Fluorescence studies of Aplysia and sperm whale apomyoglobins

Anderson, Sonia R.; Brunori, Maurizio; Weber, Gregorio

doi:10.1021/bi00826a015

Cited by 75 publications

(26 citation statements)

References 17 publications

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“…It is not possible to exclude that the overall process may partly evolve through a different pathway involving haem rotation inside the protein crevice, in particular in the case of Aplysia myoglobin which is known to be considerably more flexible than is sperm whale myoglobin (Bolognesi et al, 1985). The result presented above should be compared with earlier findings on the properties of the haem pocket in Aplysia myoglobin; it is in fact noteworthy that this protein binds haem with lower affinity than that of sperm whale myoglobin (Rossi Fanelli & Antonini, 1960), and that the spectroscopic properties of its aminonaphthalenesulphonyl derivative indicate that haem is embedded in a relatively more polar environment (Anderson et al, 1970), even though this point has been recently challenged (see Bolognesi et al, 1985, andMacgregor &Weber, 1986). Finally it appears rewarding that also for Aplysia myoglobin the c.d.…”

Section: Resultsmentioning

confidence: 75%

Haem disorder in two myoglobins: comparison of reorientation rate

Bellelli

Foon

Ascoli

et al. 1987

Biochemical Journal

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The globins from sperm whale and from Aplysia limacina myoglobins were reconstituted by addition of stoichiometric ferric protohaem and the Soret c.d. was followed as a function of time. For both reconstituted proteins, the Soret c.d. changes with time, reflecting haem reorientation inside its pocket, as previously described [Aojula, Wilson & Drake (1986) Biochem. J. 237,[613][614][615][616] for sperm whale myoglobin. The time course of the c.d. transition is found to be approx. 10 times faster in Aplysia than in sperm whale myoglobin, a result which is in agreement with the known structural and physicochemical properties of the two myoglobins; furthermore, these results confirm that c.d. and n.m.r. data on haem orientation in haemoproteins reflect the same molecular phenomenon. INTRODUCTIONSeveral haemoproteins have been described to exist in two interconvertible conformations, differing in their n.m.r. properties. La Mar and collaborators (La Mar et al., 1978) have shown that the coexistence of conformers is related to the fact that the intrinsically asymmetric haem molecule can assume two orientations differing by 180°rotation around its a-y axis. Often one of the conformers is largely prevalent at equilibrium, and constitutes the 'native' (or ordered) conformation as detected by X-ray crystallography. In the case of sperm whale myoglobin the disordered form accounts for only 10% of the total haemoprotein in solution (La Mar et al., 1983), while in other cases the disordered form can be as populated as the ordered one, as in tuna fish myoglobin (Levy et al., 1985).Immediately after reconstitution obtained by mixing sperm whale apomyoglobin with either ferrous or ferric haem, the disordered form accounts for approx. 50 % of the total pigment; this population ratio changes with time and decays slowly towards its equilibrium value. The re-equilibration has been followed by means of n.m.r. (La Mar et al., 1984) or c.d. spectroscopy (Aojula et al., 1986) and the measured half-time ranges from hours to days depending on the experimental conditions (for example, pH and temperature).To a first approximation it may be assumed that the prevalent pathway to achieve haem reorientation requires dissociation of the porphyrin from the protein, since the haem pocket appears to be too small to allow the haem to rotate freely; for this reason we compared the rate of haem reorientation in two myoglobins whose physicochemical properties, particularly with respect to haem affinity, differ widely: those from sperm whale and from Aplysia limacina; in fact myoglobin from Aplysia limacina

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Section: Resultsmentioning

confidence: 75%

Haem disorder in two myoglobins: comparison of reorientation rate

Bellelli

Foon

Ascoli

et al. 1987

Biochemical Journal

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“…1). Several attempts to separate the individual Trp contributions to the protein emission have been carried out, either using various quenchers and perturbing reagents (23,24), or tuna apoMb containing only Trp 14 (24 -26), or other apoMbs with various Trp locations (27,28). For native apoproteins it was observed that Trp 7 is more accessible to solvent * The costs of publication of this article were defrayed in part by the payment of page charges.…”

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confidence: 99%

Multisite Fluorescence in Proteins with Multiple Tryptophan Residues

Tcherkasskaya¹,

Bychkova²,

Uversky³

et al. 2000

Journal of Biological Chemistry

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Time-resolved fluorescence experiments were carried out on a variety of apomyoglobins with one or two tryptophan (Trp) residues located at invariant positions 7 and 14 in the primary sequence. In all cases, the Trp fluorescence kinetics were resolved adequately into two discrete lifetime domains, and decay-associated spectra (DAS) were obtained for each decay component. The DAS resolved for unfolded proteins were indistinguishable by position of the emission maxima and the spectral shapes. The folded proteins revealed noticeable differences in the DAS, which relate to the diverse local environments around the Trp residues in the individual proteins. Furthermore, the DAS of wild-type protein possessing two Trp residues were simulated well by that of one Trp mutants either in the native, molten globule, or unfolded states. Overall, employing Trp fluorescence and site-directed mutagenesis allowed us to highlight the conformational changes induced by the single amino acid replacement and generate novel structural information on equilibrium folding intermediates. Specifically, it was found that conformational fluctuations in the local cluster around the evolutionarily conserved Trp 14 are very similar in the native and molten globule states of apomyoglobins. This result indicates that residues in the E and B helices contributing to this cluster are most likely involved in the stabilization of the overall architecture of the structured molten globule intermediate.

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“…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].…”

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confidence: 99%

Heterogeneous motions within human apohemoglobin

Haouz

Mohsni

Zentz

et al. 1999

European Journal of Biochemistry

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Conservation of the secondary and tertiary protein organization of human apohemoglobin was observed at temperatures ranging from 7 to 25 8C using CD spectra in the far-UV (200±250 nm) and near-UV (250±300 nm) regions. The dynamics of apohemoglobin were probed using fluorescence quenching experiments on the Trp residues and an extrinsic dye (ANS or bis-ANS) located in the heme cavities. The long decay time of the dye emission (. 10 ns) reveals the dynamics of the protein matrix averaged over the whole molecule. The short decay time of the Trp residue emission (ù3 ns) probes the dynamics of their close vicinities. When the temperature rises from 10 to 20 8C, the average intraproteic motions throughout the whole apohemoglobin matrix are greatly accelerated, whereas the hydrophobic protein regions around the a14, b15 and b37 Trp residues appear much less animated. These dynamic differences between the behavior of the softer matrix and the packed rigid regions containing the tryptophans could be one of the requisites for apohemoglobin stability. We suspect that the highly rigid tryptophan domains in human apohemoglobin are likely to be knots.Keywords: apohemoglobin; dynamics; protein domains; Trp fluorescence.The ability of a protein to undergo conformational change is a direct consequence of its flexibility, the origin of which is to be found in fluctuations in the amino acid residues. Small changes in local configuration allow the great stereo-organization of macromolecules. Both immediate and indirect evidence show that all proteins are within a fluctuating system [1±3], with interconversion rates between their protein conformational substates [4,5]. Linderstro Èm-Lang and Schellman [6] were the first to suggest that a protein does not possess a unique conformation but exists as a group of structures not too different in free energy, but frequently differing in enthalpy and entropy. Klotz [7] and Weber [8] have also suggested that proteins are not rigid, but rather that a protein is dynamic and exists in many conformational states. Proteins are complex systems often exhibiting both soft regions and rigid and compact domains [9,10]. Using the fluorescence approach, many authors have reported movements of Tyr and Trp residues in various proteins [11±14]. Other studies were carried out on a time-dependent dipolar re-orientation of the environment surrounding the Trp residue [15]. The majority of these fluorescence studies focused on the dynamics of particular areas within the protein [16,17], leaving other areas within the macromolecule uninvestigated. We therefore investigated the dynamic differences of intraproteic motions as opposed to those of any one particular area.Indeed protein matrix mobility is an average of the many contributions of local protein fluctuations; the various regions cannot have the same dynamic behavior and should be more or less sensitive to thermic energy. Therefore, the present study introduces a new approach, which gives an insight into an integrated picture of these various dynamics. To ...

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Fluorescence studies of Aplysia and sperm whale apomyoglobins

Cited by 75 publications

References 17 publications

Haem disorder in two myoglobins: comparison of reorientation rate

Haem disorder in two myoglobins: comparison of reorientation rate

Multisite Fluorescence in Proteins with Multiple Tryptophan Residues

Heterogeneous motions within human apohemoglobin

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