Tryptophan Phosphorescence in Fluid Solution

Strambini, Giovanni B.; Gonnelli, Margherita

doi:10.1021/ja00134a008

Cited by 106 publications

(151 citation statements)

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“…The emission was detected with an EMI 9635 QB photomultiplier. Phosphorescence decay in fluid solution at room temperature was monitored with a homemade apparatus suitable for lifetime measurements in the s-ms range described in detail elsewhere (75). Pulsed excitation( ex ϭ 292 nm) was generated by a frequency-doubled flash-pumped dye laser (UV500 M Candela) with a pulse duration of 1 s and an energy/pulse typically of 1-10 mJ.…”

Section: Methodsmentioning

confidence: 99%

“…It should be noted, however, that the variability of these parameters can be even somewhat greater when one compares different preparations of the protein. Such variability in the decay kinetics can be traced down to different amounts of quenching impurities present in organic solvents (glycerol), and glasswares (75). For this reason, comparisons are always made between samples obtained from the same enzyme preparation.…”

Section: Methodsmentioning

confidence: 99%

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Conformational Changes of the Mitochondrial F1-ATPase ∊-Subunit Induced by Nucleotide Binding as Observed by Phosphorescence Spectroscopy

Baracca

Gabellieri

Barogi

et al. 1995

Journal of Biological Chemistry

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Changes in conformation of the ⑀-subunit of the bovine heart mitochondrial F 1 -ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg 2؉ increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the ⑀-subunit are evident at high ligand concentration added to both the nucleotide-depleted F 1 (Nd-F 1 ) and the F 1 preparation containing the three tightly bound nucleotides (F 1 (2,1)). Since the effects observed are similar in both the F 1 forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the ⑀-subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F 1 . The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F 1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F 1 (3,0)). Phosphorescence lifetime measurements on this F 1 form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the ⑀-subunit. This implies that the binding to the vacant noncatalytic site of F 1 (2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F 1 and F 1 (2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F 1 (2,1) in the absence of Mg 2؉ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the ⑀-subunit is discussed.The ATPase (ATP synthase) is the enzyme responsible for ATP synthesis during oxidative phosphorylation in all energytransducing membranes. It is composed of two main parts: F 0

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Conformational Changes of the Mitochondrial F1-ATPase ∊-Subunit Induced by Nucleotide Binding as Observed by Phosphorescence Spectroscopy

Baracca

Gabellieri

Barogi

et al. 1995

Journal of Biological Chemistry

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“…12,13 The β-pleated sheet enveloping chromophore Trp109 stretches to the subunit interface establishing hydrogen bond mediated contacts with an equivalent β-pleated sheet of the neighboring subunit. 8 Mutations introduced into the contact region that prevent hydrogen bond formation, could affect communication between active sites and the rigidity of the Trp109 environment.…”

Section: Phosphorescence Decay Measurementmentioning

confidence: 99%

“…10 Structural rigidity, conformational and dynamic properties of proteins could be assessed by monitoring the phosphorescence properties of a reporter group located in the vicinity of the protein region of interest. 11 Phosphorescence lifetime represents a sensitive probe of local protein rigidity 12,13 and could be correlated to changes in rigidity triggered by binding of substrates or allosteric effectors. [14][15][16] Tryptophan phosphorescence at room temperature could provide information regarding the dynamic changes in protein structure occurring on a millisecond to second time scale.…”

Section: Introductionmentioning

confidence: 99%

“…The exceptionally rigid and solvent-inaccessible environment in the inner core of the macromolecule provides for a very long phosphorescence lifetime, up to two seconds. [11][12][13][14][15][16][17][18][19][20][21][22][23] Trp109 is connected to the active site via a rigid α-helical rod and to the subunit interface via a β-sheet that envelopes Trp109. 8 Therefore, the triplet lifetime of this residue could be used as a sensitive monitor of the conformational state in the polypeptide region that connects the active sites of neighboring subunits.…”

Section: Introductionmentioning

confidence: 99%

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Influence of Subunit Interface Mutations on Kinetic and Dynamic Properties of Alkaline Phosphatase from E. coli

Šprung¹,

Bučević-Popović²,

Soldo³

et al. 2013

Croat. Chem. Acta

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Abstract. Mutations, replacing amino acids involved in the formation of hydrogen bonds between subunits of dimeric alkaline phosphatase, have been introduced. Influence of mutations on kinetic properties and structural stability of mutant enzymes was established. In addition, alterations in protein dynamic properties have been studied using room temperature phosphorescence. Kinetic properties of both mutant enzymes were virtually the same, differing from the wild type enzyme in the k cat value that was almost twice lower. Changes in protein dynamic properties of mutant proteins, compared to the wild type enzyme, did not parallel changes in kinetic properties suggesting that an alteration in the rigidity of the Trp109 environment is not responsible for the reduction of kinetic properties. Instead, combined kinetic and dynamic consequences of introduced mutations suggest that breaking of specific links, involved in transmission of conformational change, could be responsible for altered kinetic properties. (doi: 10.5562/cca2168)

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Fluorescence and Phosphorescence Methods to Probe Protein Structure and Stability in Ice: The Case of Azurin

Strambini¹

2010

Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals

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Tryptophan Phosphorescence in Fluid Solution

Cited by 106 publications

References 0 publications

Conformational Changes of the Mitochondrial F1-ATPase ∊-Subunit Induced by Nucleotide Binding as Observed by Phosphorescence Spectroscopy

Conformational Changes of the Mitochondrial F1-ATPase ∊-Subunit Induced by Nucleotide Binding as Observed by Phosphorescence Spectroscopy

Influence of Subunit Interface Mutations on Kinetic and Dynamic Properties of Alkaline Phosphatase from E. coli

Fluorescence and Phosphorescence Methods to Probe Protein Structure and Stability in Ice: The Case of Azurin

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