Multiple-state equilibrium unfolding of guanidino kinases

Groß, Martin; Lustig, Ariel; Wallimann, Theo; Furter, Rolf

doi:10.1021/bi00033a005

Cited by 69 publications

(76 citation statements)

References 36 publications

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“…Unfolding transitions in the secondary structure of the variants were monitored by the calculation of [] at 222 nm, as a function of urea concentration (24). The signal diminished at increasing denaturant concentration (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…With respect to the maximum peak height shown by the native enzyme, 14.0 Ϯ 1.5-, 10.4 Ϯ 1.6-, and 7.5 Ϯ 1.0-fold increases were recorded for G6PD B, G6PD A, and G6PD A Ϫ , respectively, indicating a different degree of hydrophobic surface exposure in each variant. Thus, the presence of the N126D mutation alone in G6PD A leads to reduced ANS binding, which is further reduced when the second mutation V68M in G6PD A Ϫ is present, indicating a progressive tendency toward loss in tertiary structure (24) as mutations are introduced into the protein. This is in agreement with the reduced amount of ellipticity signal at 270 nm in the double mutant.…”

Section: Table I Secondary Structural Weightsmentioning

confidence: 99%

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Structural Defects Underlying Protein Dysfunction in Human Glucose-6-phosphate Dehydrogenase A− Deficiency

Gómez‐Gallego

Garrido-Pertierra

Bautista

2000

Journal of Biological Chemistry

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The enzyme variant glucose-6-phosphate dehydrogenase (G6PD) A ؊ , which gives rise to human glucose-6-phosphate dehydrogenase deficiency, is a protein of markedly reduced structural stability. This variant differs from the normal enzyme, G6PD B, in two amino acid substitutions. A further nondeficient variant, G6PD A, bears only one of these two mutations and is structurally stable. In this study, the synergistic structural defect in recombinant G6PD A ؊ was reflected by reduced unfolding enthalpy due to loss of ␤-sheet and ␣-helix interactions where both mutations are found. This was accompanied by changes in inner spatial distances between residues in the coenzyme domain and the partial disruption of tertiary structure with no significant loss of secondary structure. However, the secondary structure of G6PD A ؊ was qualitatively affected by an increase in ␤-sheets substituting ␤-turns related to the lower unfolding enthalpy. The structural changes observed did not affect the active site of the mutant proteins, since its spatial position was unmodified. The final result is a loss of folding determinants leading to a protein with decreased intracellular stability. This is suggested as the cause of the enzyme deficiency in the red blood cell, which is unable to perform de novo protein synthesis.

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Section: Resultsmentioning

confidence: 99%

Section: Table I Secondary Structural Weightsmentioning

confidence: 99%

Structural Defects Underlying Protein Dysfunction in Human Glucose-6-phosphate Dehydrogenase A− Deficiency

Gómez‐Gallego

Garrido-Pertierra

Bautista

2000

Journal of Biological Chemistry

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“…Intrinsic tryptophan fluorescence is a sensitive probe of local and global conformation in proteins [24], and has commonly been used to investigate protein conformational stability upon addition of chemical denaturants, such as urea and guanidinium chloride [25][26][27]. We measured the intrinsic tryptophan fluorescence of EnvZ per to probe mainly for pH-sensitivity of these tryptophan residues in the refolded protein.…”

Section: Structural Characterizationmentioning

confidence: 99%

Structural characterization of Escherichia coli sensor histidine kinase EnvZ: the periplasmic C-terminal core domain is critical for homodimerization

Khorchid

Inouye

Ikura

2004

Biochemical Journal

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Escherichia coli EnvZ is a membrane sensor histidine kinase that plays a pivotal role in cell adaptation to changes in extracellular osmolarity. Although the cytoplasmic histidine kinase domain of EnvZ has been extensively studied, both biochemically and structurally, little is known about the structure of its periplasmic domain, which has been implicated in the mechanism underlying its osmosensing function. In the present study, we report the biochemical and biophysical characterization of the periplasmic region of EnvZ (Ala 38 -Arg 162 ). This region was found to form a dimer in solution, and to consist of two well-defined domains: an N-terminal α-helical domain and a C-terminal core domain (Glu 83 -Arg 162 ) containing both α-helical and β-sheet secondary structures. Our pull-down assays and analytical ultracentrifugation analysis revealed that dimerization of the periplasmic region is highly sensitive to the presence of CHAPS, but relatively insensitive to salt concentration, thus suggesting the significance of hydrophobic interactions between the homodimeric subunits. Periplasmic homodimerization is mediated predominantly by the C-terminal core domain, while a regulatory function may be attributed mainly to the N-terminal α-helical domain, whose mutations have been shown previously to produce a high-osmolarity phenotype.

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“…3, the greatest differences between R-CK and O-CK were in the region from 0 to 1 M GdnHCl, during which both R-CK and O-CK had steep burst in the spectra as was shown in the insets. The steep transition has been attributed to dimer dissociation (26). As indicated by changes in secondary structures, tertiary structures, and hydrophobic surface exposure, the steep transition in the structures of O-CK occurred at lower concentrations of GdnHCl than that of R-CK.…”

mentioning

confidence: 99%

The Generation of the Oxidized Form of Creatine Kinase Is a Negative Regulation on Muscle Creatine Kinase

Zhao

Yan

Liu

et al. 2007

Journal of Biological Chemistry

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Muscle creatine kinase (CK) is a crucial enzyme in energy metabolism, and it exists in two forms, the reduced form (R-CK) and the oxidized form (O-CK). In contrast with R-CK, O-CK contained an intrachain disulfide bond in each subunit. Here we explored the properties of O-CK and its regulatory role on muscle CK. The intrachain disulfide bond in O-CK was demonstrated to be formed between Cys 74 and Cys 146 by site-directed mutagenesis. Biophysical analysis indicated that O-CK showed decreased catalytic activity and that it might be structurally unstable. Further assays through guanidine hydrochloride denaturation and proteolysis by trypsin and protease K revealed that the tertiary structure of O-CK was more easily disturbed than that of R-CK. Surprisingly, O-CK, unlike R-CK, cannot interact with the M-line protein myomesin through biosensor assay, indicating that O-CK might have no role in muscle contraction. Through in vitro ubiquitination assay, CK was demonstrated to be a specific substrate of muscle ring finger protein 1 (MURF-1). O-CK can be rapidly ubiquitinated by MURF-1, while R-CK can hardly be ubiquitinated, implying that CK might be degraded by the ATP-ubiquitin-proteasome pathway through the generation of O-CK. The results above were further confirmed by molecular modeling of the structure of O-CK. Therefore, it can be concluded that the generation of O-CK was a negative regulation of R-CK and that O-CK might play essential roles in the molecular turnover of MM-CK.In the living cells, continuous turnover of the structural, catalytic, and regulatory proteins are required to maintain the normal function. Two pathways are responsible for degradation of most of the cellular proteins, the lysosomal proteases and the ATP-ubiquitin-proteasome system (1, 2). The ubiquitin system is the main proteolytic system involved in intracellular catabolism of most abnormal, short-lived and long-lived muscle proteins (3). Studies in various cases of atrophy indicated that the activation of this pathway is primarily responsible for the rapid loss of muscle proteins (3, 4), which was further confirmed by that the two muscle specific E3 2 ligases, MURF-1 and MURF-2, had been suggested to target a lot of myofibrillar proteins and energy metabolism enzymes for ubiquitin-dependent degradation (5). For the myofibrillar proteins, the dissociation from the myofibrillar complexes was the rate-limiting step in the degradation (3), but little is known about how the cytoplasmic proteins, including the enzymes required for ATP/energy production, in the muscle cells were recognized and degraded by the ubiquitin system. Among the energy metabolism enzymes in the muscle cells, creatine kinase (CK, EC 2.7.3.2) plays a significant role in energy homeostasis (6, 7). It catalyzes the reversible conversion from MgATP and creatine to MgADP and phosphocreatine, high energy phosphate able to supply ATP on demand. Muscle type CK (MM-CK) has the unique property to bind with the M-line of sarcomere mediated by the NH 2 -terminal lysine charge clamp...

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Multiple-state equilibrium unfolding of guanidino kinases

Cited by 69 publications

References 36 publications

Structural Defects Underlying Protein Dysfunction in Human Glucose-6-phosphate Dehydrogenase A− Deficiency

Structural Defects Underlying Protein Dysfunction in Human Glucose-6-phosphate Dehydrogenase A− Deficiency

Structural characterization of Escherichia coli sensor histidine kinase EnvZ: the periplasmic C-terminal core domain is critical for homodimerization

The Generation of the Oxidized Form of Creatine Kinase Is a Negative Regulation on Muscle Creatine Kinase

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