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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]

Accumulating evidence suggests that amyloid plaqueassociated myelin lipid loss as a result of elevated amyloid burden might also contribute to Alzheimer's disease. The amyloid fibrils are closely associated with lipids under physiological conditions; however, the progression of membrane remodeling events leading to lipid-fibril assembly remains unknown. Here we first reconstitute the interaction of amyloid Beta 40 (Aβ-40) with myelin-like model membrane and show that the binding of Aβ-40 induces extensive tubulation. To look into the mechanism of membrane tubulation, we chose a set of membrane conditions varying in lipid packing density and net charge that allows us to dissect the contribution of lipid specificity of Aβ-40 binding, aggregation kinetics, and subsequent changes in membrane parameters such as fluidity, diffusion, and compressibility modulus. We show that the binding of Aβ-40 depends predominantly on the lipid packing defect densities and electrostatic interactions and results in rigidification of the myelin-like model membrane during the early phase of amyloid aggregation. Furthermore, elongation of Aβ-40 into higher oligomeric and fibrillar species leads to eventual fluidization of the model membrane followed by extensive lipid membrane tubulation observed in the late phase. Taken together, our results capture mechanistic insights into snapshots of temporal dynamics of Aβ-40-myelin-like model membrane interaction and demonstrate how short timescale, local phenomena of binding, and fibril-mediated load generation results in the consequent association of lipids with growing amyloid fibrils. [ABSTRACT FROM AUTHOR]

An insight into the conformation and dynamics of unfolded and early intermediate states of a protein is essential to understand the mechanism of its aggregation and to design potent inhibitor molecules. Fluorescence correlation spectroscopy has been used to study the effects of several model protein stabilizers on the conformation of the unfolded state and early folding dynamics of tetramethyl rhodamine-labeled cytochrome c from Saccharomyces cerevisiae at single molecular resolution. Special attention has been given to arginine, which is a widely used stabilizer for improving refolding yield of different proteins. The value of the hydrodynamic radius (rH) obtained by analyzing the intensity fluctuations of the diffusing molecules has been found to increase in a two-state manner as the protein is unfolded by urea. The results further show that the presence of arginine and other protein stabilizers favors a relatively structured conformation of the unfolded states (rH of 29 Å) over an extended one (rH of 40 Å), which forms in their absence. Also, the time constant of a kinetic component (τR) of about 30 µs has been observed by analyzing the correlation functions, which represents formation of a collapsed state. This time constant varies with urea concentration representing an inverted Chevron plot that shows a roll-over behavior in the absence of arginine. To the best of our knowledge, this is one of the first applications of fluorescence correlation spectroscopy to study direct folding kinetics of a protein. [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]