multimer
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Von Willebrandov faktor (vWF) je multimerický proteín, ktorý plní dve kľúčové funkcie v hemostáze. Podieľa sa na interakcii medzi doštičkami a subendotelom v mieste cievneho poškodenia a interakcii medzi doštičkami navzájom. vWF tvorí komplex s faktorom VIII v pomere (1: 1), čím ho chráni pred proteolýzou aktivovaným proteínom C. vWF je proteín, ktorý je zostavený z identických podjednotiek do lineárnych reťazcov rôznej veľkosti označovaných ako multiméry. Tieto multiméry môžu mať hmotnosť > 20 miliónov daltonov a dĺžku > 2 mikrometre. Klinicky významné a najúčinnejšie pri interakcii s kolagénom a trombocytovými receptormi sú multiméry s vysokou molekulovou hmotnosťou (HMW). V článku uvádzame porovnanie dvoch dostupných metód analýzy multimérov von Willebrandovho faktora - klasickú manuálnu metódu a novú semiautomatickú metódu prístroja Hydrasys. Nová metóda využíva vopred pripravený 2% agarózový gél, výsledky sú dostupné do 6 h, metóda je štandardizovaná a reprodukovateľná. Avšak abnormality multimérov sa musia ďalej podrobnejšie skúmať pomocou klasickej - manuálnej metódy s rôznymi koncentráciami agarózových gélov.
Von Willebrand factor (vWF) is a multimeric protein that plays a principal role in two key functions in hemostasis. It participates in the interaction between the platelets and the subendothelium at the site of the vascular damage and if is also important in the interaction between the platelets. vWF is complexed with factor VIII (1: 1) to protect it from proteolysis by activated Protein C. vWF is a protein that is assembled from identical subunits into linear strings of varying size referred to as multimers. These multimers can be > 20 million daltons in mass and > 2 micrometers in length. Clinically significant and most effective in interacting with collagen and thrombocyte receptors are high molecular weight multimers. In our article we present comparing of two available methods of von Willebrand factor multimer analysis - the classical manual method and the new semi-automatic method of the Hydrasys instrument. The new method uses a prepared 2% agarose gel, the results are available within 6 h, the method is standardized and reproducible. However, abnormalities of multimers must be further investigated in more detail using the classical-manual method with different concentrations of agarose gels.
The European Clinical Laboratory and Molecular (ECLM) classification of von Willebrand disease (vWD) is based on the splitting approach which uses sensitive and specific von Willebrand factor (vWF) assays with regard to the updated molecular data on structure and function of vWF gene and protein defects. A complete set of FVIII:C and vWF ristocetine cofactor, collagen binding, and antigen, vWF multimeric analysis in low- and medium-resolution gels, and responses to desmopressin (DDAVP) of FVIII:C and vWF parameters are mandatory. The ECLM classification distinguishes recessive types 1 and 3 vWD from recessive vWD 2C due to mutations in the D1 and D2 domains and vWD 2N due to mutations in the D'-FVIII-binding domain of vWF. The ECLM classification differentiates between mild vWD type 1 with variable penetrance of bleedings from symptomatic dominant type 1 vWD secretion defect and/or clearance defect with normal vWF multimers versus vWD 1M and 2M with normal or smeary vWF multimers in low- and medium-resolution gels. High-quality multimeric analysis of vWF in medium-resolution gels based on a DDAVP challenge test clearly delineates and distinguishes each of the dominant type 2 vWDs 1/2E, 2M, 2B, 2A, and 2D caused by vWF gene mutations in the D3 multimerization domain, loss or gain-of-function mutations in the glycoprotein Ib receptor A1 domain, gene mutations in the A2 proteolytic domain, and the C-terminal dimerization domain, respectively.
- MeSH
- desmopresin farmakologie MeSH
- diferenciální diagnóza * MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- mutace MeSH
- von Willebrandova nemoc, typ 1 diagnóza MeSH
- von Willebrandova nemoc, typ 2 diagnóza MeSH
- von Willebrandův faktor analýza genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.
- MeSH
- alely * MeSH
- HEK293 buňky MeSH
- heterozygot MeSH
- histonlysin-N-methyltransferasa genetika MeSH
- histony genetika MeSH
- kompenzace dávky (genetika) MeSH
- lidé MeSH
- lokus kvantitativního znaku MeSH
- myši knockoutované MeSH
- myši MeSH
- poškození DNA MeSH
- rekombinace genetická * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
High pressure methods have become a useful tool for studying protein structure and stability. Using them, various physico-chemical processes including protein unfolding, aggregation, oligomer dissociation or enzyme-activity decrease were studied on many different proteins. Oligomeric protein dissociation is a process that can perfectly utilize the potential of high-pressure techniques, as the high pressure shifts the equilibria to higher concentrations making them better observable by spectroscopic methods. This can be especially useful when the oligomeric form is highly stable at atmospheric pressure. These applications may be, however, hindered by less intensive experimental response as well as interference of the oligomerization equilibria with unfolding or aggregation of the subunits, but also by more complex theoretical description. In this study we develop mathematical models describing different kinds of oligomerization equilibria, both closed (equilibrium of monomer and the highest possible oligomer without any intermediates) and consecutive. Closed homooligomer equilibria are discussed for any oligomerization degree, while the more complex heterooligomer equilibria and the consecutive equilibria in both homo- and heterooligomers are taken into account only for dimers and trimers. In all the cases, fractions of all the relevant forms are evaluated as functions of pressure and concentration. Significant points (inflection points and extremes) of the resulting transition curves, that can be determined experimentally, are evaluated as functions of pressure and/or concentration. These functions can be further used in order to evaluate the thermodynamic parameters of the system, i.e. atmospheric-pressure equilibrium constants and volume changes of the individual steps of the oligomer-dissociation processes.
Sedimentation equilibrium and size-exclusion chromatography experiments on Mycobacterium tuberculosis hypoxanthine-guanine phosphoribosyltransferase (MtHGPRT) have established the existence of this enzyme as a reversibly associating mixture of dimeric and tetrameric species in 0.1 M Tris-HCl-0.012 M MgCl2, pH 7.4. Displacement of the equilibrium position towards the larger oligomer by phosphate signifies the probable existence of MtHGPRT as a tetramer in the biological environment. These data thus add credibility to the relevance of considering enzyme function in the light of a published tetrameric structure deduced from X-ray crystallography. Failure of 5-phospho-α-d-ribosyl-1-pyrophosphate (PRib-PP) to perturb the dimer-tetramer equilibrium position indicates the equivalence and independence of binding for this substrate (the first to bind in an ordered sequential mechanism) to the two oligomers. By virtue of the displacement of the equilibrium position towards dimer that is affected by removing MgCl2 from the Tris-HCl buffer, it can be concluded that divalent metal ions, as well as phosphate, can affect the oligomerization. These characteristics of MtHGPRT in solution are correlated with published crystal structures of four enzyme-ligand complexes.
The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots.
- MeSH
- Escherichia coli * MeSH
- flavinmononukleotid metabolismus MeSH
- kinetika MeSH
- kvarterní struktura proteinů MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- podjednotky proteinů chemie metabolismus MeSH
- proteiny z Escherichia coli chemie metabolismus MeSH
- represorové proteiny chemie metabolismus MeSH
- roztoky MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used to study the oligomeric states of soluble proteins. A mild binding of the used protein stains to nondissociated protein oligomers imposed a charge shift on the proteins resulting into separation of protein species according to their size under physiological conditions. Adsorbed stains could be easily removed after electrophoresis by washing of polyacrylamide gel with buffer and protein complexes could be visualized either by the detection of their enzyme activity or by using a nonspecific protein stain. The specific detection of enzyme activity of glycosidases, lactate dehydrogenase, or phosphatases was shown as an example.
Dimerization of many eukaryotic transcription regulatory factors is critical for their function. Regulatory role of an epigenetic reader lens epithelium-derived growth factor/p75 (LEDGF/p75) requires at least two copies of this protein to overcome the nucleosome-induced barrier to transcription elongation. Moreover, various LEDGF/p75 binding partners are enriched for dimeric features, further underscoring the functional regulatory role of LEDGF/p75 dimerization. Here, we dissected the minimal dimerization region in the C-terminal part of LEDGF/p75 and, using paramagnetic NMR spectroscopy, identified the key molecular contacts that helped to refine the solution structure of the dimer. The LEDGF/p75 dimeric assembly is stabilized by domain swapping within the integrase binding domain and additional electrostatic "stapling" of the negatively charged α helix formed in the intrinsically disordered C-terminal region. We validated the dimerization mechanism using structure-inspired dimerization defective LEDGF/p75 variants and chemical crosslinking coupled to mass spectrometry. We also show how dimerization might affect the LEDGF/p75 interactome.
In the major peripheral plant light-harvesting complex LHCII, excitation energy is transferred between chlorophylls along an energetic cascade before it is transmitted further into the photosynthetic assembly to be converted into chemical energy. The efficiency of these energy transfer processes involves a complicated interplay of pigment-protein structural reorganization and protein dynamic disorder, and the system must stay robust within the fluctuating protein environment. The final, lowest energy site has been proposed to exist within a trimeric excitonically coupled chlorophyll (Chl) cluster, comprising Chls a610-a611-a612. We studied an LHCII monomer with a site-specific mutation resulting in the loss of Chls a611and a612, and find that this mutant exhibits two predominant overlapping fluorescence bands. From a combination of bulk measurements, single-molecule fluorescence characterization, and modeling, we propose the two fluorescence bands originate from differing conditions of exciton delocalization and localization realized in the mutant. Disruption of the excitonically coupled terminal emitter Chl trimer results in an increased sensitivity of the excited state energy landscape to the disorder induced by the protein conformations. Consequently, the mutant demonstrates a loss of energy transfer efficiency. On the contrary, in the wild-type complex, the strong resonance coupling and correspondingly high degree of excitation delocalization within the Chls a610-a611-a612 cluster dampens the influence of the environment and ensures optimal communication with neighboring pigments. These results indicate that the terminal emitter trimer is thus an essential design principle for maintaining the efficient light-harvesting function of LHCII in the presence of protein disorder.
