DE NOVO DESIGN OF FUNCTIONAL CHANNEL PROTEINS

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The ultimate goal of the program is to identify the fundamental principles that determine the biological design of channel proteins. The immediate goal is to realize the molecular design of a pore-forming structure and to use it towards understanding the molecular basis of ionic selectivity, channel blockade and voltage regulation of channel open probability. The central notion is that given the primary structure of channel proteins it may be possible to identify functional modules that will fold predictably into stable structural motifs and fulfill functional attributes of the authentic system. A first step in this endeavor is to model only the most fundamental unit of function of ion channels, namely the pore-forming structure. A plausible molecular blueprint for the pore-forming structure of channel proteins is a bundle of amphipathic alpha-helices that cluster together to generate a hydrophilic channel. Such pore structures are designed from functional modules that represent the amino acid sequence of authentic proteins and refined to accommodate specific functional characteristics. The next level of complexity incorporates the voltage- sensing device and considers the design of a minimum voltage-gated channel that would exhibit the essential pore properties of ionic selectivity with the additional regulation by transmembrane potential. The approach involves: (1) Formulation of a structural model of the protein based on sequence analysis and secondary structure predictions; (2) Conformational energy calculations to assess the validity of the proposed structural motifs, to design "computer mutations" to guide experimental design, and to obtain quantitative descriptions of the energy profile for ionic diffusion through the designed channels; [3] Synthesis of the designed structures by solid-phase peptide synthesis and by direct expression of synthetic genes encoding the designed channel proteins; [4] Functional analysis of the designed channels by reconstitution of synthetic proteins in planar lipid bilayers and by expression of corresponding cRNA in amphibian oocytes or cDNA in mammalian cells. Single channel current recordings under voltage-clamp conditions provide a detailed set of functional parameters to determine ionic selectivity, pharmacological specificity and voltage-dependent regulation of channel open probability; (5] Site-selective replacements for evaluation of structure-function relationships; [6] Protein structure determination by multidimensional NMR spectroscopy of isotopically labeled proteins in deuterated detergent micelles and by solid-state NMR in oriented phospholipid bilayer lamellae. It is anticipated that the convergence of structural information with the detailed analysis of channel protein function at the level of single molecular events, integrated with the benefits of peptide synthesis and recombinant DNA techniques, guided by molecular modeling, will provide paths for a systematic investigation of the structure-function map of channel proteins, and may provide clues about the biological design of this class of proteins that are fundamental components of living cells.