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The presence of magnetic nanoparticles (NPs) in physiological systems induces toxicity through its effects on mitochondrial function and reactive oxygen species (ROS) imbalance. Magnetic NP induced cytotoxicity has been elaborately evaluated for impending threats, however, a detailed investigation is lacking. It is shown that the interaction of Fe3O4 NPs with cytochrome c can lead to different events based on the NPs to protein ratio, the solution conditions, and the type of surface protection. At low NPs concentration, rapid binding and subsequent electron transfer are the preferred events while at higher concentration slow oxidative modification of the protein is initiated. The slow event of protein modification yields conformational disorientation, loss of stability, and formation of amyloid-like structures with cytochrome c. The possibility that the NP induced oxidative stress and age can work in concert to compromise different aspects of cellular quality control processes is discussed. Suitable surface modifications of the NPs inhibit their direct binding to the protein molecules and minimize NP induced toxicity. [ABSTRACT FROM AUTHOR]

What happens in the early stage of protein folding remains an interesting unsolved problem. Rapid kinetics measurements with cytochrome c using submillisecond continuous flow mixing devices suggest simultaneous formation of a compact collapsed state and secondary structure. These data seem to indicate that collapse formation is guided by specific short and long range interactions (heteropolymer collapse). A contrasting interpretation also has been proposed, which suggests that the collapse formation is rapid, nonspecific, and a trivial solvent related compaction, which could as well be observed by a homopolymer (homopolymer collapse). We address this controversy using fluorescence correlation spectroscopy (FCS), which enables us to monitor the salt-induced compaction accompanying collapse formation and the associated time constant directly at single molecule resolution. In addition, we follow the formation of secondary structure using far UV CD. The data presented here suggest that both these models (homopolymer and heteropolymer) could be applicable depending on the solution conditions. For example, the formation of secondary structure and compact state is not simultaneous in aqueous buffer. In aqueous buffer, formation of the compact state occurs through a two-state co-operative transition following heteropolymer formalism, whereas secondary structure formation takes place gradually. In contrast, in the presence of urea, a compaction of the protein radius occurs gradually over an extended range of salt concentration following homopolymer formalism. The salt-induced compaction and the formation of secondary structure take place simultaneously in the presence of urea. [ABSTRACT FROM AUTHOR]

Interaction of lauryl maltoside (LM) surfactant with bovine heart cytochrome c oxidase (CcO) has been studied by NMR techniques. Detailed 2-D 1H and 13C NMR techniques were used to assign the NMR signals of the surfactant nuclei. Paramagnetic dipolar shift of the surfactant 13C NMR signals were used to identify the atoms close to the enzyme. The diamagnetic carbon monoxide complex of CcO did not cause any shift in the surfactant NMR spectra suggesting that the paramagnetic centres of the native CcO cause the shifts by dipolar interactions. The results showed that the polar head groups of the surfactant comprised of two maltoside rings are more affected, while the hydrophobic tail groups did not show any significant change on binding of the surfactant to the enzyme. This indicated that surfactant head groups possibly bind to the enzyme surface and the hydrophobic tail of the surfactant forms micelles and remains away from the enzyme. Based on the results, we propose that the membrane bound enzyme is possibly stabilised in aqueous solution by association with the micelles of the neutral surfactant so that the polar heads of the micelles bind to the polar surface of the enzyme. These micelles might form a ‘belt like’ structure around the enzyme helping it to remain monodispersed in the active form. [Copyright &y& Elsevier]

The image shows the major findings of Goutam De, Krishnananda Chattopadhyay, and coworkers, who studied the interaction between iron oxide nanoparticles and cytochrome c, as reported on page 683. The triangle at the top left shows the binding of the protein onto the surface of the bare particles forming amyloid fibrils (transmission electron microscopy image is shown in the background). Appropriate surface modification inhibits protein binding and amyloid formation (shown in the triangle at the bottom right). [ABSTRACT FROM AUTHOR]

To understand how a protein folds and behaves inside living cells, the effects of synthetic crowding media on protein folding, function, stability, and association have been studied in detail. Because the effect of excluded volume is more prominent in an extended state than in the native protein, a majority of these studies have been conducted in the unfolded state of different model proteins. Here, we have used fluorescence correlation spectroscopy (FCS) and other biophysical methods to investigate the effect of crowding agents Ficoll70 and Dextran70 on the nativelike state of cytochrome c from yeast. Yeast cytochrome c (y-cytc) contains a substantial expanded state in its native folded condition, which is present in equilibrium with a compact conformer in aqueous buffer. We have found that the crowding medium affects the native state equilibrium between compact and expanded states, shifting its population toward the compact conformer. As a result, the peroxidase activity of y-cytc decreases. Urea-induced protein stability measurements show that the compaction destabilizes the protein due to charge repulsions between similar charged clusters. Interestingly, the time constant of conformational fluctuations between the compact and expanded conformers has been found to increase in the crowded milieu, suggesting a crucial role of the solution microviscosity. [ABSTRACT FROM AUTHOR]

Although the primary function of cytochrome c (cyt c) is electron transfer, the protein caries out an additional secondary function involving its interaction with membrane cardiolipin (CDL), its peroxidase activity, and the initiation of apoptosis. Whereas the primary function of cyt c is essentially conserved, its secondary function varies depending on the source of the protein. We report here a detailed experimental and computational study, which aims to understand, at the molecular level, the difference in the secondary functions of cyt c obtained from horse heart (mammalian) and Saccharomyces cerevisiae (yeast). The conformational landscape of cyt c has been found to be heterogeneous, consisting of an equilibrium between the compact and extended conformers as well as the oligomeric species. Because the determination of relative populations of these conformers is difficult to obtain by ensemble measurements, we used fluorescence correlation spectroscopy (FCS), a method that offers single-molecule resolution. The population of different species is found to depend on multiple factors, including the protein source, the presence of CDL and urea, and their concentrations. The complex interplay between the conformational distribution and oligomerization plays a crucial role in the variation of the pre-apoptotic regulation of cyt c observed from different sources. Finally, computational studies reveal that the variation in the charge distribution at the surface and the charge reversal sites may be the key determinant of the conformational stability of cyt c. [ABSTRACT FROM AUTHOR]