Reducing lambda repressor to the core.

Lambda repressor fragment λ(*)(6-85) is one of the fastest folding small protein fragments known to date. We hypothesized that removal of three out of five helices of λ(*)(6-85) would further reduce this protein to its smallest folding core. Molecular dynamics simulations singled out two energetically stable reduced structures consisting of only helices 1 and 4 connected by a short glycine/serine linker, as well as a less stable control. We investigated these three polypeptides and their fragments experimentally by using circular dichroism, fluorescence spectroscopy, and temperature jump relaxation spectroscopy to gain insight into their thermodynamic and kinetic properties. Based on the thermal melts, the order of peptide stability was in correspondence with theoretical predictions. The most stable two-helix bundle, λ(blue1), is a cooperatively folding miniprotein with the same melting temperature and folding rate as the full-length λ(*)(6-85) pseudo wild type and a well-defined computed structure.

Identification of the protein kinase A regulatory RIalpha-catalytic subunit interface by amide H/2H exchange and protein docking.

An important goal after structural genomics is to build up the structures of higher-order protein-protein complexes from structures of the individual subunits. Often structures of higher order complexes are difficult to obtain by crystallography. We have used an alternative approach in which the structures of the individual catalytic (C) subunit and RIalpha regulatory (R) subunit of PKA were first subjected to computational docking, and the top 100,000 solutions were subsequently filtered based on amide hydrogen/deuterium (H/2H) exchange interface protection data. The resulting set of filtered solutions forms an ensemble of structures in which, besides the inhibitor peptide binding site, a flat interface between the C-terminal lobe of the C-subunit and the A- and B-helices of RIalpha is uniquely identified. This holoenzyme structure satisfies all previous experimental data on the complex and allows prediction of new contacts between the two subunits.