Pressure-induced perturbation of ANS-apomyoglobin complex: frequency domain fluorescence studies on native and acidic compact states.
The pressure dependence of the flexibility of the 8-anilino-1-naphthalene sulfonate (ANS)-apomyoglobin complex was investigated in the range between atmospheric pressure and 2.4 kbar by frequency domain fluorometry. We examined two structural states: native and acidic compact. The conformational dynamics of the ANS-apomyoglobin complex were deduced by studying the emission decay of ANS, which can form a noncovalent complex with the apoprotein in both the native and the acidic compact forms. Because the free fluorophore has a very short lifetime (less than 75 ps), its contribution can be separated from the long-lived emission. The latter arises from ANS molecules bound to the protein and provides information on the structural and dynamic characteristics of the macromolecule. The fluorescence emission decay of the ANS-apomyoglobin complex at neutral pH has a broad fluorescence lifetime distribution (width at half-maximum = 4.1 ns). The small changes in the fluorescence distribution parameters that occur with changes in pressure indicate that the ANS-apomyoglobin complex at neutral pH holds its compactness even at 2.4 kbar. A small contraction of molecular volume has been detected at low pressure, followed by a slight swelling with an increase in flexibility at higher pressures. The heterogeneity of ANS fluorescence in the acidic compact state of apomyoglobin is even greater than that in the native form (distribution width = 10 ns); moreover, the acidic compact state appears more expanded and accessible to solvent molecules than the native state, as suggested by the distribution center, which is 11 ns for the former and 19 ns for the latter. The lifetime distribution center remains constant with increasing pressure, which suggests that no other binding site is formed at high pressure.
Bismuto
E
E
Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Italy.
Irace
G
G
Sirangelo
I
I
Gratton
E
E
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D009211:Myoglobin / Q000737:chemistry*; D011312:Pressure; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; C027132:1-anilino-8-naphthalenesulfonate