Design and Modular Synthesis of Proteins with Novel Functions

Wolfgang Haehnel

Institute of Biology II, Biochemistry, Albert-Ludwigs-University, Schänzlestr. 1, D-79104 Freiburg, Germany


The design of proteins with novel functions can follow different strategies. Most common is to modify or change the active site of proteins with known structure by site directed mutation. An alternative is the de novo design of proteins. This approach has the potential to create sites not known in nature. But it has to find a solution for the folding problem of proteins. A major portion of this problem is avoided by template assembled synthetic proteins (TASP) which arrange peptide building blocks in predefined orientation and position. With this strategy amphipathic helices can be assembled in an antiparallel four-helix bundle with a hydrophobic core harboring a functional group. We have used this approach in a modular way to assemble proteins in solution and by using combinatorial strategies on solid support.

1. By exchanging a single helix with a Ru(bpy)3 complex at different positions four-helix bundle proteins were synthesized capable of laser-induced electron transfer. The electron transfer pathways through the helix to a heme in the hydrophobic interior of the protein has been studied.

2. Peptide synthesis on solid support was combined with the modular assembly by chemoselective ligation of peptide building blocks. The template bound by a linker to cellulose could be cleaved to follow the synthesis by mass spectrometry. Pairs of different helices with a histidine as heme ligand were combined on the template with pairs of different shielding helices resulting in 462 new immobilized protein heme complexes. An automatic analysis of the absorbance spectra provided information of amino acid compositions forming the heme-binding pocket and tuning the redox potential.

3. New metal centers have been created by combinations of three different sets of amphiphilic helices on solid support. In the hydrophobic interior the proteins carry a cysteine and several histidines at various positions for copper-ligation. This approach was successful to synthesize for the first time copper proteins based on a four-helix bundle. Three of the copper proteins were synthesized in solution and analyzed by UV-Vis, Resonance Raman, and EPR spectroscopy. The most stable variant showed properties intermediate between those of a type 1 and a type 2 copper center.

These results demonstrate that combinatorial protein chemistry guided by rational principles, represents an efficient approach for the de novo design and synthesis of metalloproteins. In view of the functional versatility of this class of proteins, the present results constitute a significant step towards tailor-made enzymes.