New strategies for designing inexpensive but selective bioadsorbants for environmental pollutants: Selection of specific ligands and their cell surface expression. 1998 annual progress report

'The broad, long term objective of the research plan is to develop exquisitely selective polypeptide metal chelators for the remediation of aqueous systems. A variety of polypeptide chelators will be developed and optimized ranging from antibodies to small peptides. Then, through unique molecular engineering approaches developed in the laboratories, the polypeptide chelators will be anchored directly on the surface of the cells that produce them. Thus, instead of using isolated biomolecules the authors will employ inexpensive genetically engineered whole cell adsorbents. Following a simple, easily scaleable treatment, the engineered cells can be used to manufacture an inexpensive, particulate adsorbent for metal removal. The authors are currently in year two of a three year program. Work has been focused on preparing the molecular biology constructs needed to carry out the optimization of a metal complex binding antibody, and on the isolation of a metal binding peptide.

Protein Stabilization by Engineered Metal Chelation

A ligand can shift a protein's folding/unfolding equilibrium by binding with higher affinity to the native state. A metal–chelating site consisting of two histidines separated by three residues (His–X_3–His) engineered into an α–helix provides a general and easily–implemented means for protein stabilization by this mechanism. We have tested this approach with the iso–1–cytochrome c of Saccharomyces cerevisiae substituted with histidine at positions 4 and 8 in its N–terminal α–helix. One mM Cu(II) complexed to iminodiacetate stabilizes the cytochrome c variant by ca. 1 kcal/mol, as determined by guanidinium chloride–induced unfolding. The protein's folding/unfolding equilibrium is shifted by a free energy equal to that calculated from the metal ion's preferential binding to the native protein. Given the ubiquity of surface α–helices and the additional possibility of inserting di–histidine chelating sites into turns and β–structures, we conclude that this is a useful method for protein stabilization