Designed protein pores as components for biosensors.
BACKGROUND
There is a pressing need for new sensors that can detect a variety of analytes, ranging from simple ions to complex compounds and even microorganisms. The devices should offer sensitivity, speed, reversibility and selectivity. Given these criteria, protein pores, remodeled so that their transmembrane conductances are modulated by the association of specific analytes, are excellent prospects as components of biosensors.


RESULTS
Structure-based design and a separation method that employs targeted chemical modification have been used to obtain a heteromeric form of the bacterial pore-forming protein staphylococcal alpha-hemolysin, in which one of the seven subunits contains a binding site for a divalent metal ion, M(II), which serves as a prototypic analyte. The single-channel current of the heteromer in planar bilayers is modulated by nanomolar Zn(II). Other M(II)s modulate the current and produce characteristic signatures. In addition, heteromers containing more than one mutant subunit exhibit distinct responses to M(II)s Hence, a large collection of responsive pores can be generated through subunit diversity and combinatorial assembly.


CONCLUSIONS
Engineered pores have several advantages as potential sensor elements: sensitivity is in the nanomolar range; analyte binding is rapid (diffusion limited in some cases) and reversible; strictly selective binding is not required because single-channel recordings are rich in information; and for a particular analyte, the dissociation rate constant, the extent of channel block and the voltage-dependence of these parameters are distinguishing, while the frequency of partial channel block reflects the analyte concentration. A single sensor element might, therefore, be used to quantitate more than one analyte at once. The approach described here can be generalized for additional analytes.
Braha
O
O
Department of Medical Biochemistry and Genetics, Texas A&M Health Science Center, College Station 77843-1114, USA.
Walker
B
B
Cheley
S
S
Kasianowicz
J J
JJ
Song
L
L
Gouaux
J E
JE
Bayley
H
H
D001427:Bacterial Toxins / Q000737:chemistry*; D015374:Biosensing Techniques; D002413:Cations, Divalent; D006460:Hemolysin Proteins / Q000737:chemistry*; D008051:Lipid Bilayers; D008958:Models, Molecular; D016296:Mutagenesis; D015202:Protein Engineering
D001427:Bacterial Toxins; D002413:Cations, Divalent; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; C021091:staphylococcal alpha-toxin