Molecular Dynamic Simulations of Vibrational Labels in Rabbit Muscle Creatine Kinase Creatine kinase functions as a dimer with two subunits that crystallize symmetrically in the apo form but asymmetrically in a transition state analog complex (TSAC). Previous spectroscopic results from our group on the active-site cysteines in the dual active sites of the creatine kinase dimer indicate that both active sites can adopt a range of structures, but the conclusions of data from both S-H stretching bands of free cysteine and CN stretching bands of cyanylated cysteine are ambiguous as to whether the protein rests in an asymmetric configuration with one active “closed” and one “open” to reactivity with its substrate. With the goal of analyzing local cysteine orientations in creatine kinase to interpret spectroscopic results, Gromacs using (force field) was used to generate trajectories starting from both the apo and substrate-induced crystal structures. Multiple explicit water models were used including TIP3P and SPC/E. The goals were to simulate unmodified and cyanylated CYS 283 and analyze the movement of the thiol or thiocyanate vs the local protein backbone to determine whether there are specific H-bond partners or dominant geometries for either residue that might be reflected in results from vibrational spectroscopy of those residues. The results showed that unmodified Cys283 in creatine kinase adopts a single, long-lived orientation about 40% of the time, due to a specific H-bond between the thiol and a specific water molecule. Similar results suggesting a locally preferred conformation were seen for cyanylated Cys283. The re-interpretation of spectroscopic results in light of these simulations will be discussed.