[Show abstract][Hide abstract] ABSTRACT: Structural changes on LexA repressor promoted by acidic pH have been investigated. Intense protein aggregation occurred around pH 4.0 but was not detected at pH values lower than pH 3.5. The center of spectral mass of the Trp increased 400 cm(-1) at pH 2.5 relatively to pH 7.2, an indication that LexA has undergone structural reorganization but not denaturation. The Trp fluorescence polarization of LexA at pH 2.5 indicated that its hydrodynamic volume was larger than its dimer at pH 7.2. 4,4'-Dianilino-1,1'-binaphthyl-5,5'- disulfonic acid (bis-ANS) experiments suggested that the residues in the hydrophobic clefts already present at the LexA structure at neutral pH had higher affinity to it at pH 2.5. A 100 kDa band corresponding to a tetramer was obtained when LexA was subject to pore-limiting native polyacrylamide gel electrophoresis at this pH. The existence of this tetrameric state was also confirmed by small angle X-ray scattering (SAXS) analysis at pH 2.5. 1D 1H NMR experiments suggested that it was composed of a mixture of folded and unfolded regions. Although 14,000-fold less stable than the dimeric LexA, it showed a tetramer-monomer dissociation at pH 2.5 from the hydrostatic pressure and urea curves. Albeit with half of the affinity obtained at pH 7.2 (Kaff of 170 nM), tetrameric LexA remained capable of binding recA operator sequence at pH 2.5. Moreover, different from the absence of binding to the negative control polyGC at neutral pH, LexA bound to this sequence with a Kaff value of 1415 nM at pH 2.5. A binding stoichiometry experiment at both pH 7.2 and pH 2.5 showed a [monomeric LexA]/[recA operator] ratio of 2:1. These results are discussed in relation to the activation of the Escherichia coli SOS regulon in response to environmental conditions resulting in acidic intracellular pH. Furthermore, oligomerization of LexA is proposed to be a possible regulation mechanism of this regulon.
Journal of Molecular Biology 07/2006; 359(4):1059-74. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The 3rd International Conference on High Pressure Bioscience and Biotechnology was held in the city of Rio de Janeiro from September 27 to September 30, 2004. The meeting, promoted by the International Association of High Pressure Bioscience and Biotechnology (IAHPBB), congregated top scientists and researchers from all over the world. In common, they shared the use of hydrostatic pressure for research, technical development, or industrial applications. The meeting consisted of invited lectures, contributed papers and a well-attended poster session. Very exciting discussions were held inside and outside the sessions, and the goals of discussing state-of-the-art data and establishing working collaborations and co-operations were fully attained.
Brazilian Journal of Medical and Biological Research 09/2005; 38(8):1147-55. · 1.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of tumor suppressor protein p53 in cell cycle control depends on its flexible and partially unstructured conformation, which makes it crucial to understand its folding landscape. Here we report an intermediate structure of the core domain of the tumor suppressor protein p53 (p53C) during equilibrium and kinetic folding/unfolding transitions induced by guanidinium chloride. This partially folded structure was undetectable when investigated by intrinsic fluorescence. Indeed, the fluorescence data showed a simple two-state transition. On the other hand, analysis of far ultraviolet circular dichroism in 1.0 M guanidinium chloride demonstrated a high content of secondary structure, and the use of an extrinsic fluorescent probe, 4,4'-dianilino-1,1' binaphthyl-5,5'-disulfonic acid, indicated an increase in exposure of the hydrophobic core at 1 M guanidinium chloride. This partially folded conformation of p53C was plagued by aggregation, as suggested by one-dimensional NMR and demonstrated by light-scattering and gel-filtration chromatography. Dissociation by high pressure of these aggregates reveals the reversibility of the process and that the aggregates have water-excluded cavities. Kinetic measurements show that the intermediate formed in a parallel reaction between unfolded and folded structures and that it is under fine energetic control. They are not only crucial to the folding pathway of p53C but may explain as well the vulnerability of p53C to undergo departure of the native to an inactive state, which makes the cell susceptible to malignant transformation.
[Show abstract][Hide abstract] ABSTRACT: Hydrostatic pressure is a useful tool for dissecting macromolecular interactions at the molecular level. Nonpolar interactions are determining factors in protein folding, protein aggregation and protein-nucleic acid recognition. Because nonpolar interactions are entropic and compressible, they are more sensitive to pressure and low temperatures. We have studied problems of macromolecular recognition using hydrostatic pressure as the primary tool and employing several spectroscopic techniques, especially fluorescence, circular dichroism and high-resolution nuclear magnetic resonance. High pressure has the unique property of stabilizing partially folded states of a protein which degree of dissimilarity from the native state may range from drifted conformations to molten-globule states. The competition between correct folding and misfolding, which in many proteins leads to formation of insoluble aggregates is an important problem in the biotechnology industry and in human diseases such as amyloidosis, Alzheimer's, prion and tumor diseases. Because of its ability to sequester folding intermediates, pressure has been used to direct the folding in one direction or the other and to explore intermediates, which are at the junction of the routes for folding and aggregation.
Progress in Biotechnology - PROGR BIOTECHNOL. 01/2002; 19:79-86.
[Show abstract][Hide abstract] ABSTRACT: The recognition of palindromic specific DNA sequences by the human papillomavirus (HPV) E2 proteins is responsible for regulation of virus transcription. The dimeric E2 DNA-binding domain of HPV-16 (E2c) dissociates into a partially folded state under high hydrostatic pressure. We show here that pressure-induced monomers of E2c are highly structured, as evidenced by NMR hydrogen-deuterium exchange measurements. On binding to both specific and nonspecific DNA, E2c becomes stable against pressure. Competitive binding studies using fluorescence polarization of fluorescein-labeled DNA demonstrate the reversibility of the specific binding. To assess the thermodynamic parameters for the linkage between protein dissociation and DNA binding, urea denaturation curves were obtained at different pressures in the presence of specific and nonspecific DNA sequences. The change in free energy on denaturation fell linearly with increase in pressure for both protein-DNA complexes, and the measured volume change was similar to that obtained for E2c alone. The data show that the free energy of dissociation increases when E2c binds to a nonspecific DNA sequence but increases even more when the protein binds to the specific DNA sequence. Thus, specific complexes are tighter but do not entail variation in the volume change. The thermodynamic data indicate that DNA-bound E2c dissociates into monomers bound to DNA. The existence of monomeric units of E2c bound to DNA may have implications for the formation of DNA loops, as an additional target for viral and host factors binding to the loosely associated dimer of the N-terminal module of the E2 protein.
Proceedings of the National Academy of Sciences 01/2001; 97(26):14289-94. · 9.81 Impact Factor