Structural and Dynamic Response to Core Repacking Substitutions in T4 Lysozyme Detected by Site-Directed Spin Labeling

Proteins can evolve to gain new functionality via mutations that decrease stability, and presumably, increase flexibility. Particularly interesting examples are mutations that modify the hydrophobic core packing and create new functions, e.g. novel ligand binding sites created by cavity-forming mutants. Many core repacking mutants of T4 Lysozyme (T4L) have been characterized, some of which bind non-polar ligands. Overall, the crystal structures of these mutants are very similar to the WT and offer little insight into the mechanism whereby the ligand reaches the cavity. Site-Directed Spin Labeling (SDSL) has been applied to investigate the structural and dynamical response of T4L in solution to 28 core repacking mutations, many of which have known crystal structures. Remarkably, the most common response in the cavity-creating susbtitutions is the appearance of a second conformational substate, with populations as high as 70 %, showing that the cavities allows for an alternative protein fold, at least locally. Osmotic perturbation shows that the conformations are in slow exchange on the EPR time scale. Thus, the cavity mutations excite molecular flexibility, with a characteristic time scale > 100 ns, probably in the μs-ms range. Interspin distance measurements carried out for two of the cavity mutants to study the magnitude and nature of the conformational rearrangement suggest the interesting possibility that the alternative states are also present in the WT protein, but at a different ratio.In some cases, ns backbone modes were also excited by the core mutation, but the effects were subtle. Remarkably, the type and magnitude of the changes in the protein flexibility were not necessarily correlated with the extent of destabilization by the substitution. The relationship of the cavity-excited states to the mechanism of ligand entry is under study.