Relationship between the conformation of glutamate dehydrogenase, the state of association of its subunit, and catalytic function

Strambini, Giovanni B.; Cioni, Patrizia; Puntoni, Alessandro

doi:10.1021/bi00435a028

Cited by 21 publications

(8 citation statements)

References 21 publications

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“…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 . In numerous studies, measurements of have revealed subtle variations in the structure of proteins that could not be detected by other means (17)(18)(19)30). Furthermore, wherever parallel measurements have been performed, changes in the protein structure detected by other techniques have always been reflected by a substantial alteration of .…”

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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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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show abstract

“…These features have proven to exhibit an unparalleled sensitivity to the conformational state of the macromolecule with sable to monitor even subtle alterations of the native fold often undetected by ordinary spectroscopic techniques. Examples are the relatively large variations of s often induced by ligand binding (metal ions [5,10], substrates and cofactors [11][12][13]), subunit association (14), the addition of stabilizing (15) or denaturing (16) agents and cosolvents (17), dehydration (18) or ice formation (19), besides variations of temperature and pressure (20).…”

Section: Introductionmentioning

confidence: 99%

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins¶

Gonnelli

Strambini

2005

Photochem Photobiol

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The phosphorescence lifetime (tau) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of tau in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (k(obs)) in tripeptides of the form acetyl-Trp-Gly-X-CONH2 (WGX), where Trp is joined to X by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (k(obs) > 40 s(-1)), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys >> His+ > Tyr >> Phe approximately His. For the aromatic side chains the corresponding contact rate at 20 degrees C is estimated to be between 3-4 x 10(9) s(-1) for Cys (as determined by Lapidus et al.), 0.8-8 x 10(6) s(-1) for His+, 0.37-3.7 x 10(6) s(-1) for Tyr and 0.2-2 x 10(5) s(-1) for Phe and His. In the cases of His and Tyr, k(obs) drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between -20 degrees C and 20 degrees C, the reaction is characterized by activation energies of about 5 kcal.M(-1) for His+ and Tyr and 8 kcal.M(-1) for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s(-1), showing that these moieties are practically inert with respect to the triplet-state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s(-1), a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.

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“…These features have proven to exhibit an unparalleled sensitivity to the conformational state of the macromolecule with table to monitor even subtle alterations of the native fold often undetected by ordinary spectroscopic techniques. Examples are the relatively large variations of τ often induced by ligand binding (metal ions [5,10], substrates and cofactors [11‐13]), subunit association (14), the addition of stabilizing (15) or denaturing (16) agents and cosolvents (17), dehydration (18) or ice formation (19), besides variations of temperature and pressure (20).…”

Section: Introductionmentioning

confidence: 99%

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins^¶

Gonnelli

Strambini

2005

Photochem & Photobiology

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The phosphorescence lifetime (τ) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of τ in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (kobs) in tripeptides of the form acetyl‐Trp‐Gly‐X‐CONH2 (WGX), where Trp is joined to × by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (kobs > 40 s−1), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys >> His+ > Tyr >> Phe ∼ His. For the aromatic side chains the corresponding contact rate at 20°C is estimated to be between 3‐4 × 109 s−1 for Cys (as determined by Lapidus et al.), 0.8‐8 × 106 s−1 for His+, 0.37‐3.7 × 106 s−1 for Tyr and 0.2‐2 × 105 s−1 for Phe and His. In the cases of His and Tyr, kobs drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between −20°C and 20°C, the reaction is characterized by activation energies of about 5 kcal·M−1 for His+ and Tyr and 8 kcal ·M−1 for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s−1, showing that these moieties are practically inert with respect to the triplet‐state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s−1, a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.

show abstract

Relationship between the conformation of glutamate dehydrogenase, the state of association of its subunit, and catalytic function

Cited by 21 publications

References 21 publications

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

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

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins¶

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins^¶

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