Rational Knob-Socket Predictions of Alpha-Helical Stability

A construct that simplifies the complexity of residue packing would significantly impact our understanding and analysis higher order protein structure in the same way that the covalent peptide bond allows linear comparisons of protein sequences and main-chain hydrogen bonding patterns identify secondary structure. The novel knob-socket (KS) model provides a construct to interpret and analyze the direct contributions of amino acid residues to the stability in α-helical protein structures. Based on residue preferences derived from a set of protein structures, the KS construct characterizes intra- and inter-helical packing into regular patterns of simple motifs. Intra-helical interactions consist of a regular pattern of three residue triangular motifs called sockets, which contribute to helical stability. For inter-helical interactions, a single amino acid knob from one α-helix packs into a three amino acid socket within another α-helix. Therefore, sockets are defined in three categories: (1) free, unpacked and favoring intra-helical interactions, (2) filled, packed and favoring inter-helical interactions, and (3) non, unpacked and disfavoring α-helical structure. The three amino acid composition of a socket serves as a code that can be used to predict protein packing and by extension, can also be used to understand individual amino acid contributions to helical stability. The KS model was used in the de novo design of an α-helical homodimer, KSα1.1. Using site-directed mutagenesis, KSα1.1 point mutants have been rationally chosen to increase and decrease stability by relating KS propensities with changes to α-helical structure. In the KS α-helical model, each point mutation affects six surrounding sockets by altering the free/filled propensity values. By analyzing the changes in the propensities of these six sockets, KS based structure predictions were made for each mutant that relate to their stability. These predicted values are compared to the experimentally determined structure and stability of each protein from chemical and thermal denaturation studies as measured by circular dichroism spectroscopy. This study serves as a starting point to reveal how residue packing contributes to protein stability