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

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
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.

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

2006 ◽  
Vol 84 (2) ◽  
pp. 126-134 ◽  
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.

Computational investigations of protein denaturation: apomyoglobin and chaotrope-arene interactions

1993 ◽  
Vol 345 (1674) ◽  
pp. 87-96 ◽  
Keyword(s):  
Thermal Denaturation ◽  
Protein Denaturation ◽  
Molten Globule ◽  
Md Simulations ◽  
Low Ph ◽  
Explicit Representation ◽  
Folding Intermediate ◽  
Detailed Model ◽  
Energy Profiles ◽  
Helical Content

Molecular dynamics (MD) and Monte Carlo (MC) simulations are being used to investigate protein denaturation. The calculations use the AMBER/OPLS force field with explicit representation of the solvent via the TIP3P and TIP4P models of water. The thermal denaturation of apomyoglobin has been followed in two 500 ps MD simulations at 85 °C. The resultant structures provide a detailed model of a molten globule, and close agreement is obtained with experimental data on the helical content of both native apomyoglobin and the low pH folding intermediate. The mechanism of protein denaturation by chaotropic agents is also being pursued. The possibility of direct contact between the chaotropes and aromatic sidechains is supported by MC computations of free energy profiles for the approach of urea and guanidinium ion to aromatic hydrocarbons in water.

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

2004 ◽  
Vol 82 (2) ◽  
pp. 305-313 ◽  
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.

scholarly journals EROSIVE POTENTIAL OF SPORTS BEVERAGES ON HUMAN ENAMEL “IN VITRO”

2018 ◽  
Vol 24 (5) ◽  
pp. 386-390
Author(s):  
Dalila Meazza Damo ◽  
Guilherme Anziliero Arossi ◽  
Helena Alvez da Silva ◽  
Leonardo Haerter dos Santos ◽  
Diego Rafael Kappaun
Keyword(s):  
Vickers Microhardness ◽  
Tooth Enamel ◽  
Low Ph ◽  
Control Surface ◽  
Test Surface ◽  
Results Of Treatment ◽  
Sports Drinks ◽  
Human Enamel ◽  
The Mean

ABSTRACT Introduction: The low pH of sports drinks may cause tooth enamel demineralization. Objective: To measure Vickers hardness of human enamel exposed to sports drinks. Methods: Human molars were used to collect the enamel samples. Each sample had a test surface (exposed to the drinks) and a control surface (unexposed). The samples were exposed to isotonic drinks Gatorade and Powerade, and to maltodextrin drinks Malto Advanced and Malto Active, for 10 minutes every 12 hours over 30 days. The Vickers microhardness test was conducted with three indentations on each surface. The mean of the indentations within each group was considered in the statistical analysis. Sports drinks variables were analyzed with ANOVA/Tukey (p≤0.01). The independent t-test was used in the comparison between the control and test surfaces of each drink (p ≤ 0.05). Results: Enamel exposure to Gatorade (p = 0.000) Malto Advanced (p = 0.000) and Malto Active (p = 0.000) was seen to significantly reduce microhardness, while the isotonic drink Powerade had no significant effect on enamel (p = 0.248). Conclusion: It was concluded that with the exception of the isotonic drink Powerade, all the sports drinks tested caused a reduction in the microhardness of human enamel. Evidence Level III; Therapeutic studies - Investigating the Results of Treatment.

scholarly journals Improvement of Lactic Acid Production in Saccharomyces cerevisiae by Cell Sorting for High Intracellular pH

2006 ◽  
Vol 72 (8) ◽  
pp. 5492-5499 ◽  
Author(s):  
Minoska Valli ◽  
Michael Sauer ◽  
Paola Branduardi ◽  
Nicole Borth ◽  
Danilo Porro ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Intracellular Ph ◽  
Cell Sorting ◽  
Acid Production ◽  
Lactic Acid Production ◽  
Low Ph ◽  
The Mean ◽  
High Level ◽  
Lactate Dehydrogenases

ABSTRACT Yeast strains expressing heterologous l-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pHi) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pHi. During batch culture, we observed in both strains a reduction of the mean pHi and the appearance of a subpopulation of cells with low pHi. Simultaneous analysis of pHi and viability proved that the cells with low pHi were dead. Based on the observation that the better lactic-acid-producing strain had a higher pHi and that the cells with low pHi were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pHi range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pHi range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid-producing strain was identified within the screening of CEN.PK m850 mutants.

Activity and conformational changes of horseradish peroxidase in trifluoroethanol

2002 ◽  
Vol 80 (2) ◽  
pp. 205-213 ◽  
Author(s):  
Hong-Wei Zhou ◽  
Yan Xu ◽  
Hai-Meng Zhou
Keyword(s):  
Enzyme Activity ◽  
Horseradish Peroxidase ◽  
Conformational Changes ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Intrinsic Fluorescence ◽  
Two Phase ◽  
Main Effect ◽  
Α Helix ◽  
Kinetics Of

The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5–25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25–40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The α-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.Key words: horseradish peroxidase, trifluoroethanol, unfolding, Soret.

Mimicking cotranslational folding of prosubtilisin E in vitro

2020 ◽  
Vol 167 (5) ◽  
pp. 473-482 ◽  
Author(s):  
Sung-Gun Kim ◽  
Yu-Jen Chen ◽  
Liliana Falzon ◽  
Jean Baum ◽  
Masayori Inouye
Keyword(s):  
Crucial Role ◽  
Tertiary Structure ◽  
Molten Globule ◽  
Secondary Structures ◽  
Terminal Region ◽  
Folding Intermediates ◽  
C Terminus ◽  
Cotranslational Folding ◽  
N Terminus

Abstract Nascent polypeptides are synthesized on ribosomes starting at the N-terminus and simultaneously begin to fold during translation. We constructed N-terminal fragments of prosubtilisin E containing an intramolecular chaperone (IMC) at N-terminus to mimic cotranslational folding intermediates of prosubtilisin. The IMC-fragments of prosubtilisin exhibited progressive enhancement of their secondary structures and thermostabilities with increasing polypeptide length. However, even the largest IMC-fragment with 72 residues truncated from the C-terminus behaved as a molten globule, indicating the requirement of the C-terminal region to have a stable tertiary structure. Furthermore, truncation of the IMC in the IMC-fragments resulted in aggregation, suggesting that the IMC plays a crucial role to prevent misfolding and aggregation of cotranslational folding intermediates during translation of prosubtilisin polypeptide.

scholarly journals Effects of Different Working Modes of Ultrasound on Structural Characteristics of Zein and ACE Inhibitory Activity of Hydrolysates

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Xiaofeng Ren ◽  
Xi Zhang ◽  
Qiufang Liang ◽  
Ting Hou ◽  
Huiji Zhou
Keyword(s):  
Inhibitory Activity ◽  
Fluorescence Spectra ◽  
Structural Changes ◽  
Structural Characteristics ◽  
Intrinsic Fluorescence ◽  
Cd Spectra ◽  
Fixed Frequency ◽  
Ace Inhibitory Activity ◽  
Ace Inhibitory ◽  
Frequency Ultrasound

Ultrasound was used as a new technology to pretreat protein prior to proteolysis to improve enzymolysis efficiency. The effects of different working modes of ultrasound on the angiotensin I-converting enzyme (ACE) inhibitory activity of zein hydrolysates and the structural characteristics of zein were investigated. The solubility, surface hydrophobicity (H0), ultraviolet-visible (UV-Vis) spectra, intrinsic fluorescence spectra, and circular dichroism (CD) spectra of zein pretreated with ultrasound were determined. All ultrasound pretreatments significantly improved the ACE inhibitory activity of zein hydrolysates (p<0.05). The highest ACE inhibitory activity, representing an increase of 99.21% over the control, was obtained with dual sweeping frequency ultrasound of 33±2 and 68±2 kHz. The effects of single sweeping frequency and dual fixed frequency ultrasound were stronger than those of single fixed frequency ultrasound for improving the ACE inhibitory activity of zein. Structural changes in zein were induced by ultrasound, as confirmed by changes in the solubility, H0, UV-Vis spectra, intrinsic fluorescence spectra, and CD spectra of zein, and these were consistent with the corresponding ACE inhibitory activities of zein hydrolysates. Thus, ultrasound working mode and frequency have significant effects on the structure of zein and the ACE inhibitory activity of zein hydrolysates.

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