The Structure of Denatured α-Lactalbumin Elucidated by the Technique of Disulfide Scrambling

Chang, Jui‐Yoa; Li, Li

doi:10.1074/jbc.m010700200

Cited by 55 publications

(107 citation statements)

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“…Molten globules are thought to be general intermediates in protein folding and unfolding (32,33). ␤-LG, a major moiety of bovine whey proteins, is one of the most investigated models for understanding the mechanism involved in protein stability upon heating.…”

Section: Discussionmentioning

confidence: 99%

Epitope Mapping of a Monoclonal Antibody Specific to Bovine Dry Milk

Song

Chen

Yang

et al. 2005

Journal of Biological Chemistry

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␤-Lactoglobulin (␤-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured ␤-LG. In the present study, we show a dramatic increase in ␤-LG immunoreactivity when heating raw milk between 70 and 80°C. To map out the specific epitope of ␤-LG recognized by this mAb, we used a combined strategy including tryptic and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66 -76) of ␤-LG. Circular dichroic spectral analysis shows that carboxymethylation on ␤-LG not only resulted in a substantial loss of ␤-configuration but also exerted a 10 times increase in immunoreactivity as compared with heated ␤-LG. The result suggests that a further disordered structure occurred in ␤-LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated ␤-LG and its binding to retinol or palmitic acid. Most interestingly, pH 9 -10, which neutralizes the Lys groups of ␤-LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of ␤-LG participated in the thermal denaturation between 70 and 80°C and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.

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Section: Discussionmentioning

confidence: 99%

Epitope Mapping of a Monoclonal Antibody Specific to Bovine Dry Milk

Song

Chen

Yang

et al. 2005

Journal of Biological Chemistry

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“…Denaturation was carried out at 23°C for 24 h. Denatured ␣LA was then mixed with an equal volume of 4% aqueous trifluoroacetic acid and subjected directly to separation by HPLC. Under these conditions (42), denatured ␣LA consists of about 45 fractions of scrambled isomers, with two predominant species, namely X-␣LA-a and X-␣LA-d, each accounting for about 24 and 17% of the total denatured structure. X-␣LA-a and X-␣LA-d were purified by HPLC, freezedried, and used as starting materials of folding experiments.…”

Section: Methodsmentioning

confidence: 99%

“…At mild concentration of GdmCl (e.g. 1.5 M), denatured ␣LA was shown to consist of 45 fractions of scrambled species (42). None of them comprises more than 10% of the total structure of denatured ␣LA.…”

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confidence: 99%

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The Folding Pathway of α-Lactalbumin Elucidated by the Technique of Disulfide Scrambling

Chang¹

2002

Journal of Biological Chemistry

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The technique of disulfide scrambling permits reversible conversion of the native and denatured (scrambled) proteins via shuffling and reshuffling of disulfide bonds. Under strong denaturing conditions (e.g. 6 M guanidinium chloride) and in the presence of a thiol initiator, ␣-lactalbumin (␣LA) denatures by shuffling its four native disulfide bonds and converts to an assembly of 45 species of scrambled isomers. Among them, two predominant isomers, designated as X-␣LA-a and X-␣LA-d, account for about 50% of the total denatured structure of ␣LA. X-␣LA-a and X-␣LA-d, which adopt the disulfide patterns of (1-2,3-4,5-6,7-8) and (1-2,3-6,4 -5,7-8), respectively, represent the most unfolded structures among the 104 possible scrambled isomers (Chang, J.-Y., and Li, L. (2001) J. Biol. Chem. 276, 9705-9712). In this study, X-␣LA-a and X-␣LA-d were purified and allowed to refold through disulfide scrambling to form the native ␣LA. Folding intermediates were trapped kinetically by acid quenching and analyzed quantitatively by reversed phase high pressure liquid chromatography. The results revealed two major on-pathway productive intermediates, two major off-pathway kinetic traps, and at least 30 additional minor transient intermediates. Of the two major on-pathway intermediates, one takes on a native-like ␣-helical domain, and the other comprises a structured ␤-sheet, calcium binding domain. The two major kinetic traps are apparently stabilized by locally formed non-native-like structures. Overall, the folding mechanism of ␣LA is essentially congruent with the model of "folding funnel" furnished with a rather intricate energy landscape.Most nascent polypeptides need to navigate their way to form the native, biologically active structures. Elucidation of the molecular mechanism and pathway of this folding process is of fundamental importance in biological science (1-14). Experimentally, it also represents one of the most daunting challenges to protein chemists and structural biologists. At present, the pathway of protein folding can be elucidated by two general methods. The most widely adopted conventional approach is to unfold proteins in the presence of strong denaturant (e.g. 8 M urea or 6 M GdmCl) 1 or by extreme pH and temperature.Following the removal of denaturant (e.g. by gel filtration, dilution, or dialysis), pH jump, or temperature jump, denatured proteins usually refold spontaneously to form the native structure. The pathway of protein refolding is then monitored by the mechanism of restoration of some physicochemical signals that distinguish the native and unfolded states. The most commonly used signals are spectra of fluorescence, circular dichroism (15), infrared (16, 17), UV, and NMR (18 -21), coupled with amide-proton exchange. This method is most versatile and can be practically applied to study folding behaviors of any protein. However, it does not permit, in most cases, isolation of folding intermediates. To date, the vast majority of our knowledge about protein folding has been acquired by the applicatio...

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“…16 The extent to which a particular disulfide is prone to scrambling would therefore be expected to substantially vary. This was exemplified by preferential formation of disulfide isomers of a-lactalbumin and lysozyme in the presence of denaturants 20,21 and the existence of a scrambled form of sillucin, a cysteine-rich peptide. 22 In this study, we have re-examined disulfide connectivity of SVN based on isolation, N-terminal sequencing, and accurate mass determination of its purified fragments obtained under acidic conditions known to suppress the interchange.…”

Section: Discussionmentioning

confidence: 99%

Topology of the disulfide bonds in the antiviral lectin scytovirin

et al. 2010

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The antiviral lectin scytovirin (SVN) contains a total of five disulfide bonds in two structurally similar domains. Previous reports provided contradictory results on the disulfide pairing in each individual domain, and we have now re-examined the disulfide topology. N-terminal sequencing and mass spectrometry were used to analyze proteolytic fragments of native SVN obtained at acidic pH, yielding the assignment as Cys7-Cys55, Cys20-Cys32, Cys26-Cys38, Cys68-Cys80, and Cys74-Cys86. We also analyzed the N-terminal domain of SVN (SD1, residues 1-48) prepared by expression/oxidative folding of the recombinant protein and by chemical synthesis. The disulfide pairing in the chemically synthesized SD1 was forced into predetermined topologies: SD1A (Cys20-Cys26, Cys32-Cys38) or SD1B (Cys20-Cys32, Cys26-Cys38). The topology of native SVN was found to be in agreement with the SD1B and the one determined for the recombinant SD1 domain. Although the two synthetic forms of SD1 were distinct when subjected to chromatography, their antiviral properties were indistinguishable, having low nM activity against HIV. Tryptic fragments, the ''cystine clusters'' [Cys20-Cys32/Cys26-Cys38; SD1] and [Cys68-Cys80/ Disclaimer: The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.Abbreviations: Acm, acetamidomethyl; ATZ, 2-anilino-5-thiazolinone; AUFS, absorbance unit full scale; Boc-Gly-OCH 2 -PAM, t-butyloxycarbonyl-glycyl-4-(hydroxymethyl)phenylacetamidomethyl-copoly(styrene/1% divinylbenzene) resin; DIEA, diisopropylethylamine; ESI-Q-TOF, electrospray ionization quadrupole-time-of-flight mass spectrometry; HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino) methylene]-N-methyl-methanaminium hexafluorophosphate N-oxide; HFBA, heptafluorobutyric acid; MALDI-TOF, matrix-assisted laser desorption-ionization time-of-flight; MS/MS, tandem mass spectrometry; m/z, mass-to-charge ratio; pE, pyroglutamyl; PTH, phenylthiohydantoin; RP-HPLC, reversed-phase high pressure liquid chromatography; rSD1, recombinant/oxidatively folded N-terminal domain of SVN (residues 1-48Cys7Ser); R.T., retention time on HPLC; SD1A, synthetic N-terminal domain of SVN [residues 1-48Cys7Ser (Cys20-Cys26, Cys32-Cys38)]; SD1B, synthetic N-terminal domain of SVN [residues 1-48Cys7Ser (Cys20-Cys32, Cys26-Cys38)]; TCEP, tris(2-Carboxyethyl)phosphine; TFA, trifluoroacetic acid. Cys74-C-86; SD2], were found to undergo rapid disulfide interchange at pH 8. This interchange resulted in accumulation of artifactual fragments in alkaline pH digests that are structurally unrelated to the original topology, providing a rational explanation for the differences between the topology reported herein and the one reported earlier (Bokesh et al., Biochemistry 2003;42:2578-2584. Our observations emphasize the fact that proteins such as SVN, with disulfide bonds in close proximity, require consider...

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The Structure of Denatured α-Lactalbumin Elucidated by the Technique of Disulfide Scrambling

Cited by 55 publications

References 55 publications

Epitope Mapping of a Monoclonal Antibody Specific to Bovine Dry Milk

Epitope Mapping of a Monoclonal Antibody Specific to Bovine Dry Milk

The Folding Pathway of α-Lactalbumin Elucidated by the Technique of Disulfide Scrambling

Topology of the disulfide bonds in the antiviral lectin scytovirin

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