Phosphorescence and optically detected magnetic resonance studies of a class of anomalous tryptophan residues in globular proteins

Hershberger, Martin V.; Maki, August H.; Galley, William C.

doi:10.1021/bi00551a032

Cited by 94 publications

(113 citation statements)

References 24 publications

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“…The 0-0 vibrational band of the Trp spectrum is centered at 411 nm, a wavelength characteristic of hydrophobic sites in proteins and certainly red shifted with respect to 406-408 nm, the wavelength for residues exposed to the aqueous phase., it is worth noting that the width of this band is about twice that obtained for a single protein with the chromophore in a well defined site [30]. Such broadness of the phosphorescence spectrum implies environmental heterogeneity of the chromophore, a finding that can be rationalized either in terms of distinct conformers of the &-subunit or by the presence of other protein impurities.…”

Section: Low-temperature Phosphorescence Emissionmentioning

confidence: 84%

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Tryptophan phosphorescence as a structural probe of mitochondrial F₁‐ATPase ε‐subunit

Solaini

Baracca

Castelli

et al. 1993

European Journal of Biochemistry

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We report the detection of tryptophan phosphorescence emission from the sole residue in the Esubunit of the bovine heart mitochondrial F, -ATPase complex. The phosphorescence spectrum, intensity and decay kinetics have been measured over the temperature range 160-273 K. The fine structure in the phosphorescence spectrum at low temperature, with the 0-0 vibrational band centered at 411 nm, reveals the hydrophobic nature of the chromophore's environment. Both the large width of the 0-0 vibrational band and the heterogeneous decay kinetics in fluid solution emphasize the existence of multiple conformations of the &-subunit, structures which are rather stable as they do not interconvert in the millisecond time scale. Further, from the relatively long triplet lifetime at 273 K, it is possible to infer the existence of a tight, rigid core in the structure of the &-subunit. Under subunit-dissociating conditions (6 M urea), the spectrum at 160 K undergoes a slight blue shift but since the phosphorescence lifetime, at all temperatures, is similar or longer than in the absence of dissociant, we conclude that dissociation does not lead to solvent exposure of the tryptophanyl side-chain. This conclusion is supported by the results obtained at 273 K by dissociating F, in the presence of 0.3 M guanidine hydrochloride. Phosphorescence lifetimes indicate that 6 M

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Section: Low-temperature Phosphorescence Emissionmentioning

confidence: 84%

“…1. The fractions [22][23][24][25][26][27][28][29][30][31][32] were characterized by constant specific activity, indicating the presence of a single molecular species that was used in the experiments.…”

Section: Preparation Of F-atpase and Removal Of Nucleotidesmentioning

confidence: 99%

Tryptophan phosphorescence as a structural probe of mitochondrial F₁‐ATPase ε‐subunit

Solaini

Baracca

Castelli

et al. 1993

European Journal of Biochemistry

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“…At low temperature, the spectrum of Trp generally displays a pronounced vibronic structure with a relatively well resolved 0,0 vibrational band. Although the wavelength of the 0,0 band ( 0 , 0 ) is related to the polarity/polarizability of the indole environment (10), its bandwidth (BW, the width at half height) reports on the structural homogeneity of the site (11). The spectrum of Trp-72 in glycerol/phosphate buffer (20 mM, pH 7; 60/40, w/w) at 140 K is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The sharp vibrational structure of the phosphorescence spectrum gives detailed information on the polarity and conformational homogeneity of the Trp environment (10,11). In the absence of internal quenching by proximal cysteine, cystine, tyrosine, or histidine residues, the phosphorescence lifetime of proteins in fluid solutions provides a direct probe of the local flexibility of the protein matrix.…”

mentioning

confidence: 99%

Tryptophan Phosphorescence Spectroscopy Reveals That a Domain in the NAD(H)-binding Component (dI) of Transhydrogenase from Rhodospirillum rubrum Has an Extremely Rigid and Conformationally Homogeneous Protein Core

Broos

Gabellieri

Boxel

et al. 2003

Journal of Biological Chemistry

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The characteristics of tryptophan phosphorescence from the NAD(H)-binding component (dI) component ofRhodospirillum rubrum transhydrogenase are described. This enzyme couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a membrane and is only active as a dimer. Tryptophan phosphorescence spectroscopy is a sensitive technique for the detection of protein conformational changes and was used here to characterize dI under mechanistically relevant conditions. Our results indicate that the single tryptophan in dI, Trp-72, is embedded in a rigid, compact, and homogeneous protein matrix that efficiently suppresses collisional quenching processes and results in the longest triplet lifetime for Trp ever reported in a protein at ambient temperature (2.9 s). The protein matrix surrounding Trp-72 is extraordinarily rigid up to 50°C. In all previous studies on Trp-containing proteins, changes in structure were reflected in a different triplet lifetime. In dI, the lifetime of Trp-72 phosphorescence was barely affected by protein dimerization, cofactor binding, complexation with the NADP(H)-binding component (dIII), or by the introduction of two amino acid substitutions at the hydride-transfer site. It is suggested that the rigidity and structural invariance of the protein domain (dI.1) housing this Trp residue are important to the mechanism of transhydrogenase: movement of dI.1 affects the width of a cleft which, in turn, regulates the positioning of bound nucleotides ready for hydride transfer. The unique protein core in dI may be a paradigm for the design of compact and stable de novo proteins.Transhydrogenase couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a biological membrane according to Equation 1.The enzyme is found in the inner membrane of higher animal mitochondria and in the cytoplasmic membrane of many bacteria (1). Under physiological conditions, it is thought to utilize the proton electrochemical gradient to generate NADPH. Transhydrogenase comprises three components: dI 1 (ϳ380 residues), which binds NAD ϩ /NADH; dII (ϳ400 residues), the membrane-embedded component harboring the proton translocation channel; and dIII (ϳ200 residues), which binds NADP ϩ / NADPH (Fig. 1). Transhydrogenase is a "dimer" of two dI-dIIdIII "monomers", though the polypeptide composition varies in different species. One of the best characterized transhydrogenases is that from Rhodospirillum rubrum. Recombinant dI and dIII from this organism are stable proteins that bind their cognate nucleotides. Upon mixing, they readily form a dI 2 ⅐dIII 1 complex (K d Ϸ 25 nM), which catalyzes rapid hydride transfer between bound nucleotides. Crystal structures are available for isolated dI (2, 3), dIII (4, 5), and for the complex (6).In transhydrogenase, proton translocation is linked to the redox reaction by way of inter-domain conformational changes. Our current working model is that proton translocation through dII drives transhydrogenase between an "open" state, in which the nucleo...

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“…Although the wavelength of the 0,0-vibrational band, 0,0 , is related to the polarity-polarizability of the indole environment (26), its bandwidth (BW, the width at two-thirds height) reports on the structural homogeneity of the site (27). For free Trp in homogeneous solutions, 0,0 ranges from 406 nm for a polar aqueous solution, to 411 nm for a completely non-polar hydrocarbon solvent (26).…”

Section: Fluorescence and Phosphorescence Characteristics Of Single-tmentioning

confidence: 99%

Substrate-induced Conformational Changes in the Membrane-embedded IICmtl-domain of the Mannitol Permease from Escherichia coli, EnzymeIImtl, Probed by Tryptophan Phosphorescence Spectroscopy

Veldhuis

Gabellieri

Vos

et al. 2005

Journal of Biological Chemistry

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Membrane-bound transport proteins are expected to proceed via different conformational states during the translocation of a solute across the membrane. Tryptophan phosphorescence spectroscopy is one of the most sensitive methods used for detecting conformational changes in proteins. We employed this technique to study substrate-induced conformational changes in the mannitol permease, EnzymeII mtl , of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Ten mutants containing a single tryptophan were engineered in the membrane-embedded IIC mtl -domain, harboring the mannitol translocation pathway. The mutants were characterized with respect to steady-state and time-resolved phosphorescence, yielding detailed, site-specific information of the Trp microenvironment and protein conformational homogeneity. The study revealed that the Trp environments vary from apolar, unstructured, and flexible sites to buried, highly homogeneous, rigid peptide cores. The most remarkable example of the latter was observed for position 97, because its long sub-second phosphorescence lifetime and highly structured spectra in both glassy and fluid media imply a well defined and rigid core around the probe that is typical of ␤-sheet-rich structural motifs. The addition of mannitol had a large impact on most of the Trp positions studied. In the case of position 97, mannitol binding induced partial unfolding of the rigid protein core. On the contrary, for residue positions 126, 133, and 147, both steady-state and time-resolved data showed that mannitol binding induces a more ordered and homogeneous structure around these residues. The observations are discussed in context of the current mechanistic and structural model of EII mtl . A phoA fusion study and hydropathy analysis of the IIC mtl -domain resulted in a topology model with three small periplasmic loops, two large cytoplasmic loops, and six putative membrane-spanning helices (6). It has been proposed that both large cytoplasmic loops fold back into the membrane-embedded part of the protein, lining up a hydrophilic pathway for the translocation of the carbohydrate (7). New structural insight on the basis of cysteine-scanning mutagenesis in the first proposed cytoplasmic loop provided evidence for the presence of this loop protruding, at least partly, into the bilayer (8). For the subcloned IIC mtldomain, a two-dimensional projection structure at 5-Å resolution was determined by electron microscopy crystallography (9). Six regions of high density were found, possibly reflecting six membrane-spanning helices.Of the available spectroscopic techniques, Trp phosphorescence spectroscopy is one of the most sensitive approaches used to study changes in protein conformation, due to the extremely slow (radiative) de-excitation rate of the triplet excited state (ϳ0.2 s Ϫ1 ). This makes Trp phosphorescence 10 8 times more sensitive for quenching processes than Trp fluorescence. A conformational change, nearby or more remote from the Trp probe, is expected to induce ...

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Phosphorescence and optically detected magnetic resonance studies of a class of anomalous tryptophan residues in globular proteins

Cited by 94 publications

References 24 publications

Tryptophan phosphorescence as a structural probe of mitochondrial F₁‐ATPase ε‐subunit

Tryptophan phosphorescence as a structural probe of mitochondrial F₁‐ATPase ε‐subunit

Tryptophan Phosphorescence Spectroscopy Reveals That a Domain in the NAD(H)-binding Component (dI) of Transhydrogenase from Rhodospirillum rubrum Has an Extremely Rigid and Conformationally Homogeneous Protein Core

Substrate-induced Conformational Changes in the Membrane-embedded IICmtl-domain of the Mannitol Permease from Escherichia coli, EnzymeIImtl, Probed by Tryptophan Phosphorescence Spectroscopy

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Phosphorescence and optically detected magnetic resonance studies of a class of anomalous tryptophan residues in globular proteins

Cited by 94 publications

References 24 publications

Tryptophan phosphorescence as a structural probe of mitochondrial F1‐ATPase ε‐subunit

Tryptophan phosphorescence as a structural probe of mitochondrial F1‐ATPase ε‐subunit

Tryptophan Phosphorescence Spectroscopy Reveals That a Domain in the NAD(H)-binding Component (dI) of Transhydrogenase from Rhodospirillum rubrum Has an Extremely Rigid and Conformationally Homogeneous Protein Core

Substrate-induced Conformational Changes in the Membrane-embedded IICmtl-domain of the Mannitol Permease from Escherichia coli, EnzymeIImtl, Probed by Tryptophan Phosphorescence Spectroscopy

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Tryptophan phosphorescence as a structural probe of mitochondrial F₁‐ATPase ε‐subunit

Tryptophan phosphorescence as a structural probe of mitochondrial F₁‐ATPase ε‐subunit