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
In analogy with many other proteins, Na(+)/Ca(2+) exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na(+)/Ca(2+) exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na(+)or Ca(2+) binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct "noncatalytic" residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.
- MeSH
- archeální proteiny chemie genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- hmotnostní spektrometrie MeSH
- interakční proteinové domény a motivy MeSH
- iontový transport MeSH
- katalytická doména MeSH
- koncentrace vodíkových iontů MeSH
- Methanocaldococcus chemie genetika metabolismus MeSH
- molekulární modely MeSH
- pumpa pro výměnu sodíku a vápníku chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- sbalování proteinů MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- sodík chemie metabolismus MeSH
- vápník chemie metabolismus MeSH
- vazba proteinů MeSH
- vodík-deuteriová výměna MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
SMC/kleisin complexes form elongated annular structures, which are critical for chromosome segregation, genome maintenance, and the regulation of gene expression. We describe marked structural similarities between bacterial and eukaryotic SMC/kleisin partner proteins (designated here as "kite" proteins for kleisin interacting tandem winged-helix (WH) elements of SMC complexes). Kite proteins are integral parts of all prokaryotic SMC complexes and Smc5/6 but not cohesin and condensin. They are made up of tandem WH domains, form homo- or heterodimers via their amino-terminal WH domain, and they associate with the central part of a kleisin subunit. In placental mammals, the kite subunit NSE3 gave rise to several (>60) kite-related proteins, named MAGE, many of which encode tumor- and testis-specific antigens. Based on architectural rather than sequence similarity, we propose an adapted model for the evolution of the SMC protein complexes and discuss potential functional similarities between bacterial Smc/ScpAB and eukaryotic Smc5/6.
- MeSH
- archeální proteiny chemie genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- kineziny chemie metabolismus MeSH
- konzervovaná sekvence MeSH
- molekulární sekvence - údaje MeSH
- proteiny buněčného cyklu chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
A haloarchaeal strain G41 showing lipolytic activity was isolated from the saline soil of Yuncheng Salt Lake, China. Biochemical and physiological characterizations along with 16S rRNA gene sequence analysis placed the isolate in the genus Haloarcula. Lipase production was strongly influenced by the salinity of growth medium with maximum in the presence of 20% NaCl or 15% Na2SO4. The lipase was purified to homogeneity with a molecular mass of 45 kDa. Substrate specificity test revealed that it preferred long-chain p-nitrophenyl esters. The lipase was highly active and stable over broad ranges of temperature (30-80 °C), pH (6.0-11.0), and NaCl concentration (10-25%), with an optimum at 70 °C, pH 8.0, and 15% NaCl, showing thermostable, alkali-stable, and halostable properties. Enzyme inhibition studies indicated that the lipase was a metalloenzyme, with serine and cysteine residues essential for enzyme function. Moreover, it displayed high stability and activation in the presence of hydrophobic organic solvents with log Pow ≥ 2.73. The free and immobilized lipases from strain G41 were applied for biodiesel production, and 80.5 and 89.2% of yields were achieved, respectively. This study demonstrated the feasibility of using lipases from halophilic archaea for biodiesel production.
- MeSH
- archeální proteiny chemie genetika metabolismus MeSH
- biopaliva analýza MeSH
- fylogeneze MeSH
- Haloarcula klasifikace enzymologie genetika izolace a purifikace MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- lipasa chemie genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- molekulová hmotnost MeSH
- půdní mikrobiologie MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Čína MeSH
