The type IV pilus (T4P) is a dynamic long thin fiber found on the surface of many bacterial groups. T4P is a versatile nanomachine; it plays many important roles such as for surface attachment, virulence factor, and surface motility apparatus. This research focuses on understanding the kinetics of PilB, the T4P assembly ATPase. PilB crystal structure exhibits an elongated hexamer with 2-fold symmetry indicating a symmetric rotary mechanism model. Except for its structure, the symmetric rotary mechanism of PilB has not been demonstrated experimentally. Its conformation and relatively low activity constrained previous in vitro studies of PilB. This study identified PilB from thermophilic organism Chloracidobacterium thermophilum (Ct) to be a model for in vitro studies. An active CtPilB was successfully expressed and purified as a hexamer. Malachite green phosphate assay was used to examine CtPilB ATPase activity. The examination indicated that CtPilB is a robust ATPase with a complex kinetics profile. The profile has a stepwise incline in ATPase activity as a function of [ATP] that led to a decline in higher [ATP]. The decline was confirmed to be a substrate inhibition by the enzyme-coupled assay. As for the incline, the detailed mechanism is still less clear to explain the multiphasic profile. The overall incline did not conform with classical Michaelis-Menten kinetic but the first part of the incline was shown to conform with Michaelis-Menten kinetics. The complex kinetics profile of PilB is consistent with the symmetric rotary mechanism of catalysis. / Master of Science / This research was conducted to understand type IV pilus (T4P), a hair-like structure found on the surface of many bacteria groups. T4P is a versatile structure; it plays many vital roles in bacterial life such as in surface motility, surface attachment, gene transfer, and virulence factor. Pilus is a dynamic polymer composed of many small pilin proteins that can be assembled or disassembled. Structurally, pilus is supported by machinery that helps to extend and retract pilus by adding or removing pilin proteins. At the core of the machinery, two different proteins are responsible to power the assemble and disassemble process by converting the chemical energy in ATP into mechanical energy. This study focuses on the protein that powers pilus assembly, PilB. Understanding PilB will be very beneficial in elucidating how the strongest biological motor work in action. The structure of PilB was determined to be a hexamer consist of six identical copies of the same protein forming a ring structure with 2-fold symmetry. This structure suggests that PilB works using symmetric rotary mechanism. Previous studies of PilB have not been productive because the purified PilB did not behave well during the assay. In this study, PilB from Chloracidobacterium thermophilum (CtPilB) was determined to be a reasonable model for the study. CtPilB was successfully purified and it was identified to have a robust activity outside the cell allowing for further biochemistry studies. The profile of CtPilB kinetics was unique and it did not conform with the classical kinetic profile. The analysis of the profile suggests that CtPilB exhibit a complex mechanism in hydrolyzing ATP.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/83819 |
| Date | 29 June 2018 |
| Creators | Sukmana, Andreas Binar Aji |
| Contributors | Biological Sciences, Yang, Zhaomin, Schubot, Florian D., Klemba, Michael |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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