Studies on the structure of yeast phosphofructokinase

In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution.     

Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.     

A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme.

Nucleoside diphosphate kinase of Drosophila, recently identified as the product of the awd gene, is essential for larval development. The conditional lethal mutation Killer of prune maps to the same gene. We purified the nucleoside diphosphate kinases from wild-type and mutant larvae by a simple procedure involving affinity chromatography on blue Sepharose. Both proteins are purified as hexamers in their native state. The mutant protein, which carries a serine instead of proline at position 97, has structural properties and catalytic efficiency that are very similar to the wild-type protein. However, the mutant protein has a much lower stability to denaturation by heat and urea. Following dilution of urea with buffer the urea-denaturated mutant nucleoside diphosphate kinase accumulates as folded monomers and cannot recover its quaternary structure and enzymatic activity. In contrast, the wild-type enzyme recovers hexameric structure and activity. This suggests that the mutation affects the folding/assembly pathway without affecting the function of the mature protein once folded and assembled into the mature hexameric structure.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Assistance of maltose binding protein to the in vivo folding of the disulfide-rich C-terminal fragment from Plasmodium falciparum merozoite surface protein 1 expressed in Escherichia coli

The C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (F19) is a leading candidate for the development of a malaria vaccine. Successful vaccination trials on primates, immunochemistry, and structural studies have shown the importance of its native conformation for its protective role against infection. F19 is a disulfide-rich protein, and the correct pairing of its 12 half-cystines is required for the native state of the protein. F19 has been produced in the Escherichia coli periplasm, which has an oxidative environment favorable for the formation of disulfide bonds. F19 was either expressed as a fusion with the maltose binding protein (MBP) or directly addressed to the periplasm by fusing it with the MBP signal peptide. Direct expression of F19 in the periplasm led to a misfolded protein with a heterogeneous distribution of disulfide bridges. On the contrary, when produced as a fusion protein with E. coli MBP, the F19 moiety was natively folded. Indeed, after proteolysis of the fusion protein, the resulting F19 possesses the structural characteristics and the immunochemical reactivity of the analogous fragment produced either in baculovirus-infected insect cells or in yeast. These results demonstrate that the positive effect of MBP in assisting the folding of passenger proteins extends to the correct formation of disulfide bridges in vivo. Although proteins or protein fragments fused to MBP have been frequently expressed with success, our comparative study evidences for the first time the helping property of MBP in the oxidative folding of a disulfide-rich protein.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Undistorted structural analysis of soluble proteins by attenuated total reflectance infrared spectroscopy

Water from the solvent very strongly absorbs light in the frequency range of interest for studying protein structure by infrared (IR) spectroscopy. This renders handling of the observation cells painstaking and time consuming, and limits the reproducibility of the measurements when IR spectroscopy is applied to proteins in aqueous solutions. These difficulties are circumvented by the use of an Attenuated Total Reflectance (ATR) accessory. However, when protein solutions are studied, ATR spectroscopy suffers from several drawbacks, the most severe being nonproportionality of the observed absorbance with the protein concentration and spectral distortions that vary from protein to protein and from sample to sample. In this study, we show (1) that the nonproportionality is due to adsorption of the protein on the ATR crystal surface; (2) that the contribution of the crystal-adsorbed protein can easily be taken into account, rendering the corrected absorbance proportional to the protein concentration; (3) that the observed variable base line distortions, likely due to changes in the penetration depth of the light beam in solutions with the refractive index that depends on the protein concentration, can be easily eliminated; and (4) that ATR IR spectra thus corrected for protein adsorption and light penetration can be used to properly analyze the secondary structure of proteins in solution.     

In vitro denaturation-renaturation of fibronectin. Formation of multimers disulfide-linked and shuffling of intramolecular disulfide bonds

It is well established that fibronectin into extracellular matrix undergoes repeated tensions applied by cells, resulting into dramatic structural changes which reflect its elastic properties. However, there is currently no study reporting with precision the consequences of this elasticity on fibronectin structure and conformation. In the present work, we investigated fibronectin structural and conformational reorganization in vitro through a denaturation-renaturation approach. The similarities and differences between "refolded fibronectin" and "native fibronectin" were investigated using various spectroscopic methods, hydrodynamic characterization, molecular imaging and biochemical characterization. In the refolded form, secondary structure elements as well as local tyrosine and tryptophan environment are identical compared to the native form. Interestingly, some differences in global tertiary structure organization and molecular conformation were observed. These differences are due to the reactivity of the two free cysteines, which are buried in the native state but become accessible during the unfolding process. First, oxidation of these residues leading to the formation of intermolecular disulfide bonds results in formation of stabilized multimer. Second, some illegitimate intramolecular disulfide bonds are formed. The presence of iodoacetamide, the sulfhydryl alkylating agent, during the unfolding-refolding process prevents all these events. This study clearly demonstrates that, under near physiological conditions, competitive renaturation pathways occur, involving free cysteines in either multimer formation or intermolecular shuffling of disulfide bonds. These findings might have important implications for future studies and be helpful to develop a deeper understanding of fibronectin morphology.     

Characterization of an acetyltransferase that detoxifies aromatic chemicals in Legionella pneumophila

Legionella pneumophila is an opportunistic pathogen and the causative agent of Legionnaires' disease. Despite being exposed to many chemical compounds in its natural and man-made habitats (natural aquatic biotopes and man-made water systems), L. pneumophila is able to adapt and survive in these environments. The molecular mechanisms by which this bacterium detoxifies these chemicals remain poorly understood. In particular, the expression and functions of XMEs (xenobiotic-metabolizing enzymes) that could contribute to chemical detoxification in L. pneumophila have been poorly documented at the molecular and functional levels. In the present paper we report the identification and biochemical and functional characterization of a unique acetyltransferase that metabolizes aromatic amine chemicals in three characterized clinical strains of L. pneumophila (Paris, Lens and Philadelphia). Strain-specific sequence variations in this enzyme, an atypical member of the arylamine N-acetyltransferase family (EC 2.3.1.5), produce enzymatic variants with different structural and catalytic properties. Functional inactivation and complementation experiments showed that this acetyltransferase allows L. pneumophila to detoxify aromatic amine chemicals and grow in their presence. The present study provides a new enzymatic mechanism by which the opportunistic pathogen L. pneumophila biotransforms and detoxifies toxic aromatic chemicals. These data also emphasize the role of XMEs in the environmental adaptation of certain prokaryotes.