Remodelling the cavity of a transmembrane pore by genetic engineering
The cavity within the transmembrane staphylococcal ?-hemolysin (?HL) 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 ?HL. These modified ?HLs 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 ?HL pore. By introducing tandem repeats of elastin-like polypeptide sequences (VPGGG), ?HL 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 ?HL 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 ?HL pores provide numerous possibilities as tools for drug delivery, cryopreservation, or molecular sensing.
Advisor:Bayley, Hagan; Pace, Nick; Giedroc, David; Johnson, Arthur E.
School:Texas A&M University
School Location:USA - Texas
Source Type:Master's Thesis
Keywords:alpha hemolysin re design
Date of Publication:05/01/2003