established a new atomic interaction with V331, revealing that an atomic interaction cascade from T324 to residues in domains 1 and 2 controls the flexibility of the ATP binding cleft. DEAD-box proteins are RNA helicases ubiquitous in RNA metabolism. E.coli DbpA is a bacterial DEAD-box protein activated by 23S rRNA, a special and unique task important for its functions in ribosome biogenesis. However, the mechanism of the coupling between RNA binding and activities at the ATP binding site is unknown. In this study, we compared the seed alignments of the DEAD-box protein family and DbpA to determine 11 key unique residues specific to DbpA. To analyze the impact of key unique residues, each unique residue was computationally mutated and modeled. In total, 12 molecular dynamics (MD) simulations, including the one for the wild-type structure of DbpA and 11 for the single-mutation models, were conducted for 5 ns each. Results of each mutant model were compared to those of the wild-type to observe structural changes caused by the mutation of each unique residue. Model comparisons from 3 of the 11 unique residues, i.e. V170A, A333G, and V29I, revealed observable and quantifiable conformational changes at the ATP and RNA binding sites. Trajectories of minimum distances among residues, and model-to-model ratios of RMSF were used as parameters to quantify the structural effects of the mutations. Atomic interactions propagated from the mutational residues to both ATP and RNA binding sites were observed due to the changes of side chains of these mutations. We hypothesize these three residues play structural roles in the unique functions of DbpA in rRNA interactions. Future studies targeting key unique residues may reveal connected allosteric pathways within the protein structure and assist studying the diversity of function among DEAD-box proteins.
