The Native-like Tertiary Fold in Molten Globule α-Lactalbumin Appears to be Controlled by a Continuous Phase Transition

doi:10.1006/jmbi.1996.0467

Journal of Molecular Biology

Volume 261, Issue 3, 23 August 1996, Pages 341-347

https://doi.org/10.1006/jmbi.1996.0467 Get rights and content

Abstract

On account of its ability to discriminate between secondary, loop and sidegroup structure and its special sensitivity to conformational mobility, vibrational Raman optical activity (ROA) has provided new insights into the complexity of order within the molten globule state from measurements on α-lactalbumin at pH 2.0 over the temperature range 2 to 45°C. Thus while much of the secondary structure present in the native protein persists with only a small gradual decrease with increasing temperature, the tertiary backbone fold changes dramatically, being almost complete and native-like at 2°C and almost completely disordered at 35°C. The change of the tertiary fold with temperature is cooperative but has no latent heat, and so has the approximate characteristics of a continuous phase transition, being of the order-disorder type since it involves the interconversion of rigid, locally-ordered loop structure with disordered mobile backbone structure. This has implications for protein folding because the long-range correlations that exist in the critical region of a continuous (but not in a first-order) phase transition could resolve, in principle, the problem of how the protein finds its native-like folding pattern at the molten globule stage.

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PH-induced conformational transitions in α-lactalbumin investigated with two-dimensional Raman correlation variance plots and moving windows
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Raman spectroscopy is being increasingly applied to the investigation of conformational transitions in polypeptides and proteins, and in particular protein unfolding, due to its ability to monitor changes in secondary structure. Here we focus on the application of two different two-dimensional correlation methods, autocorrelation and moving windows techniques, to investigate the pH-induced conformational transitions in α-lactalbumin monitored with Raman scattering. From our results we have identified three distinct stages in the conformational transition occurring at ranges ∼pH 6.5–4.6, ∼pH 4.6–3.6 and ∼pH 3.6–1.8, suggesting the existence of three possible conformational species. The first two transition phases the conformational changes are predominantly occurring in the backbone secondary structure, while in the final transition phase changes are occurring in both secondary structure and side chain residue solvent exposure.
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HAMLET (human α-lactalbumin made lethal to tumor cells) is a protein-lipid complex that induces apoptosis-like death in tumor cells, but leaves fully differentiated cells unaffected. This review summarizes the information on the in vivo effects of HAMLET in patients and tumor models, on the tumor cell biology, and on the molecular characteristics of the complex. HAMLET limits the progression of human glioblastomas in a xenograft model and removes skin papillomas in patients. This broad anti-tumor activity includes >40 different lymphomas and carcinomas and apoptosis is independent of p53 or bcl-2. In tumor cells, HAMLET enters the cytoplasm, translocates to the perinuclear area, and enters the nuclei, where it accumulates. HAMLET binds strongly to histones and disrupts the chromatin organization. In the cytoplasm, HAMLET targets ribosomes and activates caspases. The formation of HAMLET relies on the propensity of α-lactalbumin to alter its conformation when the strongly bound Ca²⁺ ion is released and the protein adopts the apo-conformation that exposes a new fatty acid binding site. Oleic acid (C18:1,9 cis) fits this site with high specificity, and stabilizes the altered protein conformation. The results illustrate how protein folding variants may be beneficial, and how their formation in peripheral tissues may depend on the folding change and the availability of the lipid cofactor. One example is the acid pH in the stomach of the breast-fed child that promotes the formation of HAMLET This mechanism may contribute to the protective effect of breastfeeding against childhood tumors. We propose that HAMLET should be explored as a novel approach to tumor therapy.
Unfolded proteins studied by Raman optical activity
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To understand the behavior of unfolded proteins it is necessary to employ experimental techniques able to discriminate between the dynamic true random coil state and more static types of disorder, including situations in which some ordered secondary structure might be present. One such technique is a novel chiroptical spectroscopy called Raman optical activity (ROA). This chapter reviews the application of ROA to studies of unfolded proteins. Because many discrete structure-sensitive bands are present in protein ROA spectra, the technique provides a fresh perspective on the structure and behavior of unfolded proteins and of unfolded sequences in proteins such as A-gliadin and prions that contain distinct structured and unstructured domains. It also provides new insight into the complexity of order in molten globule and reduced protein states and of the more mobile sequences in fully folded proteins such as β-lactoglobulin. The power of ROA in this area derives from the fact that, like the complementary technique of vibrational circular dichroism (VCD), it is a form of vibrational optical activity and so is sensitive to chirality associated with all the 3N−6 fundamental molecular vibrational transitions, where N is the number of atoms.
Exploration of partially unfolded states of human α-lactalbumin by molecular dynamics simulation
2001, Journal of Molecular Biology
Molecular dynamics simulations are used to probe the properties of non-native states of the protein human α-lactalbumin (human α-LA) with a detailed atomistic model in an implicit aqueous solvent environment. To sample the conformational space, a biasing force is introduced that increases the radius of gyration relative to the native state and generates a large number of low-energy conformers that differ in terms of their root-mean-square deviation, for a given radius of gyration. The resulting structures are relaxed by unbiased simulations and used as models of the molten globule and partly denatured states of human α-LA, based on measured radii of gyration obtained from nuclear magnetic resonance experiments. The ensembles of structures agree in their overall properties with experimental data available for the human α-LA molten globule and its more denatured states. In particular, the simulation results show that the native-like fold of the α-domain is preserved in the molten globule. Further, a considerable proportion of the antiparallel β-strand in the β-domain are present. This indicates that the lack of hydrogen exchange protection found experimentally for the β-domain is due to rearrangement of the β-sheet involving transient populations of non-native β-structures. The simulations also provide details concerning the ensemble of structures that contribute as the molten globule unfolds and shows, in accord with experimental data, that unfolding is not cooperative; i.e. the various structural elements do not unfold simultaneously.
A view of dynamics changes in the molten globulenative folding step by quasielastic neutron scattering
2000, Journal of Molecular Biology
Citation Excerpt :
Such a growing range of cooperative motion is thought to be responsible for the dramatic change in dynamic properties toward glass transition of glass-forming liquids (Adam & Gibbs, 1965). A vibrational Raman optical activity study has shown that the native-like tertiary fold in molten globule α-lactalbumin is controlled by a continuous phase transition but not a first-order phase transition (Wilson et al., 1996), indicating that structurally the α-lactalbumin molten globule to native transition can be comparable to glass transition experimentally. We propose that a growing spatially correlated motion within a partially folded protein may assist the molten globule to native transition.
In order to understand the changes in protein dynamics that occur in the final stages of protein folding, we have used neutron scattering to probe the differences between a protein in its folded state and the molten globule states. The internal dynamics of bovine α-lactalbumin (BLA) and its molten globules (MBLA) have been examined using incoherent, quasielastic neutron scattering (IQNS). The IQNS results show length scale dependent, pico-second dynamics changes on length scales from 3.3 to 60 Å studied. On shorter-length scales, the non-exchangeable protons undergo jump motions over potential barriers, as those involved in side-chain rotamer changes. The mean potential barrier to local jump motions is higher in BLA than in MBLA, as might be expected. On longer length scales, the protons undergo spatially restricted diffusive motions with the diffusive motions being more restricted in BLA than in MBLA. Both BLA and MBLA have similar mean square amplitudes of high frequency motions comparable to the chemical bond vibrational motions. Bond vibrational motions thus do not change significantly upon folding. Interestingly, the quasielastic scattering intensities show pronounced maxima for both BLA and MBLA, suggesting that “clusters” of atoms are moving collectively within the proteins on picosecond time scales. The correlation length, or “the cluster size”, of such atom clusters moving collectively is dramatically reduced in the molten globules with the correlation length being 6.9 Å in MBLA shorter than that of 18 Å in BLA. Such collective motions may be important for the stability of the folded state, and may influence the protein folding pathways from the molten globules.
Energetic basis of structural stability in the molten globule state: α-Lactalbumin
2000, Journal of Molecular Biology
The denatured states of α-lactalbumin, which have features of a molten globule state, have been studied to elucidate the energetics of the molten globule state and its contribution to the stability of the native conformation. Analysis of calorimetric and CD data shows that the heat capacity increment of α-lactalbumin denaturation highly correlates with the degree of disorder of the residual structure of the state. As a result, the denaturational transition of α-lactalbumin from the native to a highly ordered compact denatured state, and from the native to the disordered unfolded state are described by different thermodynamic functions. The enthalpy and entropy of the denaturation of α-lactalbumin to compact denatured state are always greater than the enthalpy and entropy of its unfolding. This difference represents the unfolding of the molten globule state. Calorimetric measurements of the heat effect associated with the unfolding of the molten globule state reveal that it is negative in sign over the temperature range of molten globule stability. This observation demonstrates the energetic specificity of the molten globule state, which, in contrast to a protein with unique tertiary structure, is stabilized by the dominance of negative entropy and enthalpy of hydration over the positive conformational entropy and enthalpy of internal interactions. It is concluded that at physiological temperatures the entropy of dehydration is the dominant factor providing stability for the compact intermediate state on the folding pathway, while for the stability of the native state, the conformational enthalpy is the dominant factor.

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Journal of Molecular Biology

CommunicationThe Native-like Tertiary Fold in Molten Globule α-Lactalbumin Appears to be Controlled by a Continuous Phase Transition

Abstract

Communication
The Native-like Tertiary Fold in Molten Globule α-Lactalbumin Appears to be Controlled by a Continuous Phase Transition