Designing surface exposed sites on Bacillus subtilis lipase A for spin-labeling and hydration studies.

Spin-labeling with electron paramagnetic resonance spectroscopy (EPR) is a facile method for interrogating macromolecular flexibility, conformational changes, accessibility, and hydration. Within we present a computationally based approach for the rational selection of reporter sites in Bacillus subtilis lipase A (BSLA) for substitution to cysteine residues with subsequent modification with a spin-label that are expected to not significantly perturb the wild-type structure, dynamics, or enzymatic function. Experimental circular dichroism spectroscopy, Michaelis-Menten kinetic parameters and EPR spectroscopy data validate the success of this approach to computationally select reporter sites for future magnetic resonance investigations of hydration and hydration changes induced by polymer conjugation, tethering, immobilization, or amino acid substitution in BSLA. Analysis of molecular dynamic simulations of the impact of substitutions on the secondary structure agree well with experimental findings. We propose that this computationally guided approach for choosing spin-labeled EPR reporter sites, which evaluates relative surface accessibility coupled with hydrogen bonding occupancy of amino acids to the catalytic pocket via atomistic simulations, should be readily transferable to other macromolecular systems of interest including selecting sites for paramagnetic relaxation enhancement NMR studies, other spin-labeling EPR studies or any method requiring a tagging method where it is desirable to not alter enzyme stability or activity.