Absorption spectroscopy and circular dichroism studies of sperm whale myoglobin chemically modified on the N-terminal α-aminogroup by isothiocyanate reagents

Postnikova, G. B.; Yumakova, Elena M.; Sukhomudrenko, A G; Vol’kenshtein, Mikhail V.; Атанасов, Б.

doi:10.1016/0301-4622(80)85005-8

Cited by 5 publications

(1 citation statement)

References 18 publications

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“…2 shows the fluorescence of MITC-met-Mb as a function of pH. The acylation of the N-terminal a-amino group, which disturbs the ionic stabilization at the N-terminal [17] might primarily affect the fluorescence of the adjacent Trp7.…”

Section: Ph-dependent Fluorescence Of Ferricmyogiobinsmentioning

confidence: 99%

Fluorescence study of the conformational properties of myoglobin structure

Postnikova¹,

Komarov²,

Yumakova³

1991

European Journal of Biochemistry

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Tryptophanyl fluorescence of high-spin and low-spin complexes of sperm whale ferric-and ferrousmyoglobins, met-, azide-and cyanomyoglobins and deoxy-, oxy-and carboxymyoglobins has been studied in the pH range 2.5 -13. The pH-dependent fluorescence of sperm whale metmyoglobin acylated at the N-terminal a-amino group by methylisothiocyanate and of bovine metmyoglobin, which contains invariant Trp7 and Trpl4 but lacks Tyrl51, have also been examined. Drastic changes in the fluorescence were registered in the acidic and alkaline pH ranges which are due to denaturation of Mb. Fluorescent and CD data indicate that at pH < 4.5 and pH > 11.5 the unique spatial structure of the protein is destroyed whereas the secondary structure and integrity are essentially preserved. In all sperm whale and bovine myoglobins studied a local conformational change in the surroundings of Trp is observed which precedes alkaline denaturation. It seems to be due to deprotonation of lysine residues and breakage of the salt bridges essential for the maintenance of the native conformation of the N-terminal and the adjacent region. The parameters of this conformational transition are found to correlate with the spin state of the heme complex. However, analysis of the fluorescence behaviour of different ligand derivatives of myoglobin in the whole pH range studied enables one to conclude that the exact protein conformation depends not only on the spin state of the Fe atom but, to a greater extent, probably on the chemical nature of the ligand and its interaction with the protein groups in the heme cavity. Local conformational changes induced by the replacement of the sixth ligand or by varying pH seem to involve the same region of contacts between the A helix and GH fragment (or between the AE and GH helical complexes) though the extent of the changes may be different.The intrinsic fluorescence of heme proteins is known to be quenched due to efficient energy transfer to the prosthetic group [l, 21. However, it appears that this quenching is not complete, and it is possible to detect the fluorescence of native heme proteins. This was first demonstrated for sperm whale myoglobin (Mb) and lupine leghemoglobin (Lb) [3] and then for various hemoglobins (Hb) [4-71 by both highly sensitive right-angle optics [3, 4, 61 and front-face fluorometry [5, 71. The fluorescence quantum yield for Mb was found to be about 5% and for Hb about 1 % of that for Trp in solution.There is evidence [6-81 that the fluorescence of Hb is sensitive to ligand-induced changes in its quaternary structure, the so-called R-T transition. It is likely that conformational changes in the tertiary structure of monomeric globins induced by pH of the medium, electronic and ligand state of the heme can also be recorded by fluorescence [3]. In mammalian myoglobins, where both tryptophans are in the A-helix region adjacent to the N-terminal, this would enable one to establish a connection between the state of the heme and the conformation of the distant protein region.The aim of the prese...

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Section: Ph-dependent Fluorescence Of Ferricmyogiobinsmentioning

confidence: 99%

Fluorescence study of the conformational properties of myoglobin structure

Postnikova¹,

Komarov²,

Yumakova³

1991

European Journal of Biochemistry

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Spin-label study of conformational changes induced by pH and ligands in leghemoglobin from yellow lupine root nodules

1985

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Conformational changes induced by ligands and pH in lupine ferrileghemoglobin selectively modified at TyrEl6 by the imidazolide spin label has been studied by the method of electron spin resonance in the pH range 6-13. It is shown that in the alkaline pH region the bound spin label registers a local conformational transition which precedes the alkaline denaturation of the protein. In aquamet, cyanide and nicotinate complexes of ferrileghemoglobin this transition occurs with pK 10.5, in acetate and azide complexes with pK 11. In all these ligand derivatives the transition is induced by alteration in the ionization state of one group (AnH+ FZ l), most probably, the &-amino group of Lys-GH3. The latter is linked with the Glu-A34 residue and this bond is essential for maintaining the native conformation of leghemoglobin. The ligand-induced conformational changes in the vicinity of the label are small and consist, most likely, in some alteration of the mutual arrangement of the AE and GH helical complexes. No correlation has been revealed between the spin state of the heme iron and the conformation of leghemoglobin in the region under study.Leghemoglobin (Lb) is the only representative of the large family of globins which occurs in tissues of higher plants. It is contained in root nodules of legumes inoculated with Rhizobium bacteria and acts to transport oxygen to nitrogenfixing bacteroids.Leghemoglobin being a close structural homolog of animal globins, it substantially differs from them in its properties. It shows the highest affinity for oxygen and besides t h s possesses a unique ability to bind bulky ligands such as nicotinic acid and aliphatic acids (from acetic to valeric) [l].The highly resolved spatial structure of leghemoglobin was first established in the USSR with the second macrocomponent of yellow lupine leghemoglobin [2]. Complete amino acid sequences of the both macrocomponents of lupine Lb, Lb I and Lb 11, were also investigated in the USSR by Egorov and coworkers [3, 41. At present the structures are available of the acetate complex of lupine Lb with a 0.2-nm resolution [5] and nicotinate complex with a 0.28-nm resolution [6]. Also, the structures of the Lb complexes with nitrobenzene, nicotinic acid and acetate, fluoride and cyanide ions have been compared with that of aquamet leghemoglobin using the 0.2-nm difference Fourier synthesis [7]. The X-ray studies provided convincing evidence that leghemoglobin belongs to the globin family. At the same time they revealed distinguishing features of the Lb structure, primarily in the heme cavity, which is greater than in Mb and Hb and enables the accomodation of bulky ligands. Exact knowledge of the Lb structure gave the answer to many questions concerning the relationship between the structure of the protein and itsCorrespondence to G. B. Postnikova, Institut Biologicheskoj Fiziki, Akademiya Nauk SSSR, Pushchino, Mokovskaya Oblast', USSR 142292Ahhreviutions. Lb, leghemoglobin; SL, spin label; SL-Lb, spinlabeled leghemoglobin; ESR, electron spin resonance...

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Fluorescence study of the conformational properties of myoglobin structure

Postnikova

Komarov

Yumakova

1991

European Journal of Biochemistry

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The pH-dependent fluorescence of intact sperm whale apomyoglobin (apo-Mb) containing two tryptophans at positions 7 and 14, and of apo-Mb derivatives modified on Trp7 by 2-hydroxy-5-nitrobenzyl bromide (Koshland reagent) and o-nitrophenylsulphenyl chloride, has been studied. The fluorescence of apomyoglobins modified at His residues by iodoacetamide and bromoacetate, and at the N-terminal cr-NH2 group by methylisothiocyanate, has also been investigated. The individual fluorescent properties of both tryptophans and their contributions to the total spectrum of apo-Mb have been resolved within the pH range 2-12.5. The quantum yield of the 'buried' Trpl4 (imax at 326 nm) is shown to be twofold higher at pH >8.5 than that of the 'exposed' Trp7 (A, , , at 333 nm). At pH 8.5-5.5 the fluorescence of Trpl4 diminished approximately twofold due to quenching by the ionized His residue, most probably His1 19. The quenching is evidently dynamic because the fluorescence lifetime is shown to be linearly proportional to quantum yield in this pH range. The fluorescence of Trp7 practically does not change between pH 5.5 and 10.0 but increases 2.5-3-fold in the pH range 5.5-4.3 while the contribution of Trpl4 remains constant. The conformational changes at the N-terminal and in the region adjacent to it, as well as in the whole apo-Mb molecule in acidic, alkaline and neutral pH ranges, are considered. A relationship is revealed between conformational states of the heme crevice and the N-terminal part of apo-Mb.Knowledge of the conformational properties of a protein molecule is important for understanding the mechanism of its functioning. Studies of the conformational changes in monomeric and oligomeric globins induced by variations of pH of the medium and electron state of the heme are of particular interest because they allow one to elucidate the ways the ligand affinity is regulated.Conformational changes in the heme crevice and other parts of Mb-like proteins have been studied by many physical methods. In our work, fluorescence has been used to study pH-and ligand-induced conformational changes in myoglobins and related structures, apomyoglobin (apo-Mb) and a metal-free analog of Mb which is the complex of apo-Mb with protoporphyrin IX. The structural changes at the Nterminal and in the adjacent region of the protein were followed by the fluorescence of two tryptophan residues localized in the A-helix, Trp7 (A5) and Trpl4 (A12). Those in the heme crevice were followed by emission of protophorphyrin IX Correspondence to G. B. Postnikova, Institute of Biological Physics of the USSR Academy of Sciences, 142292, Pushchino, Moscow Region, USSR.Abbreviations. met-Mb, metmyoglobin; CA-Mb, met-Mb alkylated at His residues by iodacetamide; CM-Mb, met-Mb alkylated at His residues by bromacetate; MITC-Mb, met-Mb acylated at the N-terminal by methylisothyocyanate (MITC); apo-Mb, myoglobin containing no heme; CA-apo-Mb, apo-Mb carboxyamidated on His; CM-apo-Mb, apo-Mb carboxymethylated on His; MITC-apo-Mb, apo-Mb modified by MITC; HNB-apo-Mb...

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Absorption spectroscopy and circular dichroism studies of sperm whale myoglobin chemically modified on the N-terminal α-aminogroup by isothiocyanate reagents

Cited by 5 publications

References 18 publications

Fluorescence study of the conformational properties of myoglobin structure

Fluorescence study of the conformational properties of myoglobin structure

Spin-label study of conformational changes induced by pH and ligands in leghemoglobin from yellow lupine root nodules

Fluorescence study of the conformational properties of myoglobin structure

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