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dc.contributor.advisorBayley, Hagan
dc.contributor.advisorPace, Nick
dc.creatorJung, Yunhee
dc.date.accessioned2006-08-16T19:07:36Z
dc.date.available2006-08-16T19:07:36Z
dc.date.created2003-05
dc.date.issued2006-08-16
dc.identifier.urihttp://hdl.handle.net/1969.1/3880
dc.description.abstractThe cavity within the transmembrane staphylococcal α-hemolysin (αHL) pore is roughly a sphere of diameter ~45 Ã… (volume ~32,600 Ã…3). The alpha-hemolysin gene was modified to introduce exogenous polypeptide sequences between positions 105 and 106 of αHL. These modified αHLs were assembled either by themselves or with wild-type (W) subunits to form stable homoheptamers and heteroheptamers, respectively. First, the ability to accommodate Gly/Ser-rich polypeptide sequences in the central cavity was tested. Concatemerized Gly/Ser-containing sequences ("loops", L; L(10n + 5), n = 0 to 21) were inserted by genetic approaches. Detailed analysis of bilayer recordings and electrophoretic migration patterns of assembled pores indicate that the upper capacity of the cavity is ~175 amino acids. Then two different polypeptides were placed in the cavity to introduce novel functional properties to the αHL pore. By introducing tandem repeats of elastin-like polypeptide sequences (VPGGG), αHL pores (E101W6) that featured a temperature-responsive gating mechanism were obtained. The temperature-dependent properties of E101W6 pores were monitored by single-channel current recording in planar lipid bilayers. The amplitude and the frequency of the transient blockades increased as the temperature increased, while their duration decreased. The hydrophobic collapse of the inserted ELP loop is proposed for the source of the observed sigmoidal two-state transition for normalized closed states of E101W6 pores. Lastly, an αHL pore was designed to detect proteins from the cis side of the membrane. The heat-stable protein kinase inhibitor (PKI) sequence was inserted into the mid-position of the Gly/Ser loop, which was generated by previous project (L105 construct). The heteromeric pore with the PKI-containing loop (P1151W6) was able to detect cAMP-dependent protein kinase catalytic subunit (PKA) at single molecular level. These engineered αHL pores provide numerous possibilities as tools for drug delivery, cryopreservation, or molecular sensing.en
dc.format.extent17986753 bytesen
dc.format.mediumelectronicen
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.publisherTexas A&M Universityen
dc.subjectalpha-hemolysinen
dc.subjectre-designen
dc.titleRemodelling the cavity of a transmembrane pore by genetic engineeringen
dc.typeBooken
dc.typeThesisen
thesis.degree.departmentCollege of Medicineen
thesis.degree.disciplineMedical Sciencesen
thesis.degree.grantorTexas A&M Universityen
thesis.degree.nameDoctor of Philosophyen
thesis.degree.levelDoctoralen
dc.contributor.committeeMemberGiedroc, David
dc.contributor.committeeMemberJohnson, Arthur E.
dc.type.genreElectronic Dissertationen
dc.type.materialtexten
dc.format.digitalOriginborn digitalen


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