Na(+)/Ca(2+) exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na(+) or Ca(2+) specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.
- MeSH
- apoproteiny chemie genetika metabolismus MeSH
- archeální proteiny chemie genetika metabolismus MeSH
- buněčná membrána chemie MeSH
- cystein chemie MeSH
- interakční proteinové domény a motivy MeSH
- inzerční mutageneze MeSH
- kinetika MeSH
- konformace proteinů MeSH
- ligandy MeSH
- Methanocaldococcus metabolismus MeSH
- molekulární modely * MeSH
- mutace MeSH
- peptidové fragmenty chemie genetika metabolismus MeSH
- pumpa pro výměnu sodíku a vápníku chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- sodík metabolismus MeSH
- stabilita proteinů MeSH
- substituce aminokyselin MeSH
- vápník metabolismus MeSH
- vazebná místa MeSH
- vodík-deuteriová výměna MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ∼5 h and resulting in complete conversion at the active-site methionine with minor extents of conversion at heterogeneous second sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidated the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly.
- MeSH
- apoproteiny chemie izolace a purifikace metabolismus MeSH
- flavinmononukleotid chemie metabolismus MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- kinetika MeSH
- konformace proteinů MeSH
- methionin analogy a deriváty chemie metabolismus MeSH
- oxidace-redukce MeSH
- proteiny z Escherichia coli chemie izolace a purifikace metabolismus MeSH
- represorové proteiny chemie izolace a purifikace metabolismus MeSH
- simulace molekulární dynamiky * MeSH
- stabilita proteinů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Arginine repressor of E. coli is a multifunctional hexameric protein that provides feedback regulation of arginine metabolism upon activation by the negatively cooperative binding of L-arginine. Interpretation of this complex system requires an understanding of the protein's conformational landscape. The ~50 kDa hexameric C-terminal domain was studied by 100 ns molecular dynamics simulations in the presence and absence of the six L-arg ligands that bind at the trimer-trimer interface. A rotational shift between trimers followed by rotational oscillation occurs in the production phase of the simulations only when L-arg is absent. Analysis of the system reveals that the degree of rotation is correlated with the number of hydrogen bonds across the trimer interface. The trajectory presents frames with one or more apparently open binding sites into which one L-arg could be docked successfully in three different instances, indicating that a binding-competent state of the system is occasionally sampled. Simulations of the resulting singly-liganded systems reveal for the first time that the binding of one L-arg results in a holoprotein-like conformational distribution.
- MeSH
- apoproteiny chemie metabolismus MeSH
- arginin chemie metabolismus MeSH
- konformace proteinů MeSH
- ligandy MeSH
- proteiny z Escherichia coli chemie metabolismus MeSH
- represorové proteiny chemie metabolismus MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Díky pokrokům v molekulární genetice jsou dnes známy funkční polymorfismy různých genů, které řídí lipidový metabolizmus, a studují se jejich účinky v interakci s nutričními faktory. Vznikly nové interdisciplinární obory; zatímco nutrigenetika zkoumá genetickou variabilitu ve vztahu k příslušné metabolické odpovědi na složení stravy s cílem vypracovat individuální dietní doporučení, nutrigenomika se zabývá vlivem nutričních faktorů na regulační funkce genů a jejich produktů. V přehledovém článku jsou shrnuty poznatky o běžných polymorfismech genů pro některé apolipoproteiny, transferové proteiny, buněčné receptory a enzymy, se zřetelem na jejich předpokládanou úlohu v patogenezi aterogenních dyslipidémií. Většina referovaných výzkumných prací se týká metabolických důsledků izolovaně studovaných jednonukleotidových polymorfismů, kdežto jen malý počet se pokouší osvětlit složité vzájemné vztahy současně se vyskytujících genových variant.
With advances in molecular genetics, functional polymorphisms of various lipid-related genes have been investigated, to discern their metabolic effects and role in gene-diet interactions. New interdisciplinary concepts have emerged; while nutrigenetics examines genetic variation and associated responses to nutrients, in order to generate individual dietary recommendations, nutrigenomics focuses on the effects of nutrients on regulatory functions of genes and their products. This review summarizes recent knowledge concerning common polymorphisms of candidate genes for some apolipoproteins, transfer proteins, cell receptors, and enzymes, which presumably modulate cardiovascular risk. Most studies investigate isolated single nucleotide polymorphisms and nutrients, while only few try to elucidate complex interrelationships between coexistent multiple gene variants.
- MeSH
- apoproteiny genetika imunologie metabolismus MeSH
- arterioskleróza etiologie genetika metabolismus MeSH
- dyslipidemie etiologie genetika metabolismus MeSH
- financování organizované MeSH
- kardiovaskulární nemoci etiologie genetika metabolismus MeSH
- medicína založená na důkazech trendy MeSH
- nemoci výživy a metabolismu etiologie genetika metabolismus MeSH
- polymorfismus genetický genetika imunologie MeSH
- rizikové faktory MeSH
- transportní proteiny pro estery cholesterolu genetika imunologie metabolismus MeSH
- MeSH
- apoproteiny klasifikace krev metabolismus MeSH
- ateroskleróza etiologie patofyziologie MeSH
- cholesterol krev MeSH
- dyslipidemie diagnóza patofyziologie MeSH
- hyperlipidemie diagnóza patofyziologie MeSH
- klinická chemie * metody MeSH
- lidé MeSH
- lipidózy klasifikace MeSH
- lipoproteiny klasifikace krev metabolismus MeSH
- metabolismus lipidů * fyziologie MeSH
- poruchy metabolismu lipidů * diagnóza patofyziologie MeSH
- triglyceridy krev MeSH
- vrozené poruchy metabolismu tuků diagnóza patofyziologie MeSH
- Check Tag
- lidé MeSH
