Pressure‐induced perturbation of ANS‐apomyoglobin complex: Frequency domain fluorescence studies on native and acidic compact states

Abstract

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 ANSapomyoglobin 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.

Non‐thermal effects of electromagnetic fields at mobile phone frequency on the refolding of an intracellular protein: Myoglobin

“…At low pH and specific salt concentrations, myoglobin forms various destabilized structures (usually indicated as I and E forms) that are variably extended, depending on conditions. Thermodynamic and kinetic studies have shown that the I form contains a tight core consisting of the intersection of the AGH helices and loose solvated helical regions portions of the B-F loop [Barrick and Baldwin, 1993;Fink, 1995;Bismuto et al, 1996]. A species formed at even lower pH and low salt, the E (extended) form, contains essentially only the AGH core and is otherwise very extended [Challender et al, 1998;Baldwin and Rose, 1999a,b].…”

“…In each substate the excited tryptophan experiences a specific microenvironment that affects its fluorescence lifetime. Myoglobin molecules fluctuate fast at acidic pH and in the presence of salt among unfolded and partly folded states with characteristics of molten globule, i.e., a native-like architecture but with labile tertiary interactions [Barrick and Baldwin, 1993;Bismuto et al, 1993Bismuto et al, , 1996Fink, 1995]. These acidic forms of myoglobin with a-helix content come out during the initial stages of myoglobin refolding, and in the folded state pack against each other with large hydrophobic contact areas forming the heme binding site.…”

Determining chemical rate coefficients using time-gated fluorescence correlation spectroscopy

“…The fluorescence lifetime of TMR is 2.2 ns, whereas the lifetime of ANS in aqueous buffer is less than 100 ps. 17 With this sample, the TMR fluorescence is observed in a background from a large number of weakly fluorescent ANS molecules. In Fig.…”

“…This pronounced sensitivity of ANS to its environment makes it an excellent probe for studies of protein stability and dynamics. 17 Figure 4a shows the fluorescence autocorrelation function for dye-labeled myoglobin (diffusion only) and for the apoMb-ANS sample (diffusion and reaction) described in the Experimental section. These data were taken with the excitation beam diameter reduced from 5 to 3 mm.…”

Structural and dynamic aspects of ?-glycosidase from mesophilic and thermophilic bacteria by multitryptophanyl emission decay studies

“…9 Frequency domain fluorometry is one of the most common spectroscopic methods to elucidate the structural and dynamic aspects of tryptophan containing proteins. [10][11][12] However, the photophysics of indolic residues in proteins is complex and involves multiple nonradiative pathways, such as solvent quenching, excited-state proton transfer, excited-state electron transfer, and intersystem crossing. [13][14][15][16] The preferential de-excitation pathways are determined by the indolic conformers mainly present in the ground state, and by the composition and mobility of the different environments experienced in the excited state.…”