The conserved Cys254 plays a crucial role in creatine kinase refolding under non-reduced conditions but not in its activity or stability.

Oxidative stress is a common factor that may affect cell survival in extreme or disease-related conditions, and it is important for the cells to develop not only redox homeostatic mechanisms, but also adequate protein-protecting mechanisms to fight against oxidative stress. In this research, we investigated the role of the conserved C254 in the refolding of creatine kinase (CK), a key cytosolic enzyme involved in intracellular energetics. It was found that the conserved C254 did not contribute to the activity, structure, stability and unfolding of CK, but played a crucial role in CK refolding under non-reduced conditions by preventing off-pathway aggregation. This property of C254 might be a result of natural selection of CK to fight against oxidative stresses that are frequently encountered by vertebrate cells. The results herein not only confirmed that the reduced condition is important to the activity, structural stability and folding of cytosolic proteins, but also highlighted that it is also crucial for cytosolic proteins to maintain the ability to fold correctly under non-reduced conditions.