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

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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Intermediate studies on refolding of arginine kinase denatured by guanidine hydrochloride

Biochemistry and Cell Biology ◽

10.1139/o04-131 ◽

2005 ◽

Vol 83 (2) ◽

pp. 109-114 ◽

Cited By ~ 5

Author(s):

Hong-Min Tang ◽

Hong Yu

Keyword(s):

Fluorescence Intensity ◽

Tertiary Structure ◽

Structural Characteristics ◽

Molten Globule ◽

Guanidine Hydrochloride ◽

Arginine Kinase ◽

Fluorescence Emission ◽

Tryptophan Fluorescence ◽

Blue Shift ◽

Molten Globule State

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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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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Temperature adaptation in Gillichthys (Teleost: Gobiidae)A4-lactate dehydrogenases

Journal of Experimental Biology ◽

10.1242/jeb.205.9.1293 ◽

2002 ◽

Vol 205 (9) ◽

pp. 1293-1303 ◽

Cited By ~ 2

Author(s):

Peter A. Fields ◽

Yong-Sung Kim ◽

John F. Carpenter ◽

George N. Somero

Keyword(s):

Conformational Changes ◽

Tertiary Structure ◽

Conformational Flexibility ◽

Cd Spectroscopy ◽

Temperature Adaptation ◽

Substrate Affinity ◽

Second Derivative ◽

Structural Differences ◽

Far Ultraviolet ◽

Lactate Dehydrogenases

SUMMARY Alternative conformations of proteins underlie a variety of biological phenomena, from prion proteins that cause spongiform encephalopathies to membrane channel proteins whose conformational changes admit or exclude specific ions. In this paper, we argue that conformational differences within globular `housekeeping' enzymes may allow rapid adaptation to novel environments. Muscle-type lactate dehydrogenases (A4-LDHs) from the gobies Gillichthys seta and G. mirabilis have identical amino acid sequences but show potentially adaptive differences in substrate affinity (apparent Michaelis constants for pyruvate, KmPYR) as well as differences in thermal stability. We examined the A4-LDH of each species using fluorescence spectroscopy, near- and far-ultraviolet circular dichroism (CD)spectroscopy and hydrogen/deuterium exchange (H/D) Fourier-transform infrared spectroscopy to determine whether structural differences were apparent, the extent to which structural differences could be related to differences in conformational flexibility and whether specific changes in secondary or tertiary structure could be defined. The fluorescence spectra and far-ultraviolet CD spectra of the A4-LDH from the two species were indistinguishable, suggesting that the two conformations are very similar in secondary and tertiary structure. Apparent melting temperatures(Tm) followed by fluorescence and CD spectroscopy confirmed that the G. mirabilis A4-LDH is more thermally stable than the G. seta form. H/D exchange kinetics of Gillichthys A4-LDH was described using double-exponential regression; at 20 °C, G. seta A4-LDH has a higher exchange constant, indicating a more flexible and open structure. At 40°C, the difference in H/D exchange constants disappears. Second-derivative analysis of H/D exchange infrared spectra indicates that α-helical, but not β-sheet structure, differs in conformational flexibility between the two forms. Second-derivative ultraviolet spectra indicate that at least one of the five tyrosyl residues in the Gillichthys LDH-A monomer is located in a more hydrophobic environment in the G. mirabilis form. Homology models of A4-LDH indicate that Tyr246 is the most likely candidate to experience a modified environment because it is involved in subunit contacts within the homotetramer and sits in a hinge between a staticα-helix and one involved in catalytic conformational changes. Subtle differences in conformation around this residue probably play a role both in altered flexibility and in the potentially adaptive differences in kinetics between the two A4-LDH forms.

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Molten Globule-Like Partially Folded State ofBacillus licheniformis α-Amylase at Low pH Induced by 1,1,1,3,3,3-Hexafluoroisopropanol

The Scientific World JOURNAL ◽

10.1155/2014/824768 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Adyani Azizah Abd Halim ◽

Mohammed Suleiman Zaroog ◽

Habsah Abdul Kadir ◽

Saad Tayyab

Keyword(s):

Thermal Denaturation ◽

Tertiary Structure ◽

Molten Globule ◽

Low Ph ◽

Intrinsic Fluorescence ◽

Cd Spectra ◽

Fluorescence Measurements ◽

The Mean ◽

Partially Folded State ◽

Cooperative Transition

Effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) on acid-denaturedBacillus licheniformis α-amylase (BLA) at pH 2.0 was investigated by far-UV CD, intrinsic fluorescence, and ANS fluorescence measurements. Addition of increasing HFIP concentrations led to an increase in the mean residue ellipticity at 222 nm (MRE222 nm) up to 1.5 M HFIP concentration beyond which it sloped off. A small increase in the intrinsic fluorescence and a marked increase in the ANS fluorescence were also observed up to 0.4 M HFIP concentration, both of which decreased thereafter. Far- and near-UV CD spectra of the HFIP-induced state observed at 0.4 M HFIP showed significant retention of the secondary structures closer to native BLA but a disordered tertiary structure. Increase in the ANS fluorescence intensity was also observed with the HFIP-induced state, suggesting exposure of the hydrophobic clusters to the solvent. Furthermore, thermal denaturation of HFIP-induced state showed a non-cooperative transition. Taken together, all these results suggested that HFIP-induced state of BLA represented a molten globule-like state at pH 2.0.

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The Molten Globule State of Maltose-Binding Protein: Structural and Thermodynamic Characterization by EPR Spectroscopy and Isothermal Titration Calorimetry

Applied Magnetic Resonance ◽

10.1007/s00723-020-01232-y ◽

2020 ◽

Vol 51 (9-10) ◽

pp. 877-886

Author(s):

Chen Nickolaus ◽

Carolyn Vargas ◽

Jörg Reichenwallner ◽

Mohammed Chakour ◽

Benjamin Selmke ◽

...

Keyword(s):

Epr Spectroscopy ◽

Tertiary Structure ◽

Binding Protein ◽

Molten Globule ◽

Spin Labeling ◽

Maltose Binding Protein ◽

Titration Calorimetry ◽

Native State ◽

Paramagnetic Resonance ◽

Distance Information

Abstract Employing site-directed spin labeling (SDSL), the structure of maltose-binding protein (MBP) had previously been studied in the native state by electron paramagnetic resonance (EPR) spectroscopy. Several spin-labeled double cysteine mutants were distributed all over the structure of this cysteine-free protein and revealed distance information between the nitroxide residues from double electron–electron resonance (DEER). The results were in good agreement with the known X-ray structure. We have now extended these studies to the molten globule (MG) state, a folding intermediate, which can be stabilized around pH 3 and that is characterized by secondary but hardly any tertiary structure. Instead of clearly defined distance features as found in the native state, several additional characteristics indicate that the MG structure of MBP contains different polypeptide chain and domain orientations. MBP is also known to bind its substrate maltose even in MG state although with lower affinity. Additionally, we have now created new mutants allowing for spin labeling at or near the active site. Our data confirm an already preformed ligand site structure in the MG explaining its substrate binding capability and thus most probably serving as a nucleation center for the final native structure.

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Conformational Changes in the Reversed Phase Liquid Chromatography of Recombinant Human Growth Hormone as a Function of Organic Solvent: The Molten Globule State

Analytical Chemistry ◽

10.1021/ac00094a011 ◽

1994 ◽

Vol 66 (22) ◽

pp. 3908-3915 ◽

Cited By ~ 36

Author(s):

S. Wicar ◽

M. G. Mulkerrin ◽

G. Bathory ◽

L. H. Khundkar ◽

B. L. Karger

Keyword(s):

Growth Hormone ◽

Liquid Chromatography ◽

Conformational Changes ◽

Molten Globule ◽

Recombinant Human Growth Hormone ◽

Human Growth ◽

Reversed Phase ◽

Reversed Phase Liquid Chromatography ◽

Molten Globule State ◽

Phase Liquid

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pH-Induced Molten Globule State of Rhizopus niveus Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

Cell Biochemistry and Biophysics ◽

10.1007/s12013-011-9335-9 ◽

2012 ◽

Vol 62 (3) ◽

pp. 487-499 ◽

Cited By ~ 125

Author(s):

Gulam Rabbani ◽

Ejaz Ahmad ◽

Nida Zaidi ◽

Sadaf Fatima ◽

Rizwan Hasan Khan

Keyword(s):

Molten Globule ◽

Native State ◽

Chemical Denaturation ◽

Molten Globule State ◽

Rhizopus Niveus

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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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