Remodelling the cavity of a transmembrane pore by genetic engineering /
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| Other Authors: | , |
| Format: | Thesis eBook |
| Language: | English |
| Published: |
[College Station, Tex.] :
[Texas A&M University],
[2006]
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| Subjects: | |
| Online Access: | Link to OAK Trust copy |
| Abstract: | The cavity within the transmembrane staphylococcal a-hemolysin (aHL) pore is roughly a sphere of diameter ~45 Å (volume ~32,600 ų). The alpha-hemolysin gene was modified to introduce exogenous polypeptide sequences between positions 105 and 106 of aHL. These modified aHLs 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 aHL pore. By introducing tandem repeats of elastin-like polypeptide sequences (VPGGG), aHL 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 aHL 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 aHL pores provide numerous possibilities as tools for drug delivery, cryopreservation, or molecular sensing. |
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| Item Description: | "Major Subject: Medical Sciences" Title from author supplied metadata (automated record created on Sep. 15, 2006.) Vita. Abstract. Electronic resource. |
| Format: | Mode of access: World Wide Web. System requirements: World Wide Web access and Adobe Acrobat Reader. |
| Bibliography: | Includes bibliographical references. |