The C2 domain originally referred to the second of four constant structural motifs in protein kinase C (PKC).Now this domain represents a large structural family sharing a homologous dimensional structure in many proteins that play important roles in many organisms.The C2A domain is one of the two C2 domains of synaptotagmin I involved in the Ca2+ regulation of exocytosis.This domain is mostly composed of β-sheet except for a small fraction of α-helix, and therefore provides an ideal model for a protein folding study.In this report, the unfolding equilibrium of the C2A domain in guanidine hydrochloride (GdnHCl) containing solutions has been studied using ultraviolet (UV) difference spectrum, fluorescence spectrum, size exclusion chromatography (SEC), and circular dichroism (CD) spectrum.The results suggest that unfolding of the C2A domain occurs as a two-state process during GdnHCl titration.By examining the changes of both tertiary structure and secondary structure, no intermediates could be detected during this unfolding study.However, it has been found that the native state of the C2A domain has a large hydrophobic surface.This result suggests that as a fragment of a protein, the C2A domain itself may exist in a state with large hydrophobic surface.This hydrophobic surface may be the molecular basis for interaction between domains in the whole protein.Furthermore, the hydrophobic behavior may play a role during the oligomerization of synaptotagmin.