The orthogonal space tempering (OST) scheme is a robust and high-order generalized ensemble sampling method that can make
sampling in MD simulation much more efficiently and recover the desired thermodynamic properties of interest in complex biophysical
protein behaviors. The OST technique provides a powerful tool to study the protein dynamics and mechanistic details of biophysical and
biochemical processes that are difficult to be realized by experimental techniques. Here the orthogonal space tempering technique is
applied to explore protein dynamics and interactions of actin monomer and TrmD methyltransferase. The first system studied is nucleotide
dependent dynamics of actin monomer. Nucleotide-dependent dynamics transition of actin has been studied for a decade. However, those
models proposed were based on crystal structures of monomer actin that are typically modified with mutation or complexed with blocking
agents to prevent polymerization, and thus subject to alteration. Additionally, typically experimental techniques cannot observe the
mechanistic transition in a time-dependent manner. Here dynamics of monomer actin with different states of bound nucleotide—ATP, ADP, and
apo form of actin—are investigated. Corresponding conformations and nucleotide binding cleft width-dependent free-energy profiles are
calculated via all-atom simulations based on OST scheme. Current results suggest G-actin-ATP prefers flat and closed conformation, while
G-actin-ADP and G-actin-APO prefers open conformation. Folding and unfolding motion of D-loop in subdomain 2 is observed in both G-actin
with ADP and ATP bound simulations. The dynamic conformations of G-actin-ATP are consistent with previous experimental results. Our
simulations will provide insights for the nucleotide-dependent mechanistic transitions and native structure of G-actin with ADP bound
after the free energy profiles converge. The second system of interest is the dynamics of TrmD tRNA (m1G37) methyltransferase. m1G37
methylation prevent frameshift errors in translation and is important for bacteria growth. TrmD methyltransferase has been considered as
an important drug target. Asymmetric features have been reported in TrmD catalysis and in the TrmD structure with sinefungin and tRNA
bound. Yet mechanistic details of TrmD catalysis remains elusive since TrmD with AdoMet and tRNA bound ternary structure has not been
resolved. Here free energy calculations based on OST scheme are performed on corresponding conformational changes with perturbation of
defined AdoMet dihedral angle on both active and inactive side of TrmD dimer. The conformation of AdoMet ligands and how both sides of
TrmD dimer coordinate with each other are investigated. Current results suggest the cooperativity of TrmD dimer is not through AdoMet
ligands on both sides but through the positioning of catalytic residues instead. Another Mg[superscript 2+] dependent TrmD methylation
simulation perturbing the distance between the target guanosine G37 and Mg[superscript 2+] is performed to study the function of
Mg[superscript 2+] in the catalysis. It is found that G37 base moves out the catalytic binding pocket while Mg[superscript 2+] approaches
the carbonyl group of G37, which suggests Mg[superscript 2+] is not involved in the transition state during methylation. Further,
conserved Mg[superscript 2+] binding motif (residue 162-179) is found in the disorder linker loop. In the simulation, Mg[superscript 2+]
binding motif interacts with G37 via Mg[superscript 2+] and water hydrogen bonding network while G37 is moving into the binding site.
Those results formulate a new hypothesis that Mg[superscript 2+] and Mg[superscript 2+] binding motif helps with flipping and positioning
of G37 into the catalytic binding pocket. After simulations complete, converged free energy profiles will test this new hypothesis and
disclose the actual mechanism of TrmD methylation catalysis. / A Thesis submitted to the Department of Chemistry & Biochemistry in partial fulfillment of the
Master of Science. / Fall Semester 2016. / September 27, 2016. / G-actin, orthogonal space tempering, simulation, TrmD methyltransferase / Includes bibliographical references. / Wei Yang, Professor Directing Thesis; James Frederich, Committee Member; Timothy Logan, Committee
Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_405657 |
| Contributors | Huang, Chi-Han (authoraut), Yang, Wei (professor directing thesis), Frederich, James H. (committee member), Logan, Timothy M. (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Chemistry and Biochemistry (degree granting departmentdgg) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource (90 pages), computer, application/pdf |
| Rights | This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them. |
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