Intermediate studies on refolding of arginine kinase denatured by guanidine hydrochloride

The refolding course and intermediate of guanidine hydrochloride (GuHCl)-denatured arginine kinase (AK) were studied in terms of enzymatic activity, intrinsic fluorescence, 1-anilino-8-naphthalenesulfonte (ANS) fluorescence, and far-UV circular dichroism (CD). During AK refolding, the fluorescence intensity increased with a significantly blue shift of the emission maximum. The molar ellipticity of CD increased to close to that of native AK, as compared with the fully unfolded AK. In the AK refolding process, 2 refolding intermediates were observed at the concentration ranges of 0.8–1.0 mol/L and 0.3–0.5 mol GuHCl/L. The peak position of the fluorescence emission and the secondary structure of these conformation states remained roughly unchanged. The tryptophan fluorescence intensity increased a little. However, the ANS fluorescence intensity significantly increased, as compared with both the native and the fully unfolded states. The first refolding intermediate at the range of 0.8–1.0 mol GuHCl/L concentration represented a typical "pre-molten globule state structure" with inactivity. The second one, at the range of 0.3–0.5 mol GuHCl/L concentration, shared many structural characteristics of native AK, including its secondary and tertiary structure, and regained its catalytic function, although its activity was lower than that of native AK. The present results suggest that during the refolding of GuHCl-denatured AK there are at least 2 refolding intermediates; as well, the results provide direct evidence for the hierarchical mechanism of protein folding.Key words: arginine kinase, guanidine-denatured, refolding, intermediate, molten globule state.

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Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol

Biochemistry and Cell Biology ◽

10.1139/o05-171 ◽

2006 ◽

Vol 84 (2) ◽

pp. 126-134 ◽

Cited By ~ 3

Author(s):

Fouzia Rashid ◽

Sandeep Sharma ◽

M A Baig ◽

Bilqees Bano

Keyword(s):

Conformational Changes ◽

Tertiary Structure ◽

Molten Globule ◽

Structural Features ◽

Cd Spectroscopy ◽

Native State ◽

Molten Globule State ◽

Fluorescence Measurements ◽

Helical Content ◽

Human Placental Cystatin

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of α-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.Key words: human placental cystatin, molten globule, acid-induced state, trifluoroethanol, methanol, CD spectroscopy, ANS fluorescence, pH, protein folding.

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Differences in the Pathways of Proteins Unfolding Induced by Urea and Guanidine Hydrochloride: Molten Globule State and Aggregates

PLoS ONE ◽

10.1371/journal.pone.0015035 ◽

2010 ◽

Vol 5 (11) ◽

pp. e15035 ◽

Cited By ~ 60

Author(s):

Olga I. Povarova ◽

Irina M. Kuznetsova ◽

Konstantin K. Turoverov

Keyword(s):

Molten Globule ◽

Guanidine Hydrochloride ◽

Molten Globule State

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Microgravity-Induced Alterations in the H3.3B (H3F3B) Gene Expression and the Histone H3 Structure

Advanced Science Engineering and Medicine ◽

10.1166/asem.2020.2672 ◽

2020 ◽

Vol 12 (8) ◽

pp. 1084-1094

Author(s):

Azadeh Hekmat ◽

Mojtaba Sadeghi Manesh ◽

Zahra Hajebrahimi ◽

Shadie Hatamie

Keyword(s):

Gene Expression ◽

Simulated Microgravity ◽

Tertiary Structure ◽

Gene Expression Regulation ◽

Molten Globule ◽

Histone H3 ◽

Fluorescence Emission ◽

Surface Hydrophobicity ◽

Expression Level ◽

Visible Absorption

It has been believed that microgravity directly can modify the structure, function, and morphology of biosystems and numerous researches have been performed to recognize these alterations. Since histone H3 is an essential protein in the field of epigenetics, this research aimed to evaluate the effects of simulated microgravity on the human H3.3B (H3F3B) gene expression and histone H3 structure. The two-dimensional clinostat was applied for simulating microgravity. Analysis of the gene expression by real-time quantitative PCR revealed that simulated microgravity diminished the expression level of H3.3B considerably (P < 0.001). The UV-Visible absorption and extrinsic fluorescence emission results displayed that after 72 h of simulated microgravity the tertiary structure of histone H3 changed and the surface hydrophobicity of the protein incremented remarkably. Nevertheless, circular dichroism (CD) data showed that simulated microgravity did not perturb the secondary structure of histone H3. Collectively, microgravity can strictly affect the gene expression level of H3.3. Furthermore, histone H3 72 h after subjecting to simulated microgravity can exhibit a molten globule structure. The significance of this research lied in the fact that simulating microgravity can be an effective physical force in gene expression regulation and the protein folding process. This finding could help astrobiologists to realize major health risks for astronaut crews and space travelers and reduce these harmful effects. Furthermore, our observations can open fascinating research lines in astrobiology, biophysics, and exobiology.

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Native-like tertiary structure in the Mucor miehei lipase molten globule state obtained at low pH

IUBMB Life ◽

10.1080/15216540701335716 ◽

2007 ◽

Vol 59 (3) ◽

pp. 179-186 ◽

Cited By ~ 10

Author(s):

Sadaf Fatima ◽

Basir Ahmad ◽

Rizwan Hasan Khan

Keyword(s):

Tertiary Structure ◽

Molten Globule ◽

Low Ph ◽

Mucor Miehei ◽

Mucor Miehei Lipase ◽

Molten Globule State ◽

Miehei Lipase

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Interaction with Lipid II Induces Conformational Changes in Bovicin HC5 Structure

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00295-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4586-4593 ◽

Cited By ~ 18

Author(s):

Aline Dias Paiva ◽

Nicole Irving ◽

Eefjan Breukink ◽

Hilário Cuquetto Mantovani

Keyword(s):

Conformational Changes ◽

Acyl Chain ◽

Fluorescence Emission ◽

Tryptophan Fluorescence ◽

Blue Shift ◽

Tryptophan Residue ◽

Membrane Systems ◽

Content Type ◽

Lipid Ii ◽

Bovicin Hc5

ABSTRACTBovicin HC5 is a lantibiotic produced byStreptococcus bovisHC5 that targets the cell wall precursor lipid II. An understanding of the modes of action against target bacteria can help broadening the clinical applicability of lantibiotics in human and veterinary medicine. In this study, the interaction of bovicin HC5 with lipid II was examined using tryptophan fluorescence and circular dichroism spectroscopy with model membrane systems that do or do not allow pore formation by bovicin HC5. In the presence of lipid II, a blue-shift of 12 nm could be observed for the fluorescence emission maximum of the tryptophan residue for all of the membrane systems tested. This change in fluorescence emission was paralleled by a decrease in accessibility toward acrylamide and phospholipids carrying a spin-label at the acyl chain; the tryptophan residue of bovicin HC5 was located near the twelfth position of the membrane phospholipid acyl chains. Moreover, the binding of lipid II by bovicin HC5 induced remarkable conformational changes in the bovicin HC5 structure. The interaction of bovicin HC5 with lipid II was highly stable even at pH 2.0. These results indicate that bovicin HC5 interacts directly with lipid II and that the topology of this interaction changes under different conditions, which is relevant for the biological activity of the peptide. To our knowledge, bovicin HC5 is the only bacteriocin described thus far that is able to interact with its target in extreme pH values, and this fact might be related to its unique structure and stability.

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Conformational changes in bovine lactoferrin induced by slow or fast temperature increases

Biological Chemistry ◽

10.1515/bc.2008.116 ◽

2008 ◽

Vol 389 (8) ◽

Cited By ~ 18

Author(s):

Waleska D. Schwarcz ◽

Lorena Carnelocce ◽

Jerson L. Silva ◽

Andréa C. Oliveira ◽

Rafael B. Gonçalves

Keyword(s):

Secondary Structure ◽

Conformational Changes ◽

Tertiary Structure ◽

Fluorescence Emission ◽

Tryptophan Fluorescence ◽

Heat Stability ◽

Bovine Lactoferrin ◽

Spectroscopic Techniques ◽

Bioactive Component ◽

Physiological Properties

Abstract Lactoferrin (LF) is an iron-binding protein present in several secreted substances, such as milk, and has broad antimicrobial and physiological properties. Because high temperatures may affect protein stability and its functional properties, we investigated the effect of heat on bovine LF structure and stability. The effects of temperatures used during the pasteurization process on LF and its relationship to protein functionality were studied. Conformational changes were monitored using spectroscopic techniques, such as circular dichroism (CD) and fluorescence spectroscopy. The CD data at 70°C showed that LF's secondary structure is drastically and irreversibly affected when the temperature is gradually increased. The same effect is observed when the temperature is gradually raised from 25°C to 105°C and changes are monitored by tryptophan fluorescence emission. We also verified the effects of simulating the pasteurization process; LF remained well structured during the entire process and this result was not time-dependent. Owing to preservation of the secondary structure with changes in the tertiary structure, we thus believe that pasteurization might cause LF to change into an intermediate partially folded state. A better understanding of heat stability is important for the use of LF as a bioactive component in food.

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Comparative analysis of the QUTR transcription repressor protein and the three C-terminal domains of the pentafunctional AROM enzyme

Biochemical Journal ◽

10.1042/bj3130941 ◽

1996 ◽

Vol 313 (3) ◽

pp. 941-950 ◽

Cited By ~ 8

Author(s):

Heather K. LAMB ◽

Jonathan D. MOORE ◽

Jeremy H. LAKEY ◽

Lisa J. LEVETT ◽

Kerry A. WHEELER ◽

...

Keyword(s):

Tertiary Structure ◽

Guanidine Hydrochloride ◽

Fluorescence Emission ◽

Shikimate Dehydrogenase ◽

Sequence Alignments ◽

Repressor Protein ◽

E Coli ◽

Domain Protein ◽

Dehydroquinate Synthase ◽

Terminal Domains

The AROM protein is a pentadomain protein catalysing steps two to six in the prechorismate section of the shikimate pathway in microbial eukaryotes. On the basis of amino acid sequence alignments and the properties of mutants unable to utilize quinic acid as a carbon source, the AROM protein has been proposed to be homologous throughout its length with the proteins regulating transcription of the genes necessary for quinate catabolism. The QUTR transcription repressor protein has been proposed to be homologous with the three C-terminal domains of the AROM protein and one-fifth of the penultimate N-terminal domain. We report here the results of experiments designed to overproduce the QUTR and AROM proteins and their constituent domains in Escherichia coli, the purpose being to facilitate domain purification and (in the case of AROM), complementation of E. coli aro- mutations in order to probe the degree to which individual domains are stable and functional. The 3-dehydroquinate dehydratase domain of the AROM protein and the 3-dehydroquinate dehydratase-like domain of the QUTR protein were purified in bulk and subjected to comparative CD spectroscopy and fluorescence emission spectroscopy. The CD spectra were found to be virtually superimposable. The fluorescence emission spectra of both domains had the signal from the tryptophan residues almost completely quenched, giving a tyrosine-dominated spectrum for both the AROM- and QUTR-derived domains. This unexpected observation was demonstrated to be due to a highly unusual environment provided by the tertiary structure, as addition of the denaturant guanidine hydrochloride gave a typical tryptophan-dominated spectrum for both domains. The spectroscopy experiments had the potential to refute the biologically-based proposal for a common origin for the AROM and QUTR proteins; however, the combined biophysical data are consistent with the hypothesis. We have previously reported that the AROM dehydroquinate synthase and 3-dehydroquinate dehydratase are stable and functional as individual domains, but that the 5-enol-pyruvylshikimate-3-phosphate synthase is only active as part of the complete AROM protein or as a bi-domain fragment with dehydroquinate synthase. Here we report that the aromA gene (encoding the AROM protein) of Aspergillus nidulans contains a 53 nt intron in the extreme C-terminus of the shikimate dehydrogenase domain. This finding accounts for the previously reported observation that the AROM protein was unable to complement aroE- (lacking shikimate dehydrogenase) mutations in E. coli. When the intron is removed the correctly translated AROM protein is able to complement the E. coli aroE- mutation. An AROM-derived shikimate dehydrogenase domain is, however, non-functional, but function is restored in a bi-domain protein with 3-dehydroquinate dehydratase. This interaction is not entirely specific, as substitution of the 3-dehydroquinate dehydratase domain with the glutathione S-transferase protein partially restores enzyme activity. Similarly an AROM-derived shikimate kinase domain is non-functional, but is functional as part of the complete AROM protein, or as a bi-domain protein with 3-dehydroquinate dehydratase.

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Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-013-0163-9 ◽

2013 ◽

Vol 169 (8) ◽

pp. 2315-2325 ◽

Cited By ~ 9

Author(s):

Neelesh Singh ◽

Reetesh Kumar ◽

M. V. Jagannadham ◽

Arvind M. Kayastha

Keyword(s):

Molten Globule ◽

Guanidine Hydrochloride ◽

Molten Globule State

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Low concentration of guanidine hydrochloride induces the formation of an aggregation-prone state in α-urease

Biochemistry and Cell Biology ◽

10.1139/o03-072 ◽

2004 ◽

Vol 82 (2) ◽

pp. 305-313 ◽

Cited By ~ 12

Author(s):

F -O McDuff ◽

A Doucet ◽

M Beauregard

Keyword(s):

Tertiary Structure ◽

Molten Globule ◽

Guanidine Hydrochloride ◽

Average Diameter ◽

Secondary Structures ◽

Fluorescence Measurements ◽

Protein Fibrils ◽

Electron Microscopy Analysis ◽

Microscopy Analysis ◽

The Impact

Canavalia ensiformis (jack bean) α-urease is a hexameric protein characterized by a complex denaturation mechanism. In previous papers, we have shown that a hydrophobic 8-anilino-1-naphthalenesulfonic acid (ANSA) binding conformer could be populated in a moderate concentration of denaturant. This state was obtained under conditions that had no detectable impact on its tertiary structure, as indicated by fluorescence measurements. In the present study, we further characterized this ANSA-binding state in an attempt to understand urease behavior. Evidence presented here shows that the presence of ANSA was not required for the generation of the conformer and that its affinity for ANSA came from an increase in hydrophobicity leading to aggregation. Circular dichroism investigation of urease revealed that it had periodical secondary structure content similar to Klebsiella aerogenes urease (secondary structures calculated on the basis of crystallographic data). The impact of 0.9 M guanidine hydrochloride (GuHCl) on soluble urease secondary structures was minimal but is compatible with a slight increase in beta-sheet structures. Such modification may indicates that aggregation involves amyloid-like fibril formation. Electron microscopy analysis of urease in the absence of GuHCl revealed the presence of urease hexamers (round shape 13 nm in diameter). These particles disappeared in the presence of moderate denaturant concentration owing to the formation of aggregates and fibril-like structures. The fibrils obtained in 1.5 M GuHCl had an average diameter of 6.5 nm, suggesting that urease hexamers dissociated into smaller oligomeric forms when forming such fibrils.Key words: protein structure, protein folding, denaturation, aggregation, multimeric proteins, protein fibrils, hydrophobicity, molten globule state.

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