Remodelling the cavity of a transmembrane pore by genetic engineering

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Title: Remodelling the cavity of a transmembrane pore by genetic engineering
Author: Jung, Yunhee
Abstract: The cavity within the transmembrane staphylococcal & #945 ; -hemolysin ( & #945 ;HL ) pore is roughly a sphere of diameter ~45 ? ? (volume ~32 ,600 ? ?3 ) . The alpha -hemolysin gene was modified to introduce exogenous polypeptide sequences between positions 105 and 106 of & #945 ;HL . These modified & #945 ;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 & #945 ;HL pore . By introducing tandem repeats of elastin -like polypeptide sequences (VPGGG ) , & #945 ;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 & #945 ;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 & #945 ;HL pores provide numerous possibilities as tools for drug delivery , cryopreservation , or molecular sensing .
URI: http : / /hdl .handle .net /1969 .1 /3880
Date: 2006-08-16


Remodelling the cavity of a transmembrane pore by genetic engineering. Available electronically from http : / /hdl .handle .net /1969 .1 /3880 .

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