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51.
Jozef Hritz Tilman Läppchen Chris Oostenbrink 《European biophysics journal : EBJ》2010,39(12):1573-1580
The FtsZ protein is a self-polymerizing GTPase that plays a central role in bacterial cell division. Several C8-substituted
GTP analogs are known to inhibit the polymerization of FtsZ by competing for the same binding site as its endogenous activating
ligand GTP. Free energy calculations of the relative binding affinities to FtsZ for a set of five C8-substituted GTP analogs
were performed. The calculated values agree well with the available experimental data, and the main contribution to the free
energy differences is determined to be the conformational restriction of the ligands. The dihedral angle distributions around
the glycosidic bond of these compounds in water are known to vary considerably depending on the physicochemical properties
of the substituent at C8. However, within the FtsZ protein, this substitution has a negligible influence on the dihedral angle
distributions, which fall within the narrow range of −140° to −90° for all investigated compounds. The corresponding ensemble
average of the coupling constants 3
J(C4,H1′) is calculated to be 2.95 ± 0.1 Hz. The contribution of the conformational selection of the GTP analogs upon binding
was quantified from the corresponding populations. The obtained restraining free energy values follow the same trend as the
relative binding affinities to FtsZ, indicating their dominant contribution. 相似文献
52.
Rafael Torres Martin de Rosales Marina Faiella Erik Farquhar Lawrence Que Jr Concetta Andreozzi Vincenzo Pavone Ornella Maglio Flavia Nastri Angela Lombardi 《Journal of biological inorganic chemistry》2010,15(5):717-728
The design, synthesis, and metal-binding properties of DF3, a new de novo designed di-iron protein model are described (“DF”
represents due ferri, Italian for “two iron,” “di-iron”). DF3 is the latest member of the DF family of synthetic proteins. They consist of helix–loop–helix
hairpins, designed to dimerize and form an antiparallel four-helix bundle that encompasses a metal-binding site similar to
those of non-heme carboxylate-bridged di-iron proteins. Unlike previous DF proteins, DF3 is highly soluble in water (up to
3 mM) and forms stable complexes with several metal ions (Zn, Co, and Mn), with the desired secondary structure and the expected
stoichiometry of two ions per protein. UV–vis studies of Co(II) and Fe(III) complexes confirm a metal-binding environment
similar to previous di-Co(II)- and di-Fe(III)-DF proteins, including the presence of a μ-oxo-di-Fe(III) unit. Interestingly,
UV–vis, EPR, and resonance Raman studies suggest the interaction of a tyrosine adjacent to the di-Fe(III) center. The design
of DF3 was aimed at increasing the accessibility of small molecules to the active site of the four-helix bundle. Indeed, binding
of azide to the di-Fe(III) site demonstrates a more accessible metal site compared with previous DFs. In fact, fitting of
the binding curve to the Hill equation allows us to quantify a 150% accessibility enhancement, with respect to DF2. All these
results represent a significant step towards the development of a functional synthetic DF metalloprotein. 相似文献
53.
Gauthamadasa K Rosales C Pownall HJ Macha S Jerome WG Huang R Silva RA 《Biochemistry》2010,49(50):10656-10665
It is expected that the attendant structural heterogeneity of human high-density lipoprotein (HDL) complexes is a determinant of its varied metabolic functions. To determine the structural heterogeneity of HDL, we determined major apolipoprotein stoichiometry profiles in human HDL. First, HDL was separated into two main populations, with and without apolipoprotein (apo) A-II, LpA-I and LpA-I/A-II, respectively. Each main population was further separated into six individual subfractions using size exclusion chromatography (SEC). Protein proximity profiles (PPPs) of major apolipoproteins in each individual subfraction was determined by optimally cross-linking apolipoproteins within individual particles with bis(sulfosuccinimidyl) suberate (BS(3)), a bifunctional cross-linker, followed by molecular mass determination by MALDI-MS. The PPPs of LpA-I subfractions indicated that the number of apoA-I molecules increased from two to three to four with an increase in the LpA-I particle size. On the other hand, the entire population of LpA-I/A-II demonstrated the presence of only two proximal apoA-I molecules per particle, while the number of apoA-II molecules varied from one dimeric apoA-II to two and then to three. For most of the PPPs described above, an additional population that contained a single molecule of apoC-III in addition to apoA-I and/or apoA-II was detected. Upon composition analyses of individual subpopulations, LpA-I/A-II exhibited comparable proportions for total protein (~58%), phospholipids (~21%), total cholesterol (~16%), triglycerides (~5%), and free cholesterol (~4%) across subfractions. LpA-I components, on the other hand, showed significant variability. This novel information about HDL subfractions will form a basis for an improved understanding of particle-specific functions of HDL. 相似文献
54.
55.
von Stetten D Seibeck S Michael N Scheerer P Mroginski MA Murgida DH Krauss N Heyn MP Hildebrandt P Borucki B Lamparter T 《The Journal of biological chemistry》2007,282(3):2116-2123
The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photo-induced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc --> Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state. 相似文献
56.
Blue light induces radical formation and autophosphorylation in the light-sensitive domain of Chlamydomonas cryptochrome 总被引:1,自引:0,他引:1
Immeln D Schlesinger R Heberle J Kottke T 《The Journal of biological chemistry》2007,282(30):21720-21728
Cryptochromes are sensory blue light receptors mediating various responses in plants and animals. Studies on the mechanism of plant cryptochromes have been focused on the flowering plant Arabidopsis. In the genome of the unicellular green alga Chlamydomonas reinhardtii, a single plant cryptochrome, Chlamydomonas photolyase homologue 1 (CPH1), has been identified. The N-terminal 500 amino acids comprise the light-sensitive domain of CPH1 linked to a C-terminal extension of similar size. We have expressed the light-sensitive domain heterologously in Escherichia coli in high yield and purity. The 59-kDa protein bears exclusively flavin adenine dinucleotide in its oxidized state. Illumination with blue light induces formation of a neutral flavin radical with absorption maxima at 540 and 580 nm. The reaction proceeds aerobically even in the absence of an exogenous electron donor, which suggests that it reflects a physiological response. The process is completely reversible in the dark and exhibits a decay time constant of 200 s in the presence of oxygen. Binding of ATP strongly stabilizes the radical state after illumination and impedes the dark recovery. Thus, ATP binding has functional significance for plant cryptochromes and does not merely result from structural homology to DNA photolyase. The light-sensitive domain responds to illumination by an increase in phosphorylation. The autophosphorylation takes place although the protein is lacking its native C-terminal extension. This finding indicates that the extension is dispensable for autophosphorylation, despite the role it has been assigned in mediating signal transduction in Arabidopsis. 相似文献
57.
Padavattan S Schirmer T Schmidt M Akdis C Valenta R Mittermann I Soldatova L Slater J Mueller U Markovic-Housley Z 《Journal of molecular biology》2007,368(3):742-752
The major allergens of honeybee venom, hyaluronidase (Hyal) and phospholipase A2, can induce life-threatening IgE-mediated allergic reactions in humans. Although conventional immunotherapy is effective, up to 40% of patients develop allergic side effects including anaphylaxis and thus, there is a need for an improved immunotherapy. A murine monoclonal anti-Hyal IgG1 antibody (mAb 21E11), that competed for Hyal binding with IgEs from sera of bee venom allergic patients, was raised. The fragment of these IgG antibodies which bind to antigen (Fab) was produced and complexed (1:1) with Hyal. The crystal structure determination of Hyal/Fab 21E11 complex (2.6 A) enabled the identification of the Hyal-IgG interface which provides indirect information on the Hyal-IgE interaction (B-cell epitope). The epitope is composed of a linear array of nine residues (Arg138, His141-Arg148) located at the tip of a helix-turn-helix motive which protrudes away from the globular core and fits tightly into the deep surface pocket formed by the residues from the six complementarity determining regions (CDRs) of the Fab. The epitope is continuous and yet its conformation appears to be essential for Ab recognition, since the synthetic 15-mer peptide comprising the entire epitope (Arg138-Glu152) is neither recognized by mAb 21E11 nor by human IgEs. The structure of the complex provides the basis for the rational design of Hyal derivatives with reduced allergenic activity, which could be used in the development of safer allergen-specific immunotherapy. 相似文献
58.
The formation of oxidized proteins is one of the highlights of oxidative stress. In order not to accumulate such proteins have to be degraded. The major proteolytic system responsible for the removal of oxidized proteins is the proteasome. The proteasome is distributed throughout the cytosolic and nuclear compartment of mammalian cells, with high concentrations in the nucleus. On the other hand a major part of protein oxidation is taking place in the cytosol. The present review highlights the current knowledge on the intracellular distribution of oxidized proteins and put it into contrast with the concentration and distribution of the proteasome. 相似文献
59.
4-Hydroxynonenal (HNE) is a lipid peroxidation product that is able to modify proteins. HNE-modified proteins are degraded to a considerable extend by the proteasomal system. It is unclear whether the recognition of HNE-modified proteins is mediated by ubiquitin, or whether the ubiquitin-independent proteasomal pathway is involved. In this study we demonstrate that HNE-modified GAPDH is preferentially ubiquitinated in vitro. In an attempt to demonstrate the formation of poly-ubiquitinated HNE-modified proteins in living cells we explored E36 fibroblasts. A clear rise in HNE-protein modification could be demonstrated after HNE treatment of the cells. Using inhibitors, we could show that the ubiquitin-dependent, ubiquitin-independent, and the lysosomal pathways affect the presence of HNE-modified proteins. We conclude that, although several proteolytic pathways exist for the degradation of HNE-modified proteins, there is the possibility of involvement of ubiquitin-dependent degradation. 相似文献
60.
Uwe John Urban Tillmann Jennifer Hülsk?tter Tilman J. Alpermann Sylke Wohlrab Dedmer B. Van de Waal 《Proceedings. Biological sciences / The Royal Society》2015,282(1798)
Dinoflagellates are a major cause of harmful algal blooms (HABs), with consequences for coastal marine ecosystem functioning and services. Alexandrium fundyense (previously Alexandrium tamarense) is one of the most abundant and widespread toxigenic species in the temperate Northern and Southern Hemisphere and produces paralytic shellfish poisoning toxins as well as lytic allelochemical substances. These bioactive compounds may support the success of A. fundyense and its ability to form blooms. Here we investigate the impact of grazing on monoclonal and mixed set-ups of highly (Alex2) and moderately (Alex4) allelochemically active A. fundyense strains and a non-allelochemically active conspecific (Alex5) by the heterotrophic dinoflagellate Polykrikos kofoidii. While Alex4 and particularly Alex5 were strongly grazed by P. kofoidii when offered alone, both strains grew well in the mixed assemblages (Alex4 + Alex5 and Alex2 + Alex5). Hence, the allelochemical active strains facilitated growth of the non-active strain by protecting the population as a whole against grazing. Based on our results, we argue that facilitation among clonal lineages within a species may partly explain the high genotypic and phenotypic diversity of Alexandrium populations. Populations of Alexandrium may comprise multiple cooperative traits that act in concert with intraspecific facilitation, and hence promote the success of this notorious HAB species. 相似文献