Localized Nature of the Transition-state Structure in Goat α-Lactalbumin Folding

Saeki, Kimiko; Arai, Munehito; Yoda, Takao; Nakao, Masaharu; Kuwajima, Kunihiro

doi:10.1016/j.jmb.2004.06.010

Cited by 26 publications

(43 citation statements)

References 51 publications

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“…Previous studies have well established that I is equivalent to the equilibrium unfolding intermediate that is populated at an intermediate concentration (2.5-3 M) of GdnHCl in the apo form. 9,10,14,15,19 The CML refolding also exhibited the presence of I as shown in our previous study. 17 Kinetic unfolding reactions for the holo and apo forms of GLA were investigated by stopped-flow CD ( Fig.…”

Section: Kinetic Refolding and Unfoldingsupporting

confidence: 83%

“…Recombinant 15 N-labeled GLA was expressed in Escherichia coli (BL21(DE3)). Recombinant 15 N-labeled GLA was purified as described in Saeki et al 15 The GLA concentration was determined spectrophotometrically using an extinction coefficient of ɛ = 2.85 × 10 4 M − 1 cm − 1 at 280 nm. 92 GdnHCl was of specially prepared reagent grade for biochemical use from Nacalai Tesque, Inc. Other chemicals were of guaranteed reagent grade.…”

Section: Methodsmentioning

confidence: 99%

“…(15) and (16) for definitions of the m values]. Because the m value is approximately proportional to a change in the accessible surface area of the protein during unfolding, 43 the apo form in the N state might expose more surface area than the holo form.…”

Section: The Burst-phase Intermediate (I) In Kinetic Refoldingmentioning

confidence: 99%

“…[4][5][6][7][8] Our previous studies on the equilibria and kinetics of the folding/unfolding of GLA and CML induced by a denaturant, guanidine hydrochloride (GdnHCl), employed circular dichroism (CD) and fluorescence spectroscopy. 5,[9][10][11][12][13][14][15][16][17] Those studies showed that the two proteins exhibit a stable equilibrium unfolding intermediate that is equivalent to an early kinetic folding intermediate formed during the dead time (a few milliseconds) of a stopped-flow experiment. The intermediate has been well characterized experimentally and has characteristics of the molten globule (MG) state.…”

Section: Introductionmentioning

confidence: 99%

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Different Folding Pathways Taken by Highly Homologous Proteins, Goat α-Lactalbumin and Canine Milk Lysozyme

Nakamura

Makabe

Tomoyori

et al. 2010

Journal of Molecular Biology

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Section: Kinetic Refolding and Unfoldingsupporting

confidence: 83%

Section: Methodsmentioning

confidence: 99%

Section: The Burst-phase Intermediate (I) In Kinetic Refoldingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Different Folding Pathways Taken by Highly Homologous Proteins, Goat α-Lactalbumin and Canine Milk Lysozyme

Nakamura

Makabe

Tomoyori

et al. 2010

Journal of Molecular Biology

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“…H. pylori apoflavodoxin at acidic pH thus exhibits all the characteristics of a molten conformation (52). Molten globules often appear in proteins at low pH (53-56), but they have also been reported under physiological conditions (57) and have been proposed to play roles in protein folding kinetics (58,59), enzymatic inactivation (60,61), and membrane translocation (61,62). For Anabaena apoflavodoxin, a molten globule conformation has also been observed at low pH (3), but it aggregates and has not been characterized in detail.…”

Section: Native and Molten Globule Conformations Of H Pylorimentioning

confidence: 99%

Conformational Stability of Helicobacter pylori Flavodoxin

Cremades

Bueno

Neira

et al. 2008

Journal of Biological Chemistry

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Flavodoxin is an essential protein for Helicobacter pylori, a pathogen living in the very acidic environment of the gastric tract and responsible for several diseases. We report the conformational stability of the protein in neutral and acidic pH. The apoprotein remains native between pH 12 and 5 and adopts a monomeric molten globule conformation at more acidic pH values. The equilibrium unfolding in urea appears two-state for either conformation, but the native one coexists with a hidden equilibrium intermediate of very similar properties. The stability of H. pylori apoflavodoxin is higher than that of the Anabaena homologue throughout the entire pH interval, which may be related to better charge compensation. H. pylori apoflavodoxin is strongly stabilized by its FMN cofactor. A global analysis of apo-and holoflavodoxin equilibrium unfolding, with and without excess FMN, indicates that the cofactor only binds to the native state. Some physical-chemical properties of the protein may represent an adaptation to the acidic environment. Unlike the apoflavodoxin from Anabaena, which becomes highly insoluble at pH 5.0, that from H. pylori remains soluble to at least 40 M. This fact, together with the high stability of the apoprotein at this low pH that can arise in the bacteria cytoplasm, seems useful to allow newly synthesized apoflavodoxin molecules to fold and remain soluble to accomplish cofactor binding, which in turn increases the stability. Also, whenever the cytoplasmic pH drops to 5, preexisting flavodoxin molecules will remain folded and soluble and will retain the FMN cofactor, thus remaining functional.Flavodoxins are bacterial electron-transfer proteins that bear a noncovalently bound redox cofactor (FMN) that allows them to participate in a variety of reactions (1). There is evidence that, in vivo, the apoprotein folds first and then binds the cofactor, and it is clear that when the cofactor is removed, the apoprotein remains folded (2-4). Helicobacter pylori is a Gram-negative bacterium related with digestive diseases such as type B gastritis, stomach and duodenal ulcers (5), adenocarcinomas (6), and stomach lymphomas (7). It is found in the stomach of 50% of the human population (8), and 20% of infected individuals develop pathologies. Recently, it has been reported that the flavodoxin from H. pylori is essential for the survival of the pathogen because of its role as electron acceptor of the pyruvate-flavodoxin oxidoreductase complex, which catalyzes pyruvate oxidative decarboxylation (9). Conversely, flavodoxin is the electron donor in the reverse reaction that is used to fix CO 2 (10). The x-ray structure of the protein is available for both the apo-(11) and holo-forms (12). H. pylori flavodoxin contains 164 residues, including two tryptophans, and shares 40% sequence identity with Anabaena PCC7119 and Azotobacter vinelandii, and 30% with Desulfovibrio desulfuricans and Desulfovibrio vulgaris flavodoxins. Interestingly, H pylori flavodoxin lacks a fairly conserved tryptophan residue involved in FMN ...

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Calcium Binding Proteins

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Localized Nature of the Transition-state Structure in Goat α-Lactalbumin Folding

Cited by 26 publications

References 51 publications

Different Folding Pathways Taken by Highly Homologous Proteins, Goat α-Lactalbumin and Canine Milk Lysozyme

Different Folding Pathways Taken by Highly Homologous Proteins, Goat α-Lactalbumin and Canine Milk Lysozyme

Conformational Stability of Helicobacter pylori Flavodoxin

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