A new folding intermediate of apomyoglobin from Aplysia limacina: stepwise formation of a molten globule.

Apomyoglobin from Aplysia limacina (al-apoMb), despite having only 20 % sequence identity with the more commonly studied mammalian globins (m-apoMbs), properties which result in an increased number of hydrophobic contacts and a loss of most internal salt bridges, shares a number of features of their folding profiles. We show here that it contains an unusually stable core which resists unfolding even at 70 degrees C. The equilibrium intermediate (I(T)) at this high temperature is distinct from the acid unfolded state I(A) which has many properties in common with the acid intermediate observed for the mammalian apoproteins (I(AGH)). It contains a smaller amount of secondary structure (27 % alpha-helical instead of 35 %) and is more highly solvated as evidenced from its fluorescence spectrum (lambda(max)=344 nm instead of 338 nm). Its stability is greatly increased (DeltaDeltaG(w)=-6.75 kcal mol(-1)) in the presence of high salt (2 M KCl), lending support to the view that hydrophobic interactions are responsible for its stability. Kinetic data show classical two-state kinetics between I(A) and the folded state both in the presence and absence of salt. Both I(A) and I(T) can be populated within the dead time of the stopped-flow apparatus, since initiating the refolding reaction from I(T) or I(A) rather than the completely unfolded state does not affect the observed refolding time-course. Our conclusion is that al-apoMb, as other "apo" proteins (including for example alpha-lactalbumin in the absence of Ca(2+)), may be described as "uncoupled" with an unusually high and exploitable tendency to populate partially folded states.