Heterogeneity of protein conformation in solution from the lifetime of tryptophan phosphorescence

Cioni, Patrizia; Gabellieri, Edi; Gonnelli, Margherita; Strambini, Giovanni B.

doi:10.1016/0301-4622(94)00039-5

Cited by 24 publications

(19 citation statements)

References 38 publications

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“…As temperature imparts thermal kinetic energy to the protein structure, the conformational interconversions may become too rapid for catalysis to occur efficiently, causing a reduction in reaction velocity before any inactivation process has commenced. Cioni et al [37] reported that a large number of protein sub-states exist in solution, and the equilibrium between the different forms was affected by the surrounding environment. At high temperatures the interconversions between sub-states, which in PGK can include the ' open ' and ' closed ' domain forms, became too rapid to distinguish using this experimental method.…”

Section: Discussionmentioning

confidence: 99%

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

Thomas

Scopes

1998

Biochemical Journal

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The effects of temperature on the kinetic parameters kcat and Km, for three isolates of the highly conserved monomeric enzyme 3-phosphoglycerate kinase (PGK), were investigated in detail using a rapid automated kinetics apparatus. PGK was purified from the thermophilic bacterium Thermoanaerobacter sp. Rt8.G4 (optimum growth temperature 68 °C), the mesophile Zymomonas mobilis (optimum growth temperature 32 °C) and a second, unidentified, soil mesophile designated unid A (optimum growth temperature 27 °C). The kinetic behaviour with temperature of each PGK preparation was distinct, despite the conserved nature of the enzyme. The kcat values increased with temperature, but not as rapidly exponentially, as might be expected from the Arrhenius equation. Maximum kcat values were at much higher temperatures than the optimum growth temperatures for the mesophiles, but for the thermophile the temperature of maximum kcat was close to its optimum growth temperature. Km values were in general nearly constant through the lower temperature ranges, but increased substantially as the optimum temperature (highest kcat) was passed. Thermal irreversible denaturation of the PGK proteins was also investigated by measuring loss of activity over time. In a dilute buffer, Arrhenius plots for denaturation were linear, and the calculated apparent energy of activation (Eact) for denaturation for the thermophilic PGK was 600 kJ·mol-1, whereas for the mesophilic enzymes the values were 200-250 kJ·mol-1. In the presence of substrates, a considerable stabilization occurred, and in the case of the Z. mobilis enzyme, the apparent Eact was increased to 480 kJ·mol-1. A theoretical explanation for these observations is presented. Comparing the kinetics data with irreversible denaturation rates determined at relevant temperatures, it was clear that kcat values reached a maximum, and then decreased with higher temperature before irreversible denaturation had any significant influence.

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Section: Discussionmentioning

confidence: 99%

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

Thomas

Scopes

1998

Biochemical Journal

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“…Monoexponential fluorescence in single Trp proteins is rarely observed and is taken to indicate that the Trp side-chain adopts only one rotameric configuration. Similarly, the phosphorescence decay of individual Trp residues is often non-exponential, multiple lifetimes emphasizing that, at ambient temperature, various native-like states of the macromolecule, differing in internal flexibility, are in thermal equilibrium (28).…”

Section: Discussionmentioning

confidence: 99%

“…The phosphorescence lifetime of Trp is one of the most sensitive probes available for the detection of conformational changes in proteins. Subtle changes in the intramolecular quenching configuration, changes in accessibility to external quenchers, and changes in local flexibility will all affect the triplet lifetime (20,28,29). The large window of lifetimes, from sub-milliseconds to seconds, makes detection of conformational changes straightforward and offers very high sensitivity, particularly for buried Trp residues that exhibit a long .…”

Section: Discussionmentioning

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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“…The above mentioned RTTP techniques permit monitoring of slow processes, extending the observation time scale from the nanosecond range of fluorescence up to microsecond to second range [4]. Additional potential of a triplet state as a structural probe comes from the drastic dependence of the tryptophan phosphorescence lifetime on the differences in the rigidity of the indole environment [5,6]. Due to the fact that similarity is expected between the room temperature phosphorescence lifetime of tryptophan residues which are exposed to solvent and that of free tryptophan in solution, it is of a great importance to determine the lifetime of the latter.…”

Section: Introductionmentioning

confidence: 99%

Photophysics of indole, tryptophan and N-acetyl-l-tryptophanamide (NATA): Heavy atom effect

Kowalska-Baron

Chan

Gałęcki

et al. 2012

Spectrochimica Acta Part A: Molecular and Biomolecular Spectros

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Heterogeneity of protein conformation in solution from the lifetime of tryptophan phosphorescence

Cited by 24 publications

References 38 publications

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

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

Photophysics of indole, tryptophan and N-acetyl-l-tryptophanamide (NATA): Heavy atom effect

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