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 (Delta Delta G(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 Ca2+), may be described as "uncoupled" with an unusually high and exploitable tendency to populate partially folded states. (C) 2000 Academic Press.