Local and Global Cooperativity in the Human α-Lactalbumin Molten Globule

Quezada, C.M.; Schulman, Brenda A.; Froggatt, Joanna J.; Dobson, Christopher M.; Redfield, Christina

doi:10.1016/j.jmb.2004.02.045

Cited by 19 publications

(29 citation statements)

References 49 publications

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“…In the N states of HLA and BLA, the backbone amide protons of residues in the D-helix and the C-terminal 3 10 helix exchange too rapidly to be measured, precluding the use of hydrogen exchange methods to probe this region (11). However, the CIDNP results reveal a high degree of solvent exclusion for the side chains of Tyr-103, Trp-104, and Trp-118 in the A state of HLA, suggesting the formation of stable hydrophobic clusters in this region, a finding that confirms previous suggestions using single proline-substituted mutants (42). Another example is Tyr-36, which, by virtue of being situated in the interdomain loop between the ␣-domain B-helix and ␤-domain ␤-sheet, lacks backbone hydrogen-exchange information.…”

Section: Discussionsupporting

confidence: 74%

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: Discussionsupporting

confidence: 74%

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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“…Two variants of [28–111] α-LA, containing proline substitutions in the A-helix and the C-terminal 3 10 helix (L11P and Q117P), have been studied previously at pH 2 22 . The L11P variant showed unfolding of the A-helix and AB loop in the absence of urea.…”

Section: Resultsmentioning

confidence: 99%

The Human α-Lactalbumin Molten Globule: Comparison of Structural Preferences at pH 2 and pH 7

Rösner

Redfield

2009

Journal of Molecular Biology

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Structural investigations of molten globules provide an important contribution towards understanding protein folding pathways. A close similarity between equilibrium molten globule states and kinetic species observed during refolding has been reported for several proteins. However, the experimental conditions, and in particular the pH, under which the equilibrium and kinetic species are studied often differ significantly. For human α-lactalbumin (α-LA), the equilibrium molten globule is most often studied at pH 2, the so-called A-state, while kinetic refolding experiments are performed at neutral pH. α-LA contains a large number of acidic amino acid residues that may influence the properties of the molten globule differently at low and neutral pH. In this study, we investigate the structural preferences of the α-LA molten globule at pH 7 at the level of individual residues using nuclear magnetic resonance spectroscopy and compare these data with previous results obtained at pH 2. We show that differences exist in the conformational ensemble that describes the α-LA molten globule at these two pH values. The molten globule at pH 7 is generally less stable than that at the low pH A-state. Most notable are differences in the stability of structure for the C-helix and the calcium-binding loop that precedes it and differences in the contribution of long-range hydrophobic contacts between the N-terminal and C-terminal regions of the α-domain to the stability of the molten globule. Our results are discussed in the context of previous studies of the α-LA molten globule and can be used to reconcile apparent discrepancies in published data relating to the C-helix. In the light of our results, the low pH A-state may not be the best model for the kinetic molten globule observed during refolding of α-LA. The pH-dependent effects reported here for α-LA may be of relevance in comparisons of equilibrium and kinetic molten globules of other proteins.

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“…3 and 4). Significant work on the backbone dynamics of α-lactalbumin and its various molten globule states has been carried out with great detail, 6,15,[47][48][49][62][63][64][65][66] and similar work has been performed with other archetypal proteins such as apomyoglobin. [67][68][69] Many of these experiments used elevated temperatures to improve the quality of the heteronuclear single quantum coherence spectra.…”

Section: Discussionmentioning

confidence: 99%

“…1a). Two disulfide bonds are present in the α-domain (6-120 and 28-111), one in the β-domain (61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77), and one in the interface between the two domains (73)(74)(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91). The protein is also stabilized by a calcium ion, which, if removed, makes α-lactalbumin lose its well-defined tertiary structure and predominate as a molten globule state under certain temperatures at neutral pH.…”

Section: α-Lactalbumin and The Rhla All-ala Mutantmentioning

confidence: 99%

α-Lactalbumin, Engineered to be Nonnative and Inactive, Kills Tumor Cells when in Complex with Oleic Acid: A New Biological Function Resulting from Partial Unfolding

Pettersson-Kastberg

Mossberg

Trulsson

et al. 2009

Journal of Molecular Biology

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Local and Global Cooperativity in the Human α-Lactalbumin Molten Globule

Cited by 19 publications

References 49 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

The Human α-Lactalbumin Molten Globule: Comparison of Structural Preferences at pH 2 and pH 7

α-Lactalbumin, Engineered to be Nonnative and Inactive, Kills Tumor Cells when in Complex with Oleic Acid: A New Biological Function Resulting from Partial Unfolding

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