Specific and nonspecific collapse in protein folding funnels

Experiments with fast folding proteins are beginning to address the relationship between collapse and folding. We investigate how different scenarios for folding can arise depending on whether the folding and collapse transitions are concurrent or whether a nonspecific collapse precedes folding. Many earlier studies have focused on the limit in which collapse is fast compared to the folding time; in this work we focus on the opposite limit where, at the folding temperature, collapse and folding occur simultaneously. Real proteins exist in both of these limits. The folding mechanism varies substantially in these two regimes. In the regime of concurrent folding and collapse, nonspecific collapse now occurs at a temperature below the folding temperature (but slightly above the glass transition temperature).

Analyzing the effect of homogeneous frustration in protein folding

The energy landscape theory has been an invaluable theoretical framework in the understanding of biological processes such as protein folding, oligomerization, and functional transitions. According to the theory, the energy landscape of protein folding is funneled toward the native state, a conformational state that is consistent with the principle of minimal frustration. It has been accepted that real proteins are selected through natural evolution, satisfying the minimum frustration criterion. However, there is evidence that a low degree of frustration accelerates folding. We examined the interplay between topological and energetic protein frustration. We employed a Cα structure-based model for simulations with a controlled nonspecific energetic frustration added to the potential energy function. Thermodynamics and kinetics of a group of 19 proteins are completely characterized as a function of increasing level of energetic frustration. We observed two well-separated groups of proteins: one group where a little frustration enhances folding rates to an optimal value and another where any energetic frustration slows down folding. Protein energetic frustration regimes and their mechanisms are explained by the role of non-native contact interactions in different folding scenarios. These findings strongly correlate with the protein free-energy folding barrier and the absolute contact order parameters. These computational results are corroborated by principal component analysis and partial least square techniques. One simple theoretical model is proposed as a useful tool for experimentalists to predict the limits of improvements in real proteins. © 2013 Wiley Periodicals, Inc.

Identification of folding intermediates of streblin, the most stable serine protease: biophysical analysis

Streblin, a serine proteinase from plant Streblus asper, has been used to investigate the conformational changes induced by pH, temperature, and chaotropes. The near/far UV circular dichroism activities under fluorescence emission spectroscopy and 8-aniline-1-naphthalene sulfonate (ANS) binding have been carried out to understand the unfolding of the protein in the presence of denaturants. Spectroscopic studies reveal that streblin belongs to the alpha+beta class of proteins and exhibits stability towards chemical denaturants, guanidine hydrochloride (GuHCl). The pH-induced transition of this protein is noncooperative for transition phases between pH 0.5 and 2.5 (midpoint, 1.5) and pH 2.5 and 10.0 (midpoint, 6.5). At pH 1.0 or lower, the protein unfolds to form acid-unfolded state, and for pH 7.5 and above, protein turns into an alkaline denatured state characterized by the absence of ANS binding. At pH 2.0 (1M GuHCl), streblin exists in a partially unfolded state with characteristics of amolten globule state. The protein is found to exhibit strong and predominant ANS binding. In total, six different intermediate states has been identified to show protein folding pathways.

Visualization of Protein Folding Funnels in Lattice Models

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ab initio protein folding simulations using atomic burials as informational intermediates between sequence and structure

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Propriedades mecânicas do cimento ósseo e da poliuretana de mamona com e sem catalisador

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of low-level laser therapy on wound healing in nicotine-treated animals

Low-level laser therapy (LLLT) has been shown to have several biological effects that favor the healing process, and nicotine has been shown to delay the healing process. In this study we investigated the healing of open wounds created on the back of rats treated with nicotine with or without LLLT. of 115 animals, 59 received subcutaneous injections of saline solution, and the others received subcutaneous injections of nicotine (3 mg/kg body weight), twice a day throughout the study period. After 30 days, skin wounds were created on the back of the animals. The animals receiving saline injections were divided into two groups: group 1 (G1, n = 29), in which the wounds were left untreated, and group 2 (G2, n = 30), in which the wounds were treated with LLLT (GaAlAs, 660 nm, 30 mW, 5.57 J/cm(2) per point, 0.39 J, 13 s per point, 0.42 W/cm(2)). The animals receiving nicotine injections were also divided into two groups: group 3 (G3, n = 29), in which the wounds were left untreated, and group 4 (G4, n = 27), in which the wounds were treated with LLLT. The animals were killed 3, 7 or 14 days after surgery. Wound healing was evaluated histologically both qualitatively and semiquantitatively. Wounds of G2 showed a delay in epithelial migration and connective tissue organization compared to those of G1. Wounds of G2 showed faster healing than those of G1; similarly, wounds of G4 showed more advanced healing than those of G3. LLLT acted as a biostimulatory coadjuvant agent balancing the undesirable effects of nicotine on wound tissue healing.

Effect of photodynamic therapy on the healing of cutaneous third-degree-burn: histological study in rats

The aim of this study was to conduct a histological assessment of the effect of photodynamic therapy (PDT) on the repairing of third-degree-burn wounds made on the backs of rats with a heated scalpel. Ninety-six rats were divided into groups: G1, control (n = 24), cold scalpel; G2, burned, heated scalpel (n = 24); G3, low-level laser therapy (LLLT) (n = 24), on burns; and G4, photodynamic therapy (PDT) (n = 24), toluidine-O blue (100 A mu g/ml) and LLLT treatment on burns. The laser (685 nm) was applied in continuous mode, 50 mW, 4.5 J/cm(2), contact mode at nine points (9 s/point). Eight animals in each group were killed at 3 days, 7 days or 14 days after surgery, and tissue specimens containing the whole wounded area were removed and processed for histological analysis; the results were statistically analyzed with Kruskal-Wallis and Dunn's tests (P < 0.05). The results demonstrated significant differences between G2 and G3, and between G2 and G4, at both 3 days and 7 days, with regard to acute inflammation scores; G1 and G2 showed significant differences when compared with G4 at 3 days, with regard to neo-angiogenesis scores; G1 and G2 were statistically different from G3 and G4 at both 3 days and 7 days, with regard to re-epithelization scores; G2 showed statistically significant differences when compared with G3 and G4 with regard to collagen fiber scores at 7 days. LLLT and PDT acted as a biostimulating coadjuvant agent, balancing the undesirable effect of the burn on the wound healing process, acting mainly in the early healing stages, hastening inflammation and increasing collagen deposition.

Structural alterations in the seminiferous tubules of rats treated with immunosuppressor tacrolimus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular characterization and phylogenetic analysis of JussuMP-I: A RGD-P-III class hemorrhagic metalloprotease from Bothrops jararacussu snake venom

Snake venom metalloproteases (SVMPs) embody zinc-dependent multidomain enzymes responsible for a relevant pathophysiology in envenomation. including local and systemic hemorrhage. The molecular features responsible for hemorrhagic potency of SVMPs have been associated with their multidomains structures which can target these proteins them to several receptors of different tissues and cellular types. BjussuMP-I. a SVMP isolated from the Bothrops jararacussu venom, has been characterized as a P-III hemorrhagic metalloprotease. The complete cDNA sequence of BjussuMP-I with 1641bp encodes open reading frames of 547 amino acid residues, which conserve the common domains of P-III high molecular weight hemorrhagic metalloproteases: (i) pre-pro-peptide, (ii) metalloprotease, (iii) disintegrin-like and (iv) rich cysteine domain. BjussuMP-I induced lyses in fibrin clots and inhibited collagen- and ADP-induced platelet aggregation. We are reporting, for the first time, the primary structure of an RGD-P-III class snake venom metalloprotease. A phylogenetic analysis of the BjussuMP-1 metalloprotease/catalytic domain was performed to get new insights into the molecular evolution of the metalloproteases. A theoretical molecular model of this domain was built through folding recognition (threading) techniques and refined by molecular dynamics simulation. Then, the final BjussuMP-I catalytic domain model was compared to other SVMPs and Reprolysin family proteins in order to identify eventual structural differences, which could help to understand the biochemical activities of these enzymes. The presence of large hydrophobic areas and some conserved surface charge-positive residues were identified as important features of the SVMPs and other metalloproteases. (C) 2006 Elsevier B.V. All rights reserved.

The 5S rDNA family evolves through concerted and birth-and-death evolution in fish genomes: an example from freshwater stingrays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallographic structure of PNP from Mycobacterium tuberculosis at 1.9 angstrom resolution

Even being a bacterial purine nucleoside phosphorylase (PNP), which normally shows hexameric folding, the Mycobacterium tuberculosis PNP (MtPNP) resembles the mammalian trimeric structure. The crystal structure of the MtPNP apoenzyme was solved at 1.9 Angstrom resolution. The present work describes the first structure of MtPNP in complex with phosphate. In order to develop new insights into the rational drug design, conformational changes were profoundly analyzed and discussed. Comparisons over the binding sites were specially studied to improve the discussion about the selectivity of potential new drugs. (C) 2004 Elsevier B.V. All rights reserved.

Hydrogen/deuterium exchange mass spectrometry for characterizing phosphoenolpyruvate-induced structural transitions in Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.1)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)