Site-directed mutagenesis: Structural basis of proton transfer pathways

The coupling of protein-mediated electron and proton transfer across a membrane of a cell or organelle is essential for life, as it is the means by which biological systems establish an electrochemical gradient that can be used for the generation and storage of energy in the form of ATP. Our research – in collaboration with Marianne Schiffer and Raj Pokkuluri (ANL) and Pierre Sebban (CNRS, France) – is designed to discover fundamental structural factors that underlie the bioenergetics and efficiency of electron-coupled proton transfer in transmembrane proteins. The power of our approach comes from the ability to correlate a wealth of spectroscopic data which characterize the function of native and mutant reaction center complexes with their high-resolution structures. This strategy is complemented by the ability to manipulate the physiology of Rhodobacter to apply selective pressure for ‘repair’ of engineered reaction centers whose function is impaired.

In previous studies, we constructed site-specific mutant reaction centers that are incapable of transferring the first and/or second proton to reduced Q_B; these mutant strains are photosynthetically incompetent. From those mutant strains, we then selected photocompetent phenotypic revertants that regained the ability to grow under photosynthetic conditions. These revertant reaction centers carry the original engineered mutations but also contain second-site compensatory mutations that restore function to the complex. Biophysical studies have determined that second-site mutations restore function to the reaction centers by activating alternative proton delivery pathways. Some of the compensatory mutations occur at sites that are quite distant from Q_B or the sites of the original engineered substitutions.

The main emphasis of this project is the structural characterization of a panel of reaction centers derived from these phenotypic revertants. The set of ~10 phenotypic revertants that we are studying contains physiochemically diverse compensatory amino acid changes at sites both close to and distant from the apparent proton transfer pathway used within the native reaction center. Data collected from these crystals of mutant and revertant reaction centers resulted in excellent electron density maps that clearly showed several unexpected effects of the mutations. In each case, the single compensatory mutation influences the positions and properties of residues near Q B through correlated motions of neighboring amino acid side chains. We have observed that these changes in main chain positions and side chain orientations can extend up to 40 D from the site of the compensatory mutation.

By correlating the spectroscopic data with the crystallographic data, we will recognize the structural elements that constitute a functional, efficient proton transfer pathway. We will thereby be able to discern similar elements in other proteins for which transmembrane proton translocation is a common feature.