The long-term objectives of this project are two-fold: (1) to obtain a detailed understanding both of the - mechanism(s) by which channels are opened and closed (i.e., gated) and of the physico-chemical factors that govern transport through open channels; (2) to determine the relationship of the translocation of proteins and polypeptide chains across membranes to the formation and existence of wide-lumen channels. The methodology to be employed for achieving these goals is the study at both the macroscopic and single-channel level of the size, ion-selectivity, voltage-dependent properties, and stochastic behavior of channels incorporated into planar phospholipid bilayer membranes. These channels include those inserted into membranes by bacterial proteins such as diphtheria toxin, tetanus toxin, botulinum toxin, anthrax toxin, and colicins of the El class (El, Ia, Th and A). With respect to the first objective: the genes for the channel-forming proteins mentioned above have all been cloned and sequenced, so that detailed models of channel structure and gating can be developed. Moreover, models can be stringently tested by comparing properties of specifically modified channels (formed by site-mutated proteins) with their predicted behavior; this will be done mainly with diphtheria toxin, and colicin Ia. With respect to the second objective: all of the above-mentioned bacterial toxins consist of at least three domains, only one of which is necessary for channel formation. In diphtheria, anthrax, tetanus, and botulinum toxin, one of the other domains is an enzyme that must cross a vesicular membrane to enter the cytosol and thereby cause cell intoxication. Whether and how these or other domains of the toxin cross planar bilayers in conjunction with the opening and closing of the channels formed by their channel-forming domains will be investigated. Protein insertion into and translocation across cell membranes is crucial to cell function. An understanding of the structure of these channels and their role in protein translocation can lead to a more rational understanding and treatment of human diseases of diverse etiological origins. In addition, the elucidation of how the channel-forming domains of the toxins under study are involved in the translocation of the enzymatic (killing) domains to the cytosol should be of great help in the effective design of chimeric proteins that can function as """"""""magic bullets"""""""" in the killing of cancer cells.
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