Fluctuation domains in myoglobin

Abstract

The dynamics of two domains in the myoglobin molecule, close to the heme and inside the protein medium including the surface, are investigated through the study of the fluorescence oxygen quenchng of two probes imbedded in the heme pocket: zinc protoporphyrin IX (with a fluorescence lifetime of 2.1 ns) and metal-free protoporphyrin IX (with a fluorescence lifetime of 17.8 ns).The dynamic aspect of proteins has been well established in the last few years. Fluorescence quenching methodologies have been particularly important in t h s area (see [l] for a review). A detailed multifrequency phase and modulation study of the diffusion of oxygen in protoporphyrin IX adducts of hemoglobin and myoglobin has been published [2]. The data were analyzed in terms of a model which takes into account the rate of acquisition of quencher by the protein, the diffusional rate of quencher in the protein interior and the rate of exit of quencher from the protein [3]. However, in those studies the calculated rate of migration of quencher through the protein interior assumed isotropic diffusion and hence represented an average value. To address the question of anisotropic diffusion in the protein interior, i. e. varying diffusion rates in different protein domains, we have prepared the zinc protoporphyrin IX adduct of myoglobin. The rationale for this approach is that the shorter fluorescence lifetime of the zinc adduct (w 2 ns) compared to the metalfree adduct ( % 18 ns) would allow us to probe a region closer to the heme pocket. We report here for the first time results of oxygen quenching experiments on horse heart myoglobin labelled with these two probes. MATERIALS AND METHODS PreparationsHorse heart myoglobin was purchased from Sigma. The porphyrins were obtained from Porphyrin Products Inc. MbdesFe (myoglobin without iron) and MbFe + Zn (myoglobin in which the heme iron has been replaced by a zinc atom) were prepared by the procedure described previously for MbdesFe [2]. Incorporation of the porphyrins in the heme pocket of myoglobin was controlled by comparing the MbdesFe and absorption and emission spectra to those of porphyrins dissolved in different solvents (Tables 1 and 2). The quality and the homogeneity of the globin-porphyrins were confirmed by the homogeneous fluorescence lifetime of the first emission peak of the incorporated porphyrin in the All experiments were carried out in aqueous solvent at pH 7 with protein concentration near 3 pM for both proteins. Concentrations were determined spectrophotometrically using absorption coefficients of 122 mM-' cm-' at 427 nm for MbFe + Zn [5] and of 12.6 mM-' cm-I at 506 nm for t61. MbdesFe Absorbance and fluorescence spectraAbsorbance data were obtained with a Perkin-Elmer Lambda 5 UV/VIS spectrophotometer using 1 -cm-pathlength cuvettes. Technical fluorescence spectra were obtained with a Perkin-Elmer LS-5 spectrofluorometer. The bandwidths used for excitation and emission were 10 nm. Optical pathlengths of 1 cm and 0.4 cm were used for the mission and excitation wavelengths...

Kinetics of precursor cleavage at the dibasic sites

“…Unexpectedly, quenching of F 13 , Y 13 and W 13 peptides exhibits also the same linear dependence (Fig. 5), indicating that the fluorescence collisional quenching of peptide residues is essentially due to migration of DCA quencher molecules within the water medium in their close vicinity [15,24–26]. Such an interpretation is consistent with the fact that the slopes of the straight lines obtained for each peptide were smaller than that found for the free amino acids (see values in parentheses in Fig.…”

Involvement of protein dynamics in enzyme stability

“…Surface mobility in regions close to Trp residues was analyzed by Trp fluorescence quenching with acrylamide. Indeed, the average fluorescence lifetime of Trp residues being short, the dynamic quenching represents acrylamide migration near these residues [20,21]. This migration follows the microscopic motions of the surface regions in the vicinity of the Trp residues [19].…”

Heterogeneous motions within human apohemoglobin

“…The dynamics of the apoHb matrix were probed using the fluorescence quenching process of ANS located in the heme cavities [21]. Indeed, the fluorescence decay time of this dye is long enough (16 ns; [20] and current study) to investigate the migration of acrylamide molecules through protein far from the region where the ANS is embedded [18,19]. Therefore the kinetic constant value of the ANS fluorescence quenching by acrylamide was measured at 10 and 20 °C, and taken as an indicator of the variations in the dynamics of the protein matrix due to temperature.…”

“…To clearly show that the protein motion in the limited spaces surrounding particular residues is not representative of the global dynamic behavior of the protein bulk, the dynamics of the Trp vicinities were examined by fluorescence quenching. Indeed, the fluorescence decay time of Trp residues is so short (3–4 ns) that their fluorescence quenching process only reveals the fluctuations in the regions close to these fluorophores [18,19]. In contrast, the long fluorescence decay time of the 8‐anilinonaphthalene‐1‐sulfonate dye (ANS) embedded in the protein matrix (≅16 ns) [20] is sufficient to detect quencher molecules (situated in any part of the protein bulk at the instant of the ANS excitation), which travel through the protein matrix and reach the ANS during the persistence of the excited state [18,19].…”

“…Indeed, the fluorescence decay time of Trp residues is so short (3–4 ns) that their fluorescence quenching process only reveals the fluctuations in the regions close to these fluorophores [18,19]. In contrast, the long fluorescence decay time of the 8‐anilinonaphthalene‐1‐sulfonate dye (ANS) embedded in the protein matrix (≅16 ns) [20] is sufficient to detect quencher molecules (situated in any part of the protein bulk at the instant of the ANS excitation), which travel through the protein matrix and reach the ANS during the persistence of the excited state [18,19]. Thus the global motion of the protein matrix (therefore not restricted to a particular aromatic residue’s domain) was checked using the fluorescence quenching of the ANS, located in the heme pocket of the apohemoglobin (apoHb) [21].…”