Two steps in the transition between the native and acid states of bovine α‐lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state?

Gussakovsky, Eugene; Haas, Elisha

doi:10.1002/pro.5560041109

Cited by 33 publications

(20 citation statements)

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“…bLA is a useful model for studies of protein folding because of its lower cooperativity, reflected in alternative conformations in different crystal forms (24), its equilibrium stable MG state, and the unique structure of its apo-form revealed here. A pre-MG state that is stable at equilibrium under mildly acidic conditions has been also described (53,54), suggesting that additional near-native partially unfolded conformations may be accessible to structural analysis. Studies are in progress to determine if apo-bLA can be crystallized under more destabilizing conditions to allow the structural analysis of other conformers that illuminate additional steps in the unfolding process.…”

Section: Resultsmentioning

confidence: 93%

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Chrysina¹,

Brew²,

Acharya³

2000

Journal of Biological Chemistry

241

217

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High affinity binding of Ca2؉ to ␣-lactalbumin (LA) stabilizes the native structure and is required for the efficient generation of native protein with correct disulfide bonds from the reduced denatured state. A progressive increase in affinity of LA conformers for Ca 2؉ as they develop increasingly native structures can account for the tendency of the apo form to assume a molten globule state and the large acceleration of folding by Ca 2؉ . To investigate the effect of calcium on structure of bovine LA, x-ray structures have been determined for crystals of the apo and holo forms at 2.2-Å resolution. In both crystal forms, which were grown at high ionic strength, the protein is in a similar global native conformation consisting of ␣-helical and ␤- ␣-Lactalbumin (LA)1 is the regulatory protein of lactose synthase (1-3), modulating the affinity of the catalytic component, UDP-galactose ␤-N-acetylglucosaminide ␤1,4-galactosyltransferase I, for acceptor substrates through a reversible proteinprotein interaction (4). LA is also secreted into milk and aggregates of a partially folded form of the human protein have been found to have selective apoptotic effects on tumor cells (5). LA is homologous with the c-type lysozymes (LZs) (6) and provides an example of extreme functional divergence in homologous proteins with closely similar structures (7-9). LA binds calcium strongly and specifically but the LZs include subgroups that bind Ca 2ϩ at a site corresponding to that in LA and others ("conventional" LZs) that lack a Ca 2ϩ -binding site (10). The K d app for Ca 2ϩ binding to apo-LA under physiological conditions is of the order of 10 Ϫ7 M (11-13). The Ca 2ϩ ion in LA has a structural role, being required for folding and native disulfide bond formation in the reduced denatured protein (14, 15). At ambient temperature and low ionic strength, apo-LA undergoes a transconformation to a molten globule (MG) state, which lacks a fixed tertiary structure but retains much of the native secondary structure. The MG is also stabilized by mildly disruptive treatments such as low pH or intermediate concentrations of denaturants (16,17); it is also identical to a kinetic MG that is highly populated during the refolding of chemically denatured LA (18 -20).Crystal structures of holo-LA from various species in different crystal forms and metal complexes (7,8,(21)(22)(23)(24)(25)(26) are similar to those of the homologous LZs, with two lobes of structure separated by a cleft (Fig. 1A). The larger ␣-helical lobe is formed by the amino-and carboxyl-terminal sections of the polypeptide chain (residues 1-34 and 86 -123) while the smaller lobe, which encompasses a small three-stranded antiparallel ␤-sheet, a small 3 10 helix and some irregular structure, is formed by the central section of the polypeptide chain (residues 35-85). We refer to the lobes as "subdomains" since they lack properties normally expected of true domains such as sequence contiguity and distinct functional properties and origins. The high affinity Ca 2ϩ -binding site is l...

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Section: Resultsmentioning

confidence: 93%

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Chrysina¹,

Brew²,

Acharya³

2000

Journal of Biological Chemistry

241

217

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“…Interestingly, it has been found that some associative forms of α-LA can induce apoptosis in tumor cells [356,357]. αLA is known to adopt different partially folded conformations under mildly denaturing conditions [353][354][355][358][359][360]. In addition, disulfide bridges were shown to play a very important role in the stabilization of α-LA structure [361][362][363][364][365].…”

Section: Carboxymethylated α-Lactabuminmentioning

confidence: 99%

Amyloidogenesis of Natively Unfolded Proteins

Uversky

2008

CAR

172

152

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Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation, which shares many structural properties with the pre-molten globule state, a partially folded intermediate first found during the equilibrium and kinetic (un)folding studies of several globular proteins and later described as one of the structural forms of natively unfolded proteins. The flexibility of this structural form is essential for the conformational rearrangements driving the formation of the core cross-beta structure of the amyloid fibril. Obviously, molecular mechanisms describing amyloidogenesis of ordered and natively unfolded proteins are different. For ordered protein to fibrillate, its unique and rigid structure has to be destabilized and partially unfolded. On the other hand, fibrillogenesis of a natively unfolded protein involves the formation of partially folded conformation; i.e., partial folding rather than unfolding. In this review recent findings are surveyed to illustrate some unique features of the natively unfolded proteins amyloidogenesis.

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“…Like most denatured or partially denatured state of proteins (30,31), it comprises a large number of conformational isomers. So far, characterization of the structure of denatured ␣-LA and the molten globule state of ␣-LA have been achieved by measuring the average property of these collective isomers using a wide range of spectroscopic and physiochemical methods, including circular dichroism (1,2,19,32), fluorescence (12,32,33), NMR (5,14,15,20,29,34), disulfide replacement (35)(36)(37), limited proteolysis (38,39), light scattering (40,41), and calorimetric techniques (42). Further understanding of the denatured state of ␣-LA and the molten globule state of ␣-LA will require fractionation of diverse populations of conformational isomers that constitute the denatured ␣-LA.…”

Section: ␣-Lamentioning

confidence: 99%

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

Chang

2001

Journal of Biological Chemistry

100

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The structure of denatured ␣-lactalbumin (␣-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, ␣-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured ␣-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured ␣-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of ␣-LA. Most importantly, unique isomers of partially unfolded ␣-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic ␣-helical domain, generating a predominant isomer containing two native disulfide bonds at the ␤-sheet domain and two scrambled disulfide bonds at the ␣-helical region. Thermal denaturation selectively unfolds the ␤-sheet domain of ␣-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of ␣-LA. ␣-LA1 represents one of the most extensively investigated models for understanding the mechanism of protein stability, folding, and unfolding. Under a variety of conditions, ␣-LA adopts a partially structured conformation termed as "molten globule" (1-9), which has been observed along both the unfolding (1, 2, 5, 10 -12) and folding pathways (13-18) of ␣-LA and is considered one of the best characterized folding/unfolding intermediates of globular proteins. At low pH (1,19,20), elevated temperature (19, 21), or mild concentration of denaturant (e.g. GdmCl; Refs. 22, 23), the native ␣-LA unfolds to the state of a molten globule. During the refolding, the fully denatured ␣-LA undergoes a rapid collapse to the state of molten globule (13,14,18) as intermediate, which is followed by consolidation and search of side chain interactions to attain the native protein (14). The structure of ␣-LA molten globule is characterized by a high degree of native-like secondary structure and a fluctuated tertiary fold (1)(2)(3)(4)19). It is stabilized mainly by the core hydrophobic amino acids within the ␣-helical domain (24 -28) and may form even in the absence of its four disulfide bonds (15). The structure of the molten globule of ␣-LA is also highly heterogeneous (3, 27, 29). Like most denatured or partially denatured state of proteins (30, 31), it comprises a large number of conformational isomers. So far, characterization of the structure of denatured ␣-LA and the molten globule state of ␣-LA have been achieved by measuring the average property of these collective isomers using a wide range of spectroscopic and physiochemical methods, including circular dichroism (1, 2, ...

show abstract

Two steps in the transition between the native and acid states of bovine α‐lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state?

Cited by 33 publications

References 50 publications

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Amyloidogenesis of Natively Unfolded Proteins

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

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