Protein folding and association: In vitro studies for self-organization and targeting in the cell

Jaenicke, Rainer

doi:10.1016/s0070-2137(96)80008-2

Cited by 65 publications

(48 citation statements)

References 447 publications

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“…However, the thermal and GdmC1-induced denaturation transitions exhibit significant differences. Similar observations have been reported for other domain proteins (Jaenicke 1987(Jaenicke , 1996. Presently, no clear-cut explanation can be given for the divergent mechanisms, because the "unfolded state" of proteins is ill defined, especially comparing the effects of different denaturants.…”

Section: Thermal Denaturationsupporting

confidence: 72%

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Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: Structure and stability of the recombinant enzyme and its isolated cellulose‐binding domain

et al. 1997

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The hyperthermophilic bacterium Thennotoga maritima is capable of gaining metabolic energy utilizing xylan. XynA, one of the corresponding hydrolases required for its degradation, is a 120-kDa endo-1.4-D-xylanase exhibiting high intrinsic stability and a temperature optimum -90 "C. Sequence alignments with other xylanases suggest the enzyme to consist of five domains. The C-terminal part of XynA was previously shown to be responsible for cellulose binding In order to characterize the domain organization and the stability of XynA and its C-terminal cellulose-binding domain (CBD), the two separate proteins were expressed in Escherichia coli. CBD, because of its instability in its ligand-free form, was expressed as a glutathione S-transferase fusion protein with a specific thrombin cleavage site as linker. XynA and CBD were compared regarding their hydrodynamic and spectral properties. As taken from analytical ultracentrifugation and gel permeation chromatography, both are monomers with 116 and 22 kDa molecular masses, respectively. In the presence of glucose as a ligand, CBD shows high intrinsic stability. Denaturation/renaturation experiments with isolated CBD yield >80% renaturation, indicating that the domain folds independently. Making use of fluorescence emission and far-UV circular dichroism in order to characterize protein stability, guanidine-induced unfolding of XynA leads to biphasic transitions, with half-concentrations cl12(GdmC1) -4 M and >5 M, in accordance with the extreme thermal stability. At acid pH, XynA exhibits increased stability, indicated by a shift of the second guanidine-transition from 5 to 7 M GdmC1. This can be tentatively attributed to the cellulose-binding domain. Differences in the transition profiles monitored by fluorescence emission and dichroic absorption indicate multi-state behavior of XynA. In the case of CBD, a temperature-induced increase in negative ellipticity at 217 nm is caused by alterations in the environment of aromatic residues that contribute to the far-UV CD in the native state.Keywords: cellulose-binding domain; hyperthermophiles; stability; Thennotoga maritima; xylanase The hyperthermophilic bacterium Thennotoga maritima is capable of utilizing simple carbohydrates as well as complex polysaccharides to gain energy. This includes xylan, which, besides cellulose, is one of the main components of secondary walls of plants. The amount of xylan varies from 7% of the dry weight of gymnosperms to 35% in birchwood (Whistler & Richards, 1970). The

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Section: Thermal Denaturationsupporting

confidence: 72%

“…4). The difference in stability (cl,2(GdmC1) = 5.6 M and 4.5 M for XynA and CBD, respectively) could be easily explained by the well-established mutual stabilization of substructures in large proteins (Vita et al, 1989;Jaenicke, 1996).…”

Section: Gdmcl-induced Denaturationmentioning

confidence: 99%

Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: Structure and stability of the recombinant enzyme and its isolated cellulose‐binding domain

et al. 1997

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“…This supports the general view that protein stability is accomplished by the cumulative effect of covalent and non-covalent bonds at the various levels of the hierarchy of protein structure (Vita et al. 1989;Jaenicke, 1996).There are various enzymes from thermophilic (and hyperthermophilic) organisms that exhibit anomalously high states of association, which suggests thermal adaptation of proteins to be attributable to higher states of subunit assembly. For example, pyruvate dehydrogenase from Bacillus stearothermophilus (To,, ~6 5°C ) contains four times as many polypeptide chains comCorrespondence ta R. Jaenicke, lnstitut…”

supporting

confidence: 75%

Tetrameric and Octameric Lactate Dehydrogenase from the Hyperthermophilic Bacterium Thermotoga maritima

Dams

Ostendorp

Ott

et al. 1996

European Journal of Biochemistry

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Lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has been functionally expressed in Escherichia coli. As shown by gel-permeation chromatography, dynamic light scattering, and ultracentrifugation, the recombinant protein forms homotetrameric and homooctameric assemblies with identical spectral properties and a common subunit molecular mass (35 kDa). Dynamic light scattering and sedimentation equilibrium experiments proved that both species are monodisperse, thus excluding their interconversion in the given ranges of concentration (0.02 -50 mg/ml) and temperature (20-SOOC). Rechromatography confirms this finding : the octamer does not dissociate at low enzyme concentrations, nor do tetramers dimerize at the given upper limit of concentration. Renaturation of pure tetramers or octamers after preceding guanidine denaturation leads to redistribution of the two species ; increased temperature favors octamer formation.Thermal analysis and denaturation by chaotropic agents do not allow the free energies of stabilization of the two forms to be quantified, because heat coagulation and kinetic partitioning between reconstitution and aggregation cause irreversible side reactions. Guanidine denaturation of the octamer leads to a highly cooperative dissociation to tetramers which subsequently dissociate and unfold to yield metastable dimers and, finally, fully unfolded monomers. Evidently, there is no tight coupling of the two tetramers within the stable octameric quaternary structure. Electron microscopy clearly corroborates this conclusion : image processing shows that the dumb-bell-shaped octamer is made up of two tetramers connected via surface contacts without significant changes in the dimensions of the constituent parts.Keywords: hyperthermophiles ; lactate dehydrogenase; quaternary structure ; stability ; Thermotoga muritimu.Lactate dehydrogenases (LDH) are dimeric or tetrameric enzymes that require their native quaternary structure for catalysis (Jaenicke, 1974). As has been shown by denaturation-renaturation experiments, using domain fragments and intact subunits of porcine skeletal muscle LDH, tertiary and quaternary interactions provide increments of stability which in toto yield the exceedingly high value for the free energy of stabilization observed for the enzyme (Miiller et al., 1982;Pfeil, 1986; Opitz et al., 1987;Jaenicke, 1991). This supports the general view that protein stability is accomplished by the cumulative effect of covalent and non-covalent bonds at the various levels of the hierarchy of protein structure (Vita et al.. 1989;Jaenicke, 1996).There are various enzymes from thermophilic (and hyperthermophilic) organisms that exhibit anomalously high states of association, which suggests thermal adaptation of proteins to be attributable to higher states of subunit assembly. For example, pyruvate dehydrogenase from Bacillus stearothermophilus (To,, ~6 5°C ) contains four times as many polypeptide chains comCorrespondence ta R. Jaenicke, lnstitut

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“…As has been mentioned earlier, the central issue in the adaptation of enzymes to extremes of physical conditions is the conservation of a corresponding functional state optimized with respect to stability and flexibility [10,11]. Considering the marginal extra free energy AAGstab, it is evident that enhanced stability may be accomplished by a large number of mechanisms.…”

Section: General Propertiesmentioning

confidence: 99%

Glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: Strategies of protein stabilization

Jaenicke

1996

FEMS Microbiology Reviews

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The molecular origin of protein stability has been the subject of active research for more than a generation (R. Jaenicke (1991) Eur. J. Biochem. 202,[715][716][717][718][719][720][721][722][723][724][725][726][727][728]. Faced with the discovery of extremophiles, in recent years the problem has gained momentum, especially because of its biotechnological potential. In analyzing a number of enzymes from the hyperthermophilic bacterium Thermotoga maritima, it has become clear that the excess free energy of stabilization is equivalent to only a few weak bonds (AAGstab = 50 kJ/mol). As taken from the comparison of homologous enzymes from mesophiles, thermophiles and hyperthermophiles, these accumulate from local interactions (especially ion pairs), enhanced secondary or supersecondary structure, and improved packing of domains and/or subunits, without significantly altering the overall topology. In this review, glyceraldehyde-3-phosphate dehydrogenase will be discussed as a representative example to illustrate possible adaptive strategies to the extreme thermal stress in hydrothermal vents.

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Protein folding and association: In vitro studies for self-organization and targeting in the cell

Cited by 65 publications

References 447 publications

Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: Structure and stability of the recombinant enzyme and its isolated cellulose‐binding domain

Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: Structure and stability of the recombinant enzyme and its isolated cellulose‐binding domain

Tetrameric and Octameric Lactate Dehydrogenase from the Hyperthermophilic Bacterium Thermotoga maritima

Glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: Strategies of protein stabilization

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