Spin-echo EPR of Na,K-ATPase unfolding by urea

► Urea increases water accessibility of kidney Na,K-ATPase Class I groups relative to Class II groups. ► Urea increases conformational heterogeneity of protein substates in Na,K-ATPase. ► Urea enhances the amplitude and rate of fluctuations between substates and depresses the glasslike transition in the Na,K-ATPase.