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Conductance and Ion Selectivity of a Mesoscopic Protein Nanopore Probed with Cysteine Scanning Mutagenesis

Petr G. Merzlyak ^*, Maria-Fatima P. Capistrano , Angela Valeva , John J. Kasianowicz  and Oleg V. Krasilnikov ^*

^* Laboratory of Membrane Biophysics, Department of Biophysics and Radiobiology, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil; Department of Biophysics and Pharmacology, Federal University of Rio Grande de Norte, Natal, Rio Grande de Norte, Brazil; Institute of Medical Microbiology and Hygiene, University of Mainz, Germany; and National Institute of Standards and Technology, Electronics and Electrical Engineering Laboratory, Semiconductor Electronics Division, Gaithersburg, Maryland 20899-8120

Correspondence: Address reprint requests to Dr. Oleg V. Krasilnikov, Universidade Federal de Pernambuco, Centro de Ciências Biológicas, Depto. de Biofísica e Radiobiologia, Av. prof. Moraes Rego, S/N, Cidade Universitária, Recife, Pernambuco, Brasil, CEP 50670-901. Tel.: 55-81-2126-8535; Fax: 55-81-2126-8560; E-mail: kras{at}ufpe.br.

Nanometer-scale proteinaceous pores are the basis of ion and macromolecular transport in cells and organelles. Recent studies suggest that ion channels and synthetic nanopores may prove useful in biotechnological applications. To better understand the structure-function relationship of nanopores, we are studying the ion-conducting properties of channels formed by wild-type and genetically engineered versions of Staphylococcus aureus -hemolysin (HL) reconstituted into planar lipid bilayer membranes. Specifically, we measured the ion selectivities and current-voltage relationships of channels formed with 24 different HL point cysteine mutants before and after derivatizing the cysteines with positively and negatively charged sulfhydryl-specific reagents. Novel negative charges convert the selectivity of the channel from weakly anionic to strongly cationic, and new positive charges increase the anionic selectivity. However, the extent of these changes depends on the channel radius at the position of the novel charge (predominately affects ion selectivity) or on the location of these charges along the longitudinal axis of the channel (mainly alters the conductance-voltage curve). The results suggest that the net charge of the pore wall is responsible for cation-anion selectivity of the HL channel and that the charge at the pore entrances is the main factor that determines the shape of the conductance-voltage curves.

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