We propose an investigation of the physiology and kinetic behavior of proteins that form channels in biological membranes. Type E1 colicins will be incorporated into planar lipid bilayers to investigate two as yet unsolved problems: the mechanism of assembly of membrane-spanning proteins, and the mechanism of ion transport and selectivity of membrane channels. Colicin proteins are encoded on extrachromosomal plasmids in host bacterial cells. Colicin E1 (56,000 Daltons) is produced in large quantities in a water soluble form by induced cells and its plasmid has been sequenced. Thus, it will be accessible to chemical and genetic manipulation. These features argue for its selection for this study. In preliminary studies we have established that the aqueous form binds tightly to membranes in a voltage independent step. Functional ion channels are formed subsequently only in response to applied voltage. We propose to determine at which step the protein inserts into the membrane, thereby exposing part of its structure in the aqueous phase on the opposite side. To do this, we will examine the ability of proteolytic enzymes and group specific reagents to attack the colicin protein from the opposite side of a planar bilayer. New methods for the formation of planar bilayers will be used to determine whether the bound form of the proteins is associated exclusively with the planar membrane, or with surface monolayers or other hydrophobic surfaces as well. Properties of the ion transport pathway will be investigated using a series of test ions of differing size, charge, shape, and chemical constituent groups. Determining which of these ions can permeate the pore and which can block it will provide information regarding the topography of the aqueous pore of the channel. Competition between blocking ions and permeant ions or other blockers will reveal which types of ionic interactions are allowed in this channel. These studies will provide the basis for understanding the general conformation and assembly of colicin channels and the basis for understanding the consequences of specific amino acid substitutions and deletions on channel properties.
NIH ExPorter
NIH ExPorter