We investigated the effect of incorporation of a small aqueous peripheral membrane protein (cyt c) into the three-dimensional periodic nanochannel structures formed by the lipid monoolein (MO) on its rich phase behavior as a function of temperature, pressure, and protein concentration using synchrotron X-ray small-angle diffraction. By simultaneous use of the pressure-jump relaxation technique and time-resolved synchrotron X-ray diffraction, we also studied the kinetics of various lipid mesophase transformations of the system for understanding the mechanistic pathways of their formation influenced by the protein-lipid interactions. Cyt c incorporated into the bicontinuous cubic phase Ia3d of MO has a significant effect on the lipid structure and the pressure stability of the system already at low protein concentrations. Concentrations higher than 0.2 wt % of cyt c led to an increase in interfacial curvature due to interaction of the protein with the lipid headgroups. This promotes the formation of a new, probably partially micellar cubic phase of crystallographic space group P4(3)32. Upon pressurization, the P4(3)32 phase undergoes a phase transition to a cubic Pn3m phase with smaller partial specific volume. Increase in protein concentration increases the pressure stability of the P4(3)32 phase. The formation of this phase from the cubic phase Pn3m is a slow process taking many seconds and having a time lag in the beginning. It seems to occur as a two-state process without ordered intermediate states. At temperatures above 60 degrees C, the P4(3)32 phase is unable to accommodate the unfolded protein and transforms to a bicontinuous cubic Ia3d phase. Time-resolved small-angle X-ray scattering studies show that the L(alpha) --> Ia3d transition in pure MO dispersions under limited hydration conditions occurs within a time interval of 1 s at 35 degrees C preceded by a lag phase of 1.5 s. The Ia3d cubic phase initially forms with a much larger lattice constant due to hydration and experiences an initially lower curvature that relaxes within about 1 s. Interestingly, no other cubic phases are involved as intermediates in the transition, i.e., the gyroid cubic phase is able to form directly from the L(alpha) phase. The mechanism behind the L(alpha) --> Ia3d transition in pure MO dispersions has been discussed within the framework of recent stalk models for membrane fusion. In the presence of cyt c, the L(alpha) --> Ia3d transition is much slower. The rather long relaxation times of the order of seconds are probably due to a kinetic trapping of the system and limitation by the transport and redistribution of water and lipid in the evolving new lipid phases. We also studied the transition from the pure lamellar L(alpha) phase to the Ia3d-P4(3)32 two phase region and observed a rather complex transition behavior with transient lamellar and cubic intermediate states.

A misfolded form of the prion protein (PrP) is the primary culprit in mammalian prion diseases. It has been shown that nucleic acids catalyze the misfolding of cellular PrP into a scrapie-like conformer. It has also been observed that the interaction of PrP with nucleic acids is nonspecific and that the complex can be toxic to cultured cells. No direct correlation has yet been drawn between changes in PrP structure and toxicity due to nucleic acid binding. Here we asked whether different aggregation, stability, and toxicity effects are detected when nonrelated DNA sequences interact with recombinant PrP. Using spectroscopic techniques to analyze PrP tertiary and secondary structure and cellular assays to assess toxicity, we found that rPrP–DNA interactions lead to different aggregated species, depending on the sequence and size of the oligonucleotide tested. A 21-mer DNA sequence (D67) induced higher levels of aggregation and also dissimilar structural changes in rPrP, compared to binding to oligonucleotides with the same length and different nucleotide sequences or different GC contents. The rPrP–D67 complex induced significant cell dysfunction, which appears to be correlated with the biophysical properties of the complex. Although sequence specificity is not apparent for PrP–nucleic acid interactions, we believe that particular nucleic acid patterns, possibly related to GC content, oligonucleotide length, and structure, govern PrP recognition. Understanding the structural and cellular effects observed for PrP–nucleic acid complexes may shed light on the still mysterious pathology of the prion protein.

The main hypothesis for prion diseases proposes that the cellular protein (PrP C ) can be altered into a misfolded, ␤-sheet-rich isoform (PrP Sc ), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we compare the stability against pressure and the thermomechanical properties of the ␣-helical and ␤-sheet conformations of recombinant murine prion protein, designated as ␣-rPrP and ␤-rPrP, respectively. High temperature induces aggregates and a large gain in intermolecular antiparallel ␤-sheet (␤-rPrP), a conformation that shares structural similarity with PrP Sc . ␣-rPrP is highly stable, and only pressures above 5 kilobars (1 kilobar ‫؍‬ 100 MegaPascals) cause reversible denaturation, a process that leads to a random and turnrich conformation with concomitant loss of ␣-helix, as measured by Fourier transform infrared spectroscopy. In contrast, aggregates of ␤-rPrP are very sensitive to pressure, undergoing transition into a dissociated species that differs from the denatured form derived from ␣-rPrP. The higher susceptibility to pressure of ␤-rPrP can be explained by its less hydrated structure. Pressure perturbation calorimetry supports the view that the accessible surface area of ␣-rPrP is much higher than that of ␤-rPrP, which explains the lower degree of hydration of ␤-rPrP. Our findings shed new light on the mechanism of prion conversion and show how water plays a prominent role. Our results allow us to propose a volume and free energy diagram of the different species involved in the conversion and aggregation. The existence of different folded conformations as well as different denatured states of PrP may explain the elusive character of its conversion into a pathogenic form.

The main hypothesis for prion diseases is that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we investigate the effects of amino-terminal deletion mutations, rPrP(Delta51-90) and rPrP(Delta32-121), on the stability and the packing properties of recombinant murine PrP. The region lacking in rPrP(Delta51-90) is involved physiologically in copper binding and the other construct lacks more amino-terminal residues (from 32 to 121). The pressure stability is dramatically reduced with decreasing N-domain length and the process is not reversible for rPrP(Delta51-90) and rPrP(Delta32-121), whereas it is completely reversible for the wild-type form. Decompression to atmospheric pressure triggers immediate aggregation for the mutants in contrast to a slow aggregation process for the wild-type, as observed by Fourier-transform infrared spectroscopy. The temperature-induced transition leads to aggregation of all rPrPs, but the unfolding temperature is lower for the rPrP amino-terminal deletion mutants. The higher susceptibility to pressure of the amino-terminal deletion mutants can be explained by a change in hydration and cavity distribution. Taken together, our results show that the amino-terminal region has a pivotal role on the development of prion misfolding and aggregation.

We report on a study of insulin incorporation into cubic phases of mono-olein (MO), using synchrotron small-angle X-ray scattering and FT-IR spectroscopy. We studied the thermal stability and aggregation scenario of insulin as a function of protein concentration in the narrow water channels of the cubic lipid matrix and compared it with data for insulin unfolding and fibrillation in bulk water solutions. The concomitant effect of insulin entrapment on the structure and phase behavior of the lipid matrix itself was also examined. We show that the protein's unfolding behavior and stability are influenced by confinement due to geometrical limitations, and vice versa, the topological properties of the lipid matrix change as well. The addition of insulin already at concentrations as low as 0.1 wt % significantly alters the phase behavior of MO. Surprisingly, new cubic structures are induced by insulin incorporation into the lipid matrix. When insulin begins to partially unfold at higher temperatures, the structure of the new cubic phase changes and finally disappears around 60 degrees C, where the aggregation process sets in. The aggregation in cubo proceeds much faster and leads to the formation of medium-sized oligomers or clusters, while the formation of large fibrillar agglomerates, as observed for bulk insulin aggregation, is largely prohibited. Hence, the results yield valuable information about the use of cubic mesoporous lipid systems as a medium for long-term storage of insulin and aggregation-prone proteins in general. Furthermore, the results provide new insights into the effects of soft-matter confinement on protein aggregation and fibrillation, a situation usually met in natural cell environments.

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid "raft" mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7-9 kbar and temperature in the range from 1 to about 80 degrees C. The generalized polarization function (GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p,T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (lo+so) two-phase coexistence region below 8+/-2 degrees C at ambient pressure. With increasing temperature, a lo+ld+so three-phase region is formed, which extends up to approximately 27 degrees C, where a liquid-ordered/liquid-disordered (lo+ld) immiscibility region is formed. Finally, above 48+/-2 degrees C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, ld). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the lo+ld (+so) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 degrees C, and a pressure range, which extends up to about 2 kbar for T=37 degrees C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.

No abstract

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for several familial amyloid diseases. Here, we use FTIR to describe the secondary structural changes that take place when wt TTR undergoes heat- or high-pressure-induced denaturation, as well as fibril formation. Upon thermal denaturation, TTR loses part of its intramolecular beta-sheet structure followed by an increase in nonnative, probably antiparallel beta-sheet contacts (bands at 1,616 and 1,686 cm(-1)) and in the light scattering, suggesting its aggregation. Pressure-induced denaturation studies show that even at very elevated pressures (12 kbar), TTR loses only part of its beta-sheet structure, suggesting that pressure leads to a partially unfolded species. On comparing the FTIR spectrum of the TTR amyloid fibril produced at atmospheric pressure upon acidification (pH 4.4) with the one presented by the native tetramer, we find that the content of beta-sheets does not change much upon fibrillization; however, the alignment of beta-sheets is altered, resulting in the formation of distinct beta-sheet contacts (band at 1,625 cm(-1)). The random-coil content also decreases in going from tetramers to fibrils. This means that, although part of the tertiary- and secondary-structure content of the TTR monomers has to be lost before fibril formation, as previously suggested, there must be a subsequent reorganization of part of the random-coil structure into a well-organized structure compatible with the amyloid fibril, as well as a readjustment of the alignment of the beta-sheets. Interestingly, the infrared spectrum of the protein recovered from a cycle of compression-decompression at pD 5, 37 degrees C, is quite similar to that of fibrils produced at atmospheric pressure (pH 4.4), which suggests that high hydrostatic pressure converts the tetramers of TTR into an amyloidogenic conformation.