Structural basis of the stability of a lysozyme molten globule

Morozova, L. A.; Haynie, Donald T.; Arico-Muendel, Christopher C.; Dael, H. Van; Dobson, Christopher M.

doi:10.1038/nsb1095-871

Cited by 126 publications

(157 citation statements)

References 30 publications

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“…A detailed understanding of the structures and energetics of molten globule intermediate states are of great significance, since this is a common intermediate state in the folding pathway of many proteins in vitro and in vivo (Ptitsyn, 1995). The molten globule states of a-lactalbumin and the evolutionarily related calcium-binding equine lysozyme are well studied under equilibrium conditions (Kuwajima, 1989;Morozova et al, 1995;Schulman et al, 1995). On the contrary, in the case of hen lysozyme, the molten globule-like state does not form under equilibriumdenaturing conditions explored so far.…”

Section: Discussionmentioning

confidence: 99%

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Bhattacharjya

Balaram

1997

Protein Science

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A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced a f f i n i t y towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 'H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (250%). This transition is characterized by a dramatic loss of A N S binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. HID exchange experiments yield higher protection factors for many of the backbone amide protons from the four a-helices along with the C-terminal 3'0 helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel @-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for a-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.

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Section: Discussionmentioning

confidence: 99%

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Bhattacharjya

Balaram

1997

Protein Science

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“…It possesses a calcium-binding site similar to ␣-lactalbumins and a bacteriolytic enzymatic activity like conventional lysozymes. It combines also the structural and folding properties of both subfamilies and is viewed as an evolutionary bridge between them (13)(14)(15).…”

mentioning

confidence: 99%

Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity?

Mališauskas

Östman

Darinskas

et al. 2005

Journal of Biological Chemistry

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In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57°C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease.The molecular basis of the pathogenicity of amyloid aggregates is a central theme in understanding the causes of a wide range of amyloid-related diseases, including Alzheimer's, Parkinson's, prion diseases, type II diabetes, and familial amyloidotic polyneuropathy (1-4). There is a striking difference between the amounts of amyloid depositions in various types of amyloid disorders. In systemic lysozyme amyloidosis, for example, the deposits can grow to kilogram quantities in the liver (5, 6). In neurodegenerative diseases, by contrast, there is no clear correlation between the amount of amyloid deposition and the clinical severity of disease. Significant cognitive impairment of Alzheimer's patients was observed without noticeable amyloid deposits in the brain, although the level of readily soluble amyloid oligomeric assemblies in the Alzheimer's brain was found to be greatly elevated (7,8).The evidence is accumulating that prefibrillar aggregates are cytotoxic both in vivo and in vitro (4,8,9), although this question is still open to debate. Moreover, aggregates of the proteins, which are not related to clinical amyloidoses, such as human ␣-lactalbumin (10), SH3 domain, or HypF-N protein, have also been found to be cytotoxic, which implies a common mechanism for cytotoxicity of misfolded proteins (11). By contrast, it has been shown that the mature amyl...

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“…The CIDNP spectra of the native EL at several pH 4.5, 6.9 and 9.0 are well-resolved and were assigned by comparison with NMR chemical shifts [17,18,22]. ELOA and EL molten display less resolved CIDNP spectra, consistent with their millisecond conformational fluctuations, although it is still straight forward to distinguish tyrosine and tryptophan/histidine residues based on their emissive (negative) and absorptive (positive) polarizations, respectively.…”

Section: El Conformation In Eloamentioning

confidence: 99%

“…The calciumbinding usually increases the protein stability against denaturing treatments, however in the case of EL, the significantly lower stability and cooperatively was observed compared to non-calcium-binding lysozymes even in its holo-form, while in the apo-form its thermodynamic stability is closer of -lactalbumins than to c-type lysozymes [15,16]. EL forms a wide range of partially folded states under equilibrium conditions similar to these of -lactalbumins [16,3,17,18]. However, EL molten globule is much more structured compared to the "classical" molten globules of α-lactalbumins, possessing an extended native-like hydrophobic core stabilised by interactions between three major α-helices (A, B and D-helices) in the α-domain [17,18].…”

Section: Equine Lysozyme (El) As a Structural Homologue Of α-Lactalbuminmentioning

confidence: 99%

“…EL forms a wide range of partially folded states under equilibrium conditions similar to these of -lactalbumins [16,3,17,18]. However, EL molten globule is much more structured compared to the "classical" molten globules of α-lactalbumins, possessing an extended native-like hydrophobic core stabilised by interactions between three major α-helices (A, B and D-helices) in the α-domain [17,18]. Like c-type lysozymes, during refolding kinetics EL forms an ensemble of well-defined transient kinetic intermediates, possessing very persistent structures [19].…”

Section: Equine Lysozyme (El) As a Structural Homologue Of α-Lactalbuminmentioning

confidence: 99%

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Structural Origin of ELOA Toxicity – Implication for HAMLET-Type Protein Complexes with Oleic Acid

Vukojević¹,

Morozova‐Roche²

2012

Lipoproteins - Role in Health and Diseases

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Structural basis of the stability of a lysozyme molten globule

Cited by 126 publications

References 30 publications

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity?

Structural Origin of ELOA Toxicity – Implication for HAMLET-Type Protein Complexes with Oleic Acid

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