The dynamic properties of the native state of tuna apomyoglobin, a single tryptophan containing protein, under denaturing conditions, i.e., acid or relatively high pressure, have been investigated by frequency domain fluorometry. Although very complex because of the presence of conformational heterogeneity, the intrinsic tryptophanyl emission decay could be resolved into two distinct distributed components, both having Lorentzian shape. The two components were associated to the native-like state and the unfolded one. The results obtained from quenching experiments and anisotropy decay data indicated that the native-like conformation existing at pH 2.5 in equilibrium with the unfolded state is compact and characterized by a larger intrinsic flexibility compared to that of the native state at neutral pH.
The effect of increasing hydrostatic pressure at neutral pH was examined following the fluorescence emission decay of the non-covalent complex formed by horse apomyoglobin with the extrinsic probe 8- anilino-1-naphthalene sulfonate (ANS). The pressure induced conformational changes at neutral pH determines a disorganization of the ANS binding region with a consequent dissociation of the fluorophore, the lifetime of which is very short in water, i.e., 75 ps, compared to that of the fluorophore bound to native horse apomyoglobin, i.e., 19 ns. This permitted to resolve the emission decay of the apomyoglobin-ANS complex into two components: a long-lived distributed component originating from the molecule population that retain a native-like conformation and a short-lived discrete one corresponding to the emission of the free fluorophore. The results indicated that horse apomyoglobin undergoes a small volume contraction at 500 kbar, followed by a slight swelling with a concomitant increase of internal flexibility at higher pressure.
Buy this Article