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The binding of human alpha1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pKa' of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with alpha1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with alpha1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a "conformational trap" of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.

Circular dichroism (CD) spectroscopy is a well known technique for the study of the secondary structure of proteins. Although the presently available algorithms reliably estimate the secondary structure of the α-helical proteins, the prediction of β-sheet structure is much less accurate. In the case of proteins with unique β-structures, such as membrane proteins and amyloid fibrils, the secondary structure prediction results are unacceptable. The problem arises from the diversity of β-structures, which results large spectral differences even between proteins with similar overall β-sheet content. We have developed a new algorithm for the accurate estimation of secondary structure contents for a broader range of protein folds with special interest to β-structure rich proteins including amyloid fibrils. The reference spectrum database was expanded with high quality spectra of globular proteins and amyloid fibrils recorded down to 175 nm using synchrotron radiation CD. Our algorithm takes into account the diverse twist of the β-sheets that has a great influence on the spectral features. For the first time, we can reliably distinguish parallel and antiparallel β-structure using CD spectroscopy. Moreover, it performs better than any other algorithms for any secondary structure types. With its improved accuracy and more detailed structural information, the method is capable of predicting the protein fold to the level of topology in the CATH classification with good reliability. A web server was constructed to make the algorithm available for the protein science research community.

A boiled extract of porcine cerebral cortex was fractionated on Sephadex G-75 and LH-20 followed by paper chromatography of the active fraction having inhibitory activity towards [3H]GABA binding to rat brain synaptic membranes. A ninhydrin-negative substance migrating more quickly than authentic GABA was identified as a copper-GABA complex. The complex inhibited specific [3H]GABA binding (IC50 approximately equal to 1 microM) and antagonized the anticonvulsant effect exerted by intraamygdaloid injection of diazepam. The effect of a synthetic copper-GABA complex was compared and found to be similar to the endogenous complex. Cu2+ alone has no affinity for the GABA recognition site but antagonizes the anticonvulsant effect of diazepam. Therefore, Cu2+ is suggested to be the pharmacologically active principle of the endogenous substance. Cu2+ does not seem to function via the recognition site of the GABA receptor.

Zusammenfassung Fur die quantitative Kennzeichnung des StrukturefTektes konnen die charakteristischen Parameter der Verdampfungsprozesse verwendet werden. In Matrizes chemisch gleicher Zusammensetzung, aber abweichender Kristallstruktur kann das Mass des Struktureffektes aufgrund der relativen Verdampfungsgeschwindigkeit der Komponenten bestimmt werden. Mit dieser Behandlungsweise wird der Einfluss der Zugabematerialien auf den Struktureffekt quantitativ bewertet. Als Modellmaterial der Versuche wurde eine uberwiegend aus Silikaten bestehende Tonerde sowie eine synthetische Mischung aus Oxiden und Karbonaten gewahlt. Fur die Anregung wurde ein Gleichstrombogen benutzt. LiF, danach Li 2 B 4 O 7 und Li 2 B 2 O 4 bewies sich als geeigneteste Zugabe fur die Unterdruckung des Struktureffektes, Li 2 CO 3 hingegen erhohte diese Wirkung im Vergleich zu dem Fall ohne Zugabe.

Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes. We carried out stepwise bilateral common carotid occlusions on rats and investigated long-term mitochondrial, mitochondria-associated membrane (MAM), and cerebrospinal fluid (CSF) proteome changes. Samples were studied by gel-based and mass spectrometry-based proteomic analyses. We found 19, 35, and 12 significantly altered proteins in the mitochondria, MAM, and CSF, respectively. Most of the changed proteins were involved in protein turnover and import in all three sample types. We confirmed decreased levels of proteins involved in protein folding and amino acid catabolism, such as P4hb and Hibadh in the mitochondria by western blot. We detected reduced levels of several components of protein synthesis and degradation in the CSF as well as in the subcellular fractions, implying that hypoperfusion-induced altered protein turnover of brain tissue can be detected in the CSF by proteomic analysis.

A judicious examination of an exhaustive PDB sample of soluble globular proteins of moderate size (N

Replacement of H2O by D2O resulted in significantly higher amount of [3H]diazepam specifically bound to synaptic membranes. The isotope effect arises from increased number of binding sites in D2O and is associated with a stronger solvation of membrane receptors by heavy water.

Synthese de derives du compose du titre et d'analogues derives de benzo [a] quinolizine; etude biologique

Fractions containing non-junctional membranes (fraction 1) could be separated from those enriched in junctional complexes (fraction 2) by sucrose gradient centrifugation of osmotically disrupted rat cerebral cortex synaptosomes. [3H]flunitrazepam binding sites were found to be homogeneous characterized by Kd = 0.65 nM, Bmax = 13 pM for fraction 1; and Kd = 2 nM, Bmax = 45 pM for fraction 2. GABA-dependent benzodiazepine receptor sites were found to be localized to the junctional complex fraction, while non-junctional membranes showed GABA-independent receptor and acceptor binding sites.

Lipid-protein interactions drive core cellular signaling events and regulate the function of most membrane proteins. Despite their significance, molecular details of these interactions are scarcely known. Here we provide mechanistic insight into the binding of the lipid mediator sphingosylphosphorylcholine to calmodulin, the most central and ubiquitous regulator protein in calcium signaling, and present a crystal structure of their complex. Thermodynamic and kinetic analysis of their interaction revealed a peculiar stoichiometry-dependent binding process. At low protein-to-lipid ratios, calmodulin binds with high affinity to lipid micelles. At high protein-to-lipid ratios, however, the resulting complex adopts a compact globular conformation with calmodulin embracing a few lipid molecules, as can be seen in our crystal structure. Intriguingly, the sphingolipid occupies the binding pocket for the amphipatic alpha-helices of calmodulin's target proteins. This finding explains the competitive inhibition of calmodulin function by the signaling lipid, and proposes an utterly novel type of endogenous regulation for the calcium sensor protein. The binding model presented here might be widely applicable to other lipid-protein interactions as well.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Human Rad51 is a key protein component of homologous recombination, the error free repair process of double strand DNA breaks. It is a 37 kDa protein of two domains. The full structure of the protein is not known in atomic details, X-ray crystallography has been successful only in the case of the larger C-domain in complex with a regulatory protein, BRCA2. The structure of the N-domain has been determined by NMR. It is known that in the repair process, the recombinases form a helical filament around ssDNA overhangs - the presynaptic filament (PSF) - at the double strand breaks, the structure of which is of vital significance in the proceeding of the repair. It is also known that the recombinases possess ATPase activity, and ATP is needed for the successful repair. While in the case of the bacterial-, archeal,- and yeast - homologues of HsRad51, data were presented concerning the parameters of their ATP-dependent PSF structures in crystalline conditions, such results are non-existent for HsRad. In this study our goal was to provide evidence for the purely structural role of ATP in forming the PSF structure by HsRad51. Therefore, the hydrolysis of ATP has been excluded by adding Ca to the solutions instead of Mg. The topology of HsRad structures without and with added ATP/Ca and 75mer ssDNA has been determined by transmission electron microscopy. The formations were labeled by the fluorescent dye ANS, and pressure perturbation fluorescence spectroscopy has been applied to characterize the strength of interaction at protomeric interfaces in the Rad51 filaments. We found that the formation of the ordered filamentous structure clearly requires the presence of ATP/Ca, and that the interface binding strength is the highest in the presynaptic filament of helical structure.

The maintenance and stability of the genetic material of cells is of vital significance. This task is fulfilled by complex mechanisms involving the associates of specific proteins that through several steps recognize and repair DNA damages. In this study we focus our attention on an early step in the process of homologous recombination of human cells that is related to the function of the protein Rad51. The repair action of the protein is initiated by a supposedly unspecific helical filament formation of its monomers around single stranded DNA overhangs at double strand breaks. We study the conditions that regulate the structure and thus the biological activity of this, presynaptic” filament. Now we report results of pressure tuning fluorescence spectroscopy (Schay et al.(2006) JBC, 281, 25972) concerning the strength of interaction at the monomer-monomer interfaces in the filament structures and results of electronmicroscopy concerning their topology and structural parameters. The fluorescence signal is based on the dye ANS that we show being bound near the monomer interfaces. The measurements yield new data concerning the role of ATP, Mg, and Ca as cofactors of the presynaptic filament formation and show the unique effect of bound K-ions significantly influencing both the stability and the topology of the presynaptic as well as of the self-aggregate filaments.Acknowledgements: the authors are grateful to Prof. Kovacs of Eotvos University for help and valuable advices in electron microscopy. The work was supported by the Hungarian grant OTKA K 84271.