PH RESPONSIVE MICRODOMAIN FORMATION IN A DE-NOVO POLYPEPTIDE

Recombinant DNA technology has been employed to produce a polypeptide capable of forming pH responsive hydrophobic microdomains. The design of this peptide is based upon an idealized conceptual model in which e lectrostatic, hydrophobic, and hydration forces are responsible far th e association of amphipathic ct-helical elements. Reduction in solutio n pH is responsible for reducing electrostatic repulsions between simi lar-ly charged residues, promoting the hydrophobic collapse of helical elements. A polymerizable synthetic element (dn31) has been synthesiz ed and inserted into an appropriate expression vector. A clone contain ing a single copy of the dn31 gene (designated dn31x1) was isolated an d the corresponding gene product DN3Lx1 isolated. The physical propert ies of DN3Lx1 were examined in solution by gel filtration chromatograp hy, CD, and fluorescence probe analysis. It was determined that DN3Lx1 self-associates in solution with the degree of aggregation dependent on pH and ionic strength. An initial objective of this work was to exa mine domain organization in higher molecular weight species containing ten or more repetitive sequences. However, attempts to express multip le repeats of DN3Lxn from concatemers were unsuccessful. (C) 1997 John Wiley & Sons, Inc.