Phosphorescence studies of environmental heterogeneity for tryptophyl residues in proteins

Purkey, Robert M.; Galley, William C.

doi:10.1021/bi00820a010

Cited by 128 publications

(82 citation statements)

References 19 publications

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“…less than one third. This finding is in agreement with the data of Purkey and Galley [3] on phosphorescence quenching by NADH in a rigid medium. They found that emission of the exposed tryptophan residues is quenched about three times less than that of the buried ones.…”

Section: Quenching Of the Protein Fluorescence By Iodidesupporting

confidence: 93%

“…~ Since the measured quantum yield of the protein, @ is an average from four tryptophan residues (two 'red' and two 'blue'j and assuming that the quantum yield of the red tryptophyl residue to be 33% of the total protein quantum yield, then Our finding, that the fluorescence quenching by iodide is interpretable in terms of two types of tryptophan emissions, is in agreement with the already quoted work by Purkey and Galley [3]. These authors, investigating the quenching by bromide of the phosphorescence of horse-liver alcohol dehydrogenase in a rigid medium, found that one type of tryptophan was more exposed to the solvent than the other and they were able to resolve the tryptophan phosphorescence decay curves in a glass containing 3 M sodium bromide into a sum of two exponential decays with different lifetimes.…”

Section: Quenching Of the Protein Fluorescence By Iodidesupporting

confidence: 90%

“…Purkey and Galley [3] have been able to establish from observations in ethylene glycol buffer glass at 77 K that the heterogeneity of protein phosphorescence arises from emission from independent tryptophan classes and they have also demonstrated a corresponding difference in fluorescence.…”

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confidence: 99%

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Fluorescence Quenching and Energy Transfer in Complexes between Horse‐Liver Alcohol Dehydrogenase and Coenzymes

Abd-Allah

Biellmann

Wiget

et al. 1978

European Journal of Biochemistry

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The fluorescence properties of horse-liver alcohol dehydrogenase were investigated with the aim of separating the contribution of Trp-15 (which is close to the protein surface) from that of Trp-314 (buried in the interior of the protein). Quenching of the protein fluorescence by iodide involves, to a larger degree, the longer wavelength region of the protein emission spectrum and is interpreted to involve only , , at 340 nm and a quantum yield of 0.19). It is shown that quenching by NAD and NADH involves both types of tryptophan, but the 'blue' one to a larger extent. In the case of NADH, radiationless energy transfer between enzyme and reduced nicotinamide ring accounts for less than one half of the total protein fluorescence quenching. Energy transfer between the tryptophan and adenine rings is also possible, but it cannot account for the rest of the protein quenching. Thus, it is suggested that protein conformational changes, following NADH binding, are the cause of part of the fluorescence quenching. The extent to which quenching by NAD can be ascribed to radiationless energy transfer processes is also calculated. It is shown that despite the small spectral overlapping between coenzyme absorption and protein emission, the energy transfer contribution cannot be neglected. However, it is very likely that also in this case a sizeable part of the protein fluorescence quenching comes from protein conformational changes following coenzyme binding. The possible nature of these conformational changes is discussed, taking into account recent X-ray data of enzyme-coenzyme complexes.Horse-liver alcohol dehydrogenase has four tryptophan residues, two per subunit of molecular weight 40000 [l]. Recent X-ray data locate them spatially: Trp-15 is close to the surface and probably exposed to water, while Trp-314 is buried inside the protein, close to the interaction domain of the two subunits property has been used in a number of ways in order to study the interaction between the apoenzyme and coenzyme [4-81. The quenching produced by binding of NADH is usually attributed to energy transfer [5,8] whereas the quenching upon binding of NAD, analogous to the cases of octopine dehydrogenase [9] and yeast alcohol dehydrogenase [I 01, has been ascribed to a protein conformational change [7]. The present work analyzes the overall protein fluorescence in terms of the contribution of the two types of tryptophan residues. It will be shown that it

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Section: Quenching Of the Protein Fluorescence By Iodidesupporting

confidence: 93%

Section: Quenching Of the Protein Fluorescence By Iodidesupporting

confidence: 90%

See 1 more Smart Citation

Fluorescence Quenching and Energy Transfer in Complexes between Horse‐Liver Alcohol Dehydrogenase and Coenzymes

Abd-Allah

Biellmann

Wiget

et al. 1978

European Journal of Biochemistry

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“…Spectra were recorded on an emission spectrometer described elsewhere. 4 The exciting band width was 6.3 nm, while the emission monochromator band width was 3.3 nm.…”

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confidence: 99%

Role of Heterogeneity of the Solvation Site in Electronic Spectra in Solution

Galley

Purkey

1970

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

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175

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Abstract. The emission spectra of polar aromatic molecules in rigid, polar solution are shown to depend on the exciting wavelength. Occurrence of the phenomenon depends on both the excited-state lifetime of the chromophore and the degree of rigidity of the medium. The results are interpreted in terms of a model which stresses the contribution of micro-environmental heterogeneity to electronic absorption and emission spectra.Shifts in chromophore electronic absorption and emission spectra as a function of the polarity and polarizability of the solvent are well known and have been interpreted by Bayliss and McRae in terms of permanent and induced dipolar interactions between a molecule in its ground and excited states and the solvent.' The interactions have been qualitatively treated as occurring between the chromophore and a general molecular environment. For aromatic amino acid residues in proteins2-4 and for aromatic hydrocarbons trapped in polycrystalline matrices' a considerable body of evidence exists that indicates that chromophores of the same species can have different electronic energies as a consequence of the existence of several distinct local environments. Even in a homogeneous phase, solute molecules can be expected to occupy a variety of solvation sites at any given time, generating an array of electronic transition energies whose summation comprises the absorption or emission spectrum of the sample as a whole. Such variation in solute-solvent local interactions is thought to be a significant source of the broadening of the absorption and emission spectra of a chromophore in a condensed phase.In the present article we provide evidence for the contribution of microenvironmental heterogeneity to solution spectra through our observation of an exciting-wavelength dependence for fluorescence and phosphorescence spectra of aromatic molecules in dilute rigid solution. In exploring the consequences of this environmental heterogeneity we show that it provides a rationalization for the failure of fluorescence concentration depolarization at the red edge of the solute absorption band7-9-the so-called "Weber red edge effect." 10 The Model. No attempt is made in this paper to present a quantitative model for the contribution of environmental heterogeneity to electronic spectra. We only emphasize three qualitative assumptions: (1) that electronic energies of chromophores in solution are a function of geometry-dependent solute-solvent interactions, (2) that at any instant in time there is an ensemble of interac-1116

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“…1) of the aromatic residue provides evidence for a phosphate-induced change within the local environment of the emitting residue. The red shift derives from either an increase in local polarizability, or an alteration in a specific asymmetric polar interaction with the aromatic side chain (Purkey and Galley, 1970). An increase in polarizability could arise, for example, from the approach of Tyr84 toward the aromatic side chain of Trpl09 on phosphate binding.…”

Section: Discussionmentioning

confidence: 99%

Room Temperature Phosphorescence Study of Phosphate Binding in Escherichia Coli Alkaline Phosphatase

Sun¹,

Kantrowitz²,

Galley³

1997

European Journal of Biochemistry

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The phosphorescence spectrum and decay of TrplO9 in Escherichia coli alkaline phosphatase was measured for the enzyme in 10 mM Tris/HCl, pH 7.4, at 21 "C. Changes in the spectrum and decay from the steady-state in response to non-covalent phosphate binding suggested a phosphate-induced alteration in the local environment surrounding Trpl09 which lies buried below the active site. The seemingly inflexible structure in the region of Trpl09, as judged by its very long phosphorescence lifetime, appeared unaltered when the enzyme was symmetrically bound with phosphate. However, the protein with phosphate bound to only one site displayed a marked increase in flexibility that extended over both subunits. For ratios of phosphatelenzyme (mollniol) between 1.0 and 2.0, the observation of exponential phosphorescence decays with lifetimes that are a function of dilution provided evidence for the rapid exchange between phosphate half-saturated and fully-saturated enzymes consistent with observed enzyme turnover rates. The lifetimes under these conditions result in the calculation of a Kd for the dissociation of phosphate from the doubly occupied enzyme of 1.1 ?0.1 pM. The non-exponential decays at P/E, (phosphate/ dimeric enzyme) ratios less than 1.0 revealed that the exchange of phosphate between phosphate-free and half-saturated enzymes was not occurring on the timescale of the phosphorescence decay times, which implied that the half-saturated molecule cannot be contributing significantly to catalysis under steadystate conditions. The observation that the phosphorescence decay at a P/Ed ratio of 1.0 is exponential with a lifetime characteristic of the half-saturated species indicates that the binding of the first phosphate is significantly greater than the second, or that the binding exhibits negative cooperativity.Keywords: alkaline phosphatase; phosphorescence ; phosphate binding ; conformational flexibility ; negative cooperativity.Escherichia coli alkaline phosphatase (AP) is usually considered as a model for the alkaline phosphatases found in a wide variety of species (Wyckoff et al., 1983;Kim and Wyckoff, 1989). The amino acid sequences of the alkaline phosphatases are highly conserved in evolution and the kinetic properties are similar among the enzymes that are widely distributed from bacteria to mammalian tissues . The enzyme is a nonspecific phosphomonoesterase that catalyzes the hydrolysis of phosphate monoesters to the corresponding alcohol, and in addition in the presence of a phosphate acceptor, a transphosphorylation reaction involving a phosphoseryl intermediate (Schwartz and Lipmann, 1961;Dayan and Wilson, 1964;Bradshaw et al., 1981). The dimeric AP molecule has a molecular mass of 95 kDa with two tightly bound zinc ions, and one magnesium ion in each of the active centers of the two identical subunits. Its kinetic properties have been well characterized and it has been established that phosphate dissociation from the enzyme is the rate-limiting step in catalysis at alkaline pH (Reid

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Phosphorescence studies of environmental heterogeneity for tryptophyl residues in proteins

Cited by 128 publications

References 19 publications

Fluorescence Quenching and Energy Transfer in Complexes between Horse‐Liver Alcohol Dehydrogenase and Coenzymes

Fluorescence Quenching and Energy Transfer in Complexes between Horse‐Liver Alcohol Dehydrogenase and Coenzymes

Role of Heterogeneity of the Solvation Site in Electronic Spectra in Solution

Room Temperature Phosphorescence Study of Phosphate Binding in Escherichia Coli Alkaline Phosphatase

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