Denaturation properties and folding transition states of leghemoglobin and other heme proteins.
TL;DR: The lifetime data clearly indicate the presence of an intermediate state during denaturation, and the unfolding transition can modulate the conformation, stability, and surface exposure of these biologically important proteins.
Abstract: This work reports unfolding transitions of monomeric heme proteins leghemoglobin (Lb), myoglobin (Mb), and cytochrome c (Cyt c) utilizing UV-Vis spectral and steady-state and time-resolved fluorescence methods. Conformational stabilities of the native "folded" state of the proteins and their "unfolded" states were investigated in the light of a two-state transition model. Two-state transition values for ΔGD (298K) were obtained by denaturation with the chaotropic agents urea and guanidium hydrochloride (GdnHCl). The free energy value of Lb is the lowest compared to Cyt c and Mb along the denaturation pathway. The m value is also the lowest for Lb compared to Cyt c and Mb. The m value (a measure of dependence of ΔGD on denaturant concentration) for Cyt c and Mb is lower when it is denatured with urea compared to GdnHCl. The UV-Vis absorbance maximum and steady state fluorescence emission maximum were drastically red shifted in the presence of a certain denaturant concentration both in cases of Mb and Lb, but the scenario is different for Cyt c. The results are analyzed using a two-state transition model. The lifetime data clearly indicate the presence of an intermediate state during denaturation. The unfolding transition can modulate the conformation, stability, and surface exposure of these biologically important proteins.
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TL;DR: The results suggest that α‐crystallin is susceptible to unfolding in the presence of chaotropic agents like urea and GdnHCl, whereas only aggregate‐like structures were observed in α‐ Crystallin treated with urea.
Abstract: α-Crystallin is a member of small heat shock proteins and is believed to play an exceptional role in the stability of eye lens proteins. The disruption or denaturation of the protein arrangement or solubility of the crystallin proteins can lead to vision problems including cataract. In the present study, we have examined the effect of chemical denaturants urea and guanidine hydrochloride (GdnHCl) on α-crystallin aggregation, with special emphasis on protein conformational changes, unfolding, and amyloid fibril formation. GdnHCl (4 M) induced a 16 nm red shift in the intrinsic fluorescence of α-crystallin, compared with 4 nm shift by 8 M urea suggesting a major change in α-crystallin structure. Circular dichroism analysis showed marked increase in the ellipticity of α-crystallin at 216 nm, suggesting gain in β-sheet structure in the presence of GdnHCl (0.5-1 M) followed by unfolding at higher concentration (2-6 M). However, only minor changes in the secondary structure of α-crystallin were observed in the presence of urea. Moreover, 8-anilinonaphthalene-1-sulfonic acid fluorescence measurement in the presence of GdnHCl and urea showed changes in the hydrophobicity of α-crystallin. Amyloid studies using thioflavin T fluorescence and congo red absorbance showed that GdnHCl induced amyloid formation in α-crystallin, whereas urea induced aggregation in this protein. Electron microscopy studies further confirmed amyloid formation of α-crystallin in the presence of GdnHCl, whereas only aggregate-like structures were observed in α-crystallin treated with urea. Our results suggest that α-crystallin is susceptible to unfolding in the presence of chaotropic agents like urea and GdnHCl. The destabilized protein has increased likelihood to fibrillate. Copyright © 2016 John Wiley & Sons, Ltd.
10 citations
Cites background from "Denaturation properties and folding..."
...Urea and guanidine hydrochloride (GdnHCl) have been widely used to study protein folding dynamics (Basak et al., 2015)....
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TL;DR: Stable nano-composite with enhanced peroxidase activity is successfully constructed by modulating the interfacial interaction forces between GO and cyt c by combination of solution chemistry and spectroscopy.
Abstract: Graphene oxide (GO) has drawn worldwide attention in various biomedical fields because of its unique properties, and great progress has been made in the past years. Probing the interaction between ...
6 citations
TL;DR: Hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain, and this work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors.
6 citations
TL;DR: In this paper, the effects of Cu2+ on the structure, peroxidase activity, and antibacterial ability of Tg-HbI were investigated in a noncompetitive manner, following first-order reaction kinetics.
Abstract: Beyond its role as an oxygen transport protein, the homodimer hemoglobin of blood clam Tegillarca granosa (Tg-HbI) has been found to possess antibacterial activity However，the mechanism of antibacterial activity of Tg-HbI remain to be investigated In this study, we investigated the effects of Cu2+ on the structure, peroxidase activity, and antibacterial ability of Tg-HbI Tg-HbI was significantly inactivated by Cu2+ in a noncompetitive inhibition manner, following first-order reaction kinetics The Spectroscopy results showed that Cu2+ changed the iron porphyrin ring and the coordination of heme with proximal histidine of Tg-HbI, and increased the hydrophobicity of heme pocket We found that proline could stabilize the heme pocket of Tg-HbI, hence, protect peroxidase activity and antimicrobial activity of Tg-HbI against damage by Cu2+ Our results suggest that Cu2+ inhibits the peroxidase and antibacterial activity of Tg-HbI by destroying its heme pocket structure and Tg-HbI probably plays an antibacterial role through its peroxidase activity This result could provide insights into the antibacterial mechanism of Tg-HbI
1 citations
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TL;DR: High-resolution proton nuclear magnetic resonance studies of protein hydration in aqueous solution show that there are two qualitatively different types of hydration sites, including well-ordered water in the x-ray structures of protein single crystals.
Abstract: High-resolution proton nuclear magnetic resonance studies of protein hydration in aqueous solution show that there are two qualitatively different types of hydration sites. A well-defined, small number of water molecules in the interior of the protein are in identical locations in the crystal structure and in solution, and their residence times are in the range from about 10(-2) to 10(-8) second. Hydration of the protein surface in solution is by water molecules with residence times in the subnanosecond range, even when they are located in hydration sites that contain well-ordered water in the x-ray structures of protein single crystals.
724 citations
TL;DR: Not received Cytochrome c Protein denaturation Compact denaturation Nh4R spectroscopy Viscosity Side chain mobility of protein
713 citations
TL;DR: In this article, the results of an investigation of acid and some similar forms of bovine and human a-lactalbumins are presented, which show that these forms are compact, have secondary structure similar to the native one but their tertiary structure can slowly fluctuate.
582 citations
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TL;DR: Simulations are presented to show that certain fluorescence observables directly track the population of states in an unfolding transition, whereas other observables skew the transition toward the dominant fluorescing species.
470 citations
Journal Article•
TL;DR: In this article, the advantages and limitations of the use of fluorescence methods for the quantitative determination of the thermodynamics of protein unfolding transitions (i.e., induced by temperature or chemical denaturant) are discussed.
Abstract: The advantages and some limitations of the use of fluorescence methods for the quantitative determination of the thermodynamics of protein unfolding transitions (i.e., induced by temperature or chemical denaturant) are discussed. Advantages include the sensitivity, multi-dimensional nature of the data, wide amenable concentration range, high signal-to-noise, rapidity of measurement, and adaptability to a variety of sample compartments. Aside from the need for a probe, some problems associated with the method involve the handling of baselines for the pre- and post-transition regions and the difficulty (shared by most other methods) of discerning whether the transition is two-state or multi-state.
440 citations