title: Engineering K+ channels using semisynthesis.
abstract: Potassium channels conduct K(+) ions selectively and at very high rates. Central to the function of K(+) channels is a structural unit called the selectivity filter. In the selectivity filter, a row of four K(+) binding sites are created using mainly the backbone carbonyl oxygen atoms. Due to the involvement of the protein backbone, site-directed mutagenesis is of limited utility in investigating the selectivity filter. In order to overcome this limitation, we have developed a semisynthetic approach, which permits the use of chemical synthesis to manipulate the selectivity filter. In this chapter, we describe the protocols that we have developed for the semisynthesis of the K(+) channel, KcsA. We anticipate that the protocols described in this chapter will also be applicable for the semisynthesis of other integral membrane proteins of interest.
authors.affiliation: Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR, USA.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Komarov
authors.forename: Corey A
authors.initials: CA
authors.lastname: Costantino
authors.forename: Francis I
authors.initials: FI
authors.lastname: Valiyaveetil
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000302:isolation & purification; D004926:Escherichia coli; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000302:isolation & purification; D060327:Solid-Phase Synthesis Techniques
chemical_list: D001426:Bacterial Proteins; D015221:Potassium Channels; D011994:Recombinant Proteins; C107790:prokaryotic potassium channel