Intrinsic fluorescence emission of intact oxy hemoglobins

Hirsch, Rhoda Elison; Zukin, R. Suzanne; Nagel, Ronald L.

doi:10.1016/0006-291x(80)91096-7

Cited by 106 publications

(51 citation statements)

References 8 publications

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“…Three tyrosines (Tyr-103, Tyr-146, and Tyr-151) and one tryptophan (Trp-7) are located at the protein surface, while one tryptophan (Trp-14) is buried inside the protein. Since the fluorescence from Trp-14 is readily quenched by the heme group, the emission from the three Tyr residues and one Trp-7 contribute to the fluorescence spectra (16). The emission maximum in fluorescence spectra is roughly correlated with the distance and orientation between heme and Tyr/Trp in heme proteins (17).…”

Section: Effects Of Insertion and Expansion Of Polyglutamine On Othermentioning

confidence: 92%

Intra- and Intermolecular β-Pleated Sheet Formation in Glutamine-repeat Inserted Myoglobin as a Model for Polyglutamine Diseases

Tanaka¹,

Morishima²,

Akagi³

et al. 2001

Journal of Biological Chemistry

100

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An aberrant structure of the expanded polyglutamine might be involved in the formation of aggregates in CAG repeat diseases. To elucidate structural properties of the expanded polyglutamine, we prepared sperm whale myoglobin (Mb) mutants, in which 12, 28, 35, and 50 repeats of glutamine were inserted at the corner between the C and D helices (Gln 12 , Gln 28 , Gln 35 , and Gln 50 , respectively). Circular dichroism and IR spectroscopies showed that the expanded polyglutamine, which was recognized by the monoclonal antibody 1C2 in Gln 28 , Gln 35 , and Gln 50 Mb forms an antiparallel ␤-pleated sheet structure. Gln 50 Mb aggregates were found to comprise an intermolecular antiparallel ␤-pleated sheet. Fluorescence together with 1 H NMR spectra revealed partial unfolding of the protein surface in Gln 35 and Gln 50 Mb, although the structural changes in the protein core were rather small. The present results indicate that the fluctuating ␤-pleated sheet of the expanded polyglutamine exposed on the protein surface facilitates the formation of aggregates through intermolecular interactions. The present study has first established and characterized structural properties of a molecular model for polyglutamine diseases in which various lengths of polyglutamine including a pathologically expanded glutamine repeat were inserted into a structurally known protein.

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Section: Effects Of Insertion and Expansion Of Polyglutamine On Othermentioning

confidence: 92%

Intra- and Intermolecular β-Pleated Sheet Formation in Glutamine-repeat Inserted Myoglobin as a Model for Polyglutamine Diseases

Tanaka¹,

Morishima²,

Akagi³

et al. 2001

Journal of Biological Chemistry

100

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“…Nad Acad Sci. USA 81 (1984) (33,34), this long-lived emission is also likely to have been the dominant contribution to Hb fluorescence measured in the steady state (35)(36)(37)(38).…”

Section: Resultsmentioning

confidence: 99%

Picosecond fluorescence decay of tryptophans in myoglobin.

Hochstrasser¹,

Negus²

1984

Proc. Natl. Acad. Sci. U.S.A.

122

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The fluorescence decay characteristics of Mb, MbCO, metMb (sperm whale), metMb (yellowfin tuna), and their apo derivatives were determined by using a picosecond streak camera and time-correlated single photon counting. The emission is dominated by tryptophans that transfer their energy to the heme on a subnanosecond time scale. Sperm whale Mb and derivatives have two tryptophans and their decays can be interpreted mainly as two exponentials, one of ca. 20 ps and the other of 130 ps, whereas tuna Mb has one tryptophan and its emission is nonexponential but dominated by one component of 31 ps. These results along with F6rster energy transfer calculations allow us to assign the ca. 30-ps emission to Trp-14 and the 130-ps emission to Mb. The streak camera was modified to determine the decay of the fluorescence anisotropy. In metMb (tuna) the fluorescence anisotropy decays in 100 ps, which is postulated to result from rapid motion of the Trp-14. Because energy transfer was used to gate the anisotropy, the fast motion of Trp-14 is proposed to correspond to only 10% of the equilibrium distribution of molecules.Tryptophans are useful optical probes of protein structure because of their relatively long wavelength absorption compared with polypeptides and other common amino acid residues. A first step in realizing the full potential of such probes is the development of techniques to distinguish the different tryptophans in a protein by means of their optical responses. We have therefore carried out picosecond laser experiments on Mb aimed at characterizing the fluorescent behavior of each of the tryptophans. Previous picosecond laser experiments aimed at detecting rapid conformational changes and relaxation processes in heme proteins have involved studying changes in the heme electronic structure as a result of photodeligation of CO (1-6), 02 (1,(4)(5)(6), and NO (6, 7). A goal of the present research is to pave the way for the study of the transmission of structural changes throughout the protein, and as a first step we have attempted in the present work to chart the picosecond anatomy of the tryptophans of Mb.Pulsed laser techniques are needed to study tryptophan fluorescence in heme proteins as a result of very efficient energy transfer to the heme. The low fluorescence quantum yields for tryptophan in heme proteins (8) suggest that the corresponding lifetimes are in the picosecond range. Thus, to obtain direct information on the lifetimes and motional characteristics of such tryptophans it was necessary to devise experiments by which the fluorescence and its polarization could be measured with picosecond accuracy.Sperm whale (SW) Mb is known to contain the two tryptophan residues, Trp-7 and Trp-14 (9). Thus, it is expected that the emission will be dominated by two decays. The heme is considerably further from Trp-7 than from Trp-14 (20 A compared with 15 A) so that the effects of energy transfer on these excited states are expected to be different. Tuna Mb has an x-ray structure very similar to SW Mb (10) bu...

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“…4 illustrates the fluorescence spectra of the ␣-, ␤-, chimeric ␣␣␤-, and chimeric ␤␤␣-subunits. The ␣-subunit showed a small peak around 310 nm originating from tyrosines (Tyr 24 , Tyr 42 , and Tyr 140 ) (29), while the fluorescence peak of the ␤-subunit around 335 nm is characteristic of Trp 37 (30). In the chimeric proteins, the ␣␣␤-subunit indicated a large peak around 345 nm, in addition to the peak at 310 nm, although the chimeric subunit contains only one tryptophan (Trp 14 ) in the A-helix.…”

Section: Circular Dichroism Spectra-inmentioning

confidence: 99%

“…In the native ␣-subunit, since Trp 14 is fully buried near the center of the molecule, the fluorescence of Trp 14 is quenched due to resonance energy transfer to the heme moiety and low intensity (30). Upon substitution of module M4, the remarkable increase in the intensity of the fluorescence peak around 345 nm is observed in the chimeric ␣␣␤-subunit, suggesting drastic changes in the microenvironment of Trp 14 and heme moiety.…”

Section: Figmentioning

confidence: 99%

Structural and Functional Roles of Modules in Hemoglobin

Inaba

Wakasugi

Ishimori

et al. 1997

Journal of Biological Chemistry

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The ␣-and ␤-subunits of human hemoglobin consist of the modules M1, M2 ؉ M3, and M4, which correspond to the exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90 -92). To gain further insight into functional and structural significance of the modules, we designed two kinds of chimeric hemoglobin subunits (chimeric ␣␣␤-and ␤␤␣-subunits), in which the module M4 was replaced by the partner subunits. CD spectra in the far-UV region showed that the secondary structure of the chimeric ␣␣␤-subunit drastically collapsed, while the chimeric ␤␤␣-subunit conserved the native globin structure (Wakasugi, K., Ishimori, K., Imai, K., Wada, Y., and Morishima, I. (1994) J. Biol. Chem. 269, 18750 -18756). SAXS data also suggested a partially disordered structure of the chimeric ␣␣␤-subunit. Based on tryptophan fluorescence spectra and computer modeling from x-ray structures of native globins, steric constraint between Trp 14 and Tyr 125 would be induced in the chimeric ␣␣␤-subunit, which would perturb the packing of the A-and H-helices and destabilize the globule structure. On the other hand, such a steric constraint was not found for the counterpart chimeric subunit, the ␤␤␣-subunit. The different stabilities of these module-substituted globins imply that modules would not always be stable "structural" units, and interactions between modules are crucial to construct stable globin subunits.Recent structural studies on proteins have revealed that some are constructed by "modules," which are compact structural units (1, 3). The exon shuffling in protein evolution with the correspondence of the module to the exon on the gene structure strongly suggested that the module structure is one of the key factors in evolution of structure and function of protein (4, 5). The module structure was first identified in the globin family (1), and their diagonal plots have clearly shown that both of the Hb subunits (␣-and ␤-subunits) consist of the four modular structures, the modules M1, M2, M3, and M4 (1), each of which is coded by an exon with the exception of the large central exon comprising two compact units.The analysis of hemoglobin functions showed that the amino acid residues associated with the heme contacts are concentrated in the central modules (M2 ϩ M3) as illustrated in Fig. 1 (6). In 1980, by using a protease, Craik et al. (7,8) isolated the central region of the ␤-subunit, which corresponds to the module M2 ϩ M3 and is coded by the central exon of the ␤-subunit. They showed that the fragment from the central region bound heme stoichiometrically and tightly, generating a characteristic strong Soret absorption band as found for the intact subunits. De Sanctis et al. (9 -12) also prepared "minimyoglobin," which is constructed by the peptide fragment from 32 (Leu) to 139 (Asn) of horse heart myoglobin. The heme binding property of the minimyoglobin peptide was almost the same as that of the intact myoglobin, and the heme binding induced the recovery of secondary structure of the minimyoglobin. They concluded that the remova...

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Intrinsic fluorescence emission of intact oxy hemoglobins

Cited by 106 publications

References 8 publications

Intra- and Intermolecular β-Pleated Sheet Formation in Glutamine-repeat Inserted Myoglobin as a Model for Polyglutamine Diseases

Intra- and Intermolecular β-Pleated Sheet Formation in Glutamine-repeat Inserted Myoglobin as a Model for Polyglutamine Diseases

Picosecond fluorescence decay of tryptophans in myoglobin.

Structural and Functional Roles of Modules in Hemoglobin

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