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991.
Alicia G. Serrano 《生物化学与生物物理学报:生物膜》2006,1758(10):1621-1632
SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid-protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-BΔC, a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-BΔC has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-BΔC indicated that the protein is properly folded, showing around 32% α-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-BΔC that were indicative of lipid-protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air-liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-BΔC in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes. 相似文献
992.
Kyoungwon Cho Nilka Lineth Torres Subhashree Subramanyam Saligrama A Deepak Nagesh Sardesai Oksoo Han Christie E. Williams Hideo Ishii Hitoshi Iwahashi Randeep Rakwal 《Journal of Plant Biology》2006,49(6):413-420
Sample preparation in plant proteomics is tedious, requiring modifications depending on the type of tissue involved. Here,
we describe a protein extraction protocol for both monocotyledonous (monocot) and dicotyledonous (dicot) species, which significantly
improves the solubilization of total proteins. For example, we used the primary leaf tissue and seeds from rice, a cereal
crop and genome model system. Total protein was first precipitated with trichloroacetic acid/acetone extraction buffer (TCAAEB)
and subsequently solubilized with a modified O’Farrell lysis buffer (LB) containing thiourea and tris (LB-TT). Separation
of total leaf proteins by two-dimensional gel electrophoresis (2-DGE) revealed improved solubilization, as determined by an
increased number of spots detected with Coomassie brilliant blue (CBB) staining. In addition, the resolution was better than
when LB-TT was used alone for protein extraction. Seed proteins could be extracted in LB-TT itself without the need for TCAAEB,
which resulted in a highly insoluble precipitate. Our CBB-stained 2-D gel protein profiles also demonstrated the efficacy
of this protocol for total protein extraction/solubilization from the dicot genome model (Arabidopsis), a dicot disease model (cucumber), and two other important monocot cereal crop models (maize and wheat). Moreover, this
is the first report on generating a 2-D gel proteome profile for wheat crown and cucumber leaf tissues. Finally, as examples
of proteome reference maps, we obtained silver nitrate-stained, large-format 2-D gels for rice leaf and wheat crown LB-TT
solubilized proteins. 相似文献
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995.
The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors 总被引:1,自引:0,他引:1
Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR?, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease. 相似文献
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998.
Priya Ramaswamy 《Journal of molecular biology》2009,392(3):666-842
Ribosomal proteins stabilize the folded structure of the ribosomal RNA and enable the recruitment of further proteins to the complex. Quantitative hydroxyl radical footprinting was used to measure the extent to which three different primary assembly proteins, S4, S17, and S20, stabilize the three-dimensional structure of the Escherichia coli 16S 5′ domain. The stability of the complexes was perturbed by varying the concentration of MgCl2. Each protein influences the stability of the ribosomal RNA tertiary interactions beyond its immediate binding site. S4 and S17 stabilize the entire 5′ domain, while S20 has a more local effect. Multistage folding of individual helices within the 5′ domain shows that each protein stabilizes a different ensemble of structural intermediates that include nonnative interactions at low Mg2+ concentration. We propose that the combined interactions of S4, S17, and S20 with different helical junctions bias the free-energy landscape toward a few RNA conformations that are competent to add the secondary assembly protein S16 in the next step of assembly. 相似文献
999.
High grade gliomas are the most common brain tumors in adults and their malignant nature makes them the fourth biggest cause of cancer death. Major efforts in neuro-oncology research are needed to reach similar progress in treatment efficacy as that achieved for other cancers in recent years. In addition to the urgent need to identify novel effective drug targets against malignant gliomas, the search for glioma biomarkers and grade specific protein signatures will provide a much needed contribution to diagnosis, prognosis, treatment decision and assessment of treatment response. Over the past years glioma proteomics has been attempted at different levels, including proteome analysis of patient biopsies and bodily fluids, of glioma cell lines and animal models. Here we provide an extensive review of the outcome of these studies in terms of protein identifications (protein numbers and regulated proteins), with an emphasis on the methods used and the limitations of the studies with regard to biomarker discovery. This is followed by a perspective on novel technologies and on the potential future contribution of proteomics in a broad sense to understanding glioma biology. 相似文献
1000.