Contribution of individual residues to formation of the native-like tertiary topology in the α-lactalbumin molten globule

Song, Jianxing; Bai, Ping; Luo, Lei; Peng, Zhengyu

doi:10.1006/jmbi.1998.1826

Cited by 77 publications

(61 citation statements)

References 45 publications

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“…In all cases, a variable subset of a small number of side chains appears to remain highly solvent-inaccessible within the core of the MG state, presumably stabilizing the overall fold. Mutagenesis studies on the A state of HLA to discern which residues are most important in generating the native topology have indicated that a relatively small number of residues is sufficient for stabilizing the fold (49). And, as determined previously with a stable HLA variant containing a multiple leucine-substituted ␣-domain (50), the hydrophobic residues involved in the stabilization of the fold need not be the native ones.…”

Section: Discussionmentioning

confidence: 85%

Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

Mok

Nagashima

Day

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Photochemically induced dynamic nuclear polarization NMR pulselabeling techniques have been used to obtain detailed information about side-chain surface accessibilities in the partially folded (molten globule) states of bovine and human ␣-lactalbumin prepared under a variety of well defined conditions. Pulse labeling involves generating nuclear polarization in the partially folded state, rapidly refolding the protein within the NMR sample tube, then detecting the polarization in the well dispersed native-state spectrum. Differences in the solvent accessibility of specific side chains in the various molten globule states indicate that the hydrophobic clusters involved in stabilizing the ␣-lactalbumin fold can be formed from interactions between a variety of different hydrophobic residues in both native and nonnative environments. The multiple subsets of hydrophobic clusters are likely to result from the existence of distinct but closely related local minima on the free-energy landscape of the protein and show that the fold and topology of a given protein may be formed from degenerate groups of side chains.hydrophobic cluster ͉ protein folding ͉ pulse labeling ͉ chemically induced dynamic nuclear polarization ͉ molten globule V ery significant advances, both theoretical and experimental, have been made in recent years in elucidating the principles that govern the folding of proteins (1, 2). In particular, the concept of an energy landscape has provided a general framework for interpreting the thermodynamics and kinetics of the folding process through which a polypeptide chain converts from a disorganized unfolded state into the tightly packed native state (N state) at the global energy minimum (3, 4). Recent approaches, involving a combination of computer-simulation techniques with experimental constraints, have enabled three-dimensional structural ensembles of various partially folded species and folding intermediates to be defined and provide a coarse-grained description of the development of interresidue interactions and chain topologies involved in attaining the final native structure (5-7). Of particular importance in this context are compact denatured states, often known as molten globules (MGs), which are commonly characterized by the presence of native-like secondary structure and hydrodynamic radii but lack both native-like packing of the internal amino acid side chains and exclusion of solvent molecules from the hydrophobic core of the protein (8, 9). The detailed experimental description of such states will undoubtedly provide important information about the specific factors stabilizing the overall chain topology at a residue-specific side-chain level (10). However, MG states are very difficult to characterize by using conventional x-ray crystallography or NMR techniques because of their heterogeneous character (11,12).We have recently developed NMR techniques to characterize aromatic side chains in transient, kinetic intermediate species present in real-time protein folding experiments (13, 14) or in p...

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Section: Discussionmentioning

confidence: 85%

Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

Mok

Nagashima

Day

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Because a random amino acid polymer does not form any specific structure, the formation of the nativelike tertiary topology is likely driven by favorable side-chain-side-chain interactions, specified by the particular arrangement of different types of amino acid residues along the primary sequence. This notion is supported by site-directed mutagenesis studies, showing that the alanine substitutions of hydrophobic residues, in particular those that are buried and participate in multiple packing interactions, destabilize the molten globule of ␣-lactalbumin (␣-LA) and apomyoglobin (apoMb; Kay and Baldwin 1996;Song et al 1998;Wu and Kim 1998;Kay et al 1999). On the other hand, various biophysical studies have failed to detect specific side-chain-side-chain interactions in the classic molten globules.…”

mentioning

confidence: 81%

“…Second, none of the residues are less than three amino acids apart from each other. Six of the selected residues (L8 and L12 on the A-helix, L23 and I27 on the B-helix, Y36 on the loop between the ␣-helical and ␤-sheet domain, and W118 on the C-terminal 3 10 helix) are critical residues that have been identified earlier (Song et al 1998). Alanine substitutions of these residues reduce the C eff for formation of the 28-111 disulfide bond by more than 40%.…”

Section: Additivity Of Double-alanine Substitutions In the ␣-La Moltementioning

confidence: 99%

“…In addition, the simple disulfide bond configuration in ␣-LA-[28-111] allows a straightforward separation and quantitative assessment of the amount of oxidized and reduced proteins. Using this system, we have identified a number of hydrophobic residues for which alanine substitutions significantly reduce the C eff for formation of the 28-111 disulfide bond (Song et al 1998). In the current study, we have extended our work to include proteins with double-alanine substitutions.…”

mentioning

confidence: 99%

“…These conditions include low pH, the presence of low concentrations of denaturant, removal of the bound calcium in a low-ionic strength buffer, and partial or complete reduction of the disulfide bonds (for reviews, see Ptitsyn 1996;Arai and Kuwajima 2000). Previously, we have used the effective concentration (C eff ) for formation of the 28-111 disulfide bond as a probe for the nativelike tertiary topology in the ␣-LA molten globule Song et al 1998). ␣-LA-[28-111] is a single disulfide variant of human ␣-LA, in which all cysteines except Cys28 and Cys111 have been replaced by alanine.…”

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confidence: 99%

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A model of dynamic side‐chain–side‐chain interactions in the α‐lactalbumin molten globule

Bai

Song

Luo³

et al. 2001

Protein Science

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Proteins in the molten globule state contain high levels of secondary structure, as well as a rudimentary, nativelike tertiary topology. Thus, the structural similarity between the molten globule and native proteins may have a significant bearing in understanding the protein-folding problem. To explore the nature of side-chain-side-chain interactions in the ␣-lactalbumin (␣-LA) molten globule, we determined the effective concentration for formation of the 28-111 disulfide bond in 14 double-mutant proteins, each containing two hydrophobic core residues replaced by alanine. We compared our results with those of single-alanine substitutions using the framework of double-mutant cycle analysis and found that, in the majority of cases, the effects of two alanine substitutions are additive. Based on these results, we propose a model of side-chain-side-chain interactions in the ␣-LA molten globule, which takes into consideration the dynamic nature of this partially folded species.Keywords: ␣-Lactalbumin; molten globule; effective concentration; side-chain interaction; double-mutant cycle analysis; protein folding Proteins with a partially folded structure as exemplified by the molten globule state have been considered to be intermediates in protein folding as well as precursors for membrane translocation, ligand binding, and protein-protein or protein-DNA interactions (for reviews, see Ptitsyn 1996; Arai and Kuwajima 2000; see also van der Goot et al. 1991;Gursky and Atkinson 1996;Seeley et al. 1996;Carroll et al. 1997;Lo et al. 1998;Zhang and Matthews 1998). Most partially folded proteins have high levels of secondary structure and a rudimentary, nativelike tertiary topology, but the amino acid side chains in these species remain largely disordered. Despite intensive studies, the mechanism by which a protein folds into the molten globule state is still relatively unclear. Because a random amino acid polymer does not form any specific structure, the formation of the nativelike tertiary topology is likely driven by favorable side-chain-side-chain interactions, specified by the particular arrangement of different types of amino acid residues along the primary sequence. This notion is supported by site-directed mutagenesis studies, showing that the alanine substitutions of hydrophobic residues, in particular those that are buried and participate in multiple packing interactions, destabilize the molten globule of ␣-lactalbumin (␣-LA) and apomyoglobin (apoMb; Kay and Baldwin 1996;Song et al. 1998;Wu and Kim 1998;Kay et al. 1999). On the other hand, various biophysical studies have failed to detect specific side-chain-side-chain interactions in the classic molten globules. For example, the loss of the near-UV circular dichroism (CD) signal in the ␣-LA molten globule suggests that the aromatic side chains are located in a symmetrical environment (Kuwajima et al. 1976;Kuwajima et al. 1985). The NMR spectra acquired in various molten globule-like proteins often display a narrow range Abbreviations: ␣-LA, ␣-lactalbumin; ␣...

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Cold denaturation of α‐lactalbumin

et al. 2000

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We have investigated the thermal unfolding of bovine alpha-lactalbumin by means of circular dichroism spectroscopy in the far- and near-ultraviolet regions, and shown that the native alpha-lactalbumin undergoes heat and cold denaturation. The guanidine hydrochloride-induced unfolding of alpha-lactalbumin was also investigated by circular dichroism spectroscopy at various temperatures from 261 to 318 K. It is shown that the population of the molten globule state is strongly dependent on temperature and that the molten globule state does not accumulate during the guanidine hydrochloride-induced unfolding transition at 261 K. Our results indicate that the molten globule state of alpha-lactalbumin undergoes cold denaturation as the native alpha-lactalbumin does, and that the heat capacity change of unfolding from the molten globule to the unfolded state is positive and significant. The present results further support the idea that the molten globule and the unfolded states do not belong to the same thermodynamic state, and that the native, molten globule and unfolded states are sufficient for interpreting the guanidine hydrochloride-induced unfolding behavior of alpha-lactalbumin.

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Contribution of individual residues to formation of the native-like tertiary topology in the α-lactalbumin molten globule

Cited by 77 publications

References 45 publications

Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

Multiple subsets of side-chain packing in partially folded states of α-lactalbumins

A model of dynamic side‐chain–side‐chain interactions in the α‐lactalbumin molten globule

Cold denaturation of α‐lactalbumin

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