Detecting Substrates Bound to the Secondary Multidrug Efflux Pump EmrE by DNP-Enhanced Solid-State NMR

Escherichia coli EmrE, a homodimeric multidrug antiporter, has been suggested to offer a convenient paradigm for secondary transporters due to its small size. It contains four transmembrane helices and forms a functional dimer. We have probed the specific binding of substrates TPP⁺ and MTP⁺ to EmrE reconstituted into 1,2-dimyrist­oyl-<i>sn</i>-glycero-3-phospho­choline liposomes by ³¹P MAS NMR. Our NMR data show that both substrates occupy the same binding pocket but also indicate some degree of heterogeneity of the bound ligand population, reflecting the promiscuous nature of ligand binding by multidrug efflux pumps. Direct interaction between ¹³C-labeled TPP⁺ and key residues within the EmrE dimer has been probed by through-space ¹³C–¹³C correlation spectroscopy. This was made possible by the use of solid-state NMR enhanced by dynamic nuclear polarization (DNP) through which a 19-fold signal enhancement was achieved. Our data provide clear evidence for the long assumed direct interaction between substrates such as TPP⁺ and the essential residue E14 in transmembrane helix 1. Our work also demonstrates the power of DNP-enhanced solid-state NMR at low temperatures for the study for secondary transporters, which are highly challenging for conventional NMR detection

Photocycle and Vectorial Proton Transfer in a Rhodopsin from the Eukaryote <i>Oxyrrhis marina</i>

Retinylidene photoreceptors are ubiquitously present in marine protists as first documented by the identification of green proteorhodopsin (GPR). We present a detailed investigation of a rhodopsin from the protist <i>Oxyrrhis marina</i> (OR1) with respect to its spectroscopic properties and to its vectorial proton transport. Despite its homology to GPR, OR1’s features differ markedly in its pH dependence. Protonation of the proton acceptor starts at pH below 4 and is sensitive to the ionic conditions. The mutation of a conserved histidine H62 did not influence the p<i>K</i>_a value in a similar manner as in other proteorhodopsins where the charged histidine interacts with the proton acceptor forming the so-called His-Asp cluster. Mutational and pH-induced effects were further reflected in the temporal behavior upon light excitation ranging from femtoseconds to seconds. The primary photodynamics exhibits a high sensitivity to the environment of the proton acceptor D100 that are correlated to the different initial states. The mutation of the H62 does not affect photoisomerization at neutral pH. This is in agreement with NMR data indicating the absence of the His-Asp cluster. The subsequent steps in the photocycle revealed protonation reactions at the Schiff base coupled to proton pumping even at low pH. The main electrogenic steps are associated with the reprotonation of the Schiff base and internal proton donor. Hence, OR1 shows a different theme of the His-Asp organization where the low p<i>K</i>_a of the proton acceptor is not dominated by this interaction, but by other electrostatic factors

Unexpected Disproportionation of Tetramethylethylenediamine-Supported Perchlorodisilane Cl₃SiSiCl₃

The addition compound Cl₃SiSiCl₃·TMEDA was formed quantitatively by treatment of Cl₃SiSiCl₃ with tetramethylethylenediamine (TMEDA) in pentane at room temperature. The crystal structure of Cl₃SiSiCl₃·TMEDA displays one tetrahedrally and one octahedrally bonded Si atom (monoclinic, <i>P</i>2₁/<i>n</i>). ²⁹Si CP/MAS NMR spectroscopy confirms this structure. Density functional theory (DFT) calculations have shown that the structure of the <i>meridional</i> isomer of Cl₃SiSiCl₃·TMEDA is 6.3 kcal lower in energy than that of <i>facial</i> coordinate species. Dissolving of Cl₃SiSiCl₃·TMEDA in CH₂Cl₂ resulted in an immediate reaction by which oligochlorosilanes Si_<i>n</i>Cl_2<i>n</i> (<i>n</i> = 4, 6, 8, 10; precipitate) and the Cl^–-complexed dianions [Si_<i>n</i>Cl_2<i>n</i>+2]^2– (<i>n</i> = 6, 8, 10, 12; CH₂Cl₂ extract) were formed. The constitutions of these compounds were confirmed by MALDI mass spectrometry. Additionally, single crystals of [Me₃NCH₂CH₂NMe₂]₂[Si₆Cl₁₄] and [Me₃NCH₂CH₂NMe₂]₂[Si₈Cl₁₈] were obtained from the CH₂Cl₂ extract. We found that Cl₃SiSiCl₃·TMEDA reacts with MeCl, forming MeSiCl₃ and the products that had been formed in the reaction of Cl₃SiSiCl₃·TMEDA with CH₂Cl₂. X-ray structure analysis indicates that the structures of [Me₃NCH₂CH₂NMe₂]₂[Si₆Cl₁₄] (monoclinic, <i>P</i>2₁/<i>n</i>) and [Me₃NCH₂CH₂NMe₂]₂[Si₈Cl₁₈] (monoclinic, <i>P</i>2₁/<i>n</i>) contain dianions adopting an “inverse sandwich” structure with inverse polarity and [Me₃NCH₂CH₂NMe₂]⁺ as countercations. Single crystals of SiCl₄·TMEDA (monoclinic, <i>Cc</i>) could be isolated by thermolysis reaction of Cl₃SiSiCl₃·TMEDA (50 °C) in tetrahydrofuran (THF)

Unexpected Disproportionation of Tetramethylethylenediamine-Supported Perchlorodisilane Cl₃SiSiCl₃

The addition compound Cl₃SiSiCl₃·TMEDA was formed quantitatively by treatment of Cl₃SiSiCl₃ with tetramethylethylenediamine (TMEDA) in pentane at room temperature. The crystal structure of Cl₃SiSiCl₃·TMEDA displays one tetrahedrally and one octahedrally bonded Si atom (monoclinic, <i>P</i>2₁/<i>n</i>). ²⁹Si CP/MAS NMR spectroscopy confirms this structure. Density functional theory (DFT) calculations have shown that the structure of the <i>meridional</i> isomer of Cl₃SiSiCl₃·TMEDA is 6.3 kcal lower in energy than that of <i>facial</i> coordinate species. Dissolving of Cl₃SiSiCl₃·TMEDA in CH₂Cl₂ resulted in an immediate reaction by which oligochlorosilanes Si_<i>n</i>Cl_2<i>n</i> (<i>n</i> = 4, 6, 8, 10; precipitate) and the Cl^–-complexed dianions [Si_<i>n</i>Cl_2<i>n</i>+2]^2– (<i>n</i> = 6, 8, 10, 12; CH₂Cl₂ extract) were formed. The constitutions of these compounds were confirmed by MALDI mass spectrometry. Additionally, single crystals of [Me₃NCH₂CH₂NMe₂]₂[Si₆Cl₁₄] and [Me₃NCH₂CH₂NMe₂]₂[Si₈Cl₁₈] were obtained from the CH₂Cl₂ extract. We found that Cl₃SiSiCl₃·TMEDA reacts with MeCl, forming MeSiCl₃ and the products that had been formed in the reaction of Cl₃SiSiCl₃·TMEDA with CH₂Cl₂. X-ray structure analysis indicates that the structures of [Me₃NCH₂CH₂NMe₂]₂[Si₆Cl₁₄] (monoclinic, <i>P</i>2₁/<i>n</i>) and [Me₃NCH₂CH₂NMe₂]₂[Si₈Cl₁₈] (monoclinic, <i>P</i>2₁/<i>n</i>) contain dianions adopting an “inverse sandwich” structure with inverse polarity and [Me₃NCH₂CH₂NMe₂]⁺ as countercations. Single crystals of SiCl₄·TMEDA (monoclinic, <i>Cc</i>) could be isolated by thermolysis reaction of Cl₃SiSiCl₃·TMEDA (50 °C) in tetrahydrofuran (THF)

Unexpected Disproportionation of Tetramethylethylenediamine-Supported Perchlorodisilane Cl₃SiSiCl₃

The addition compound Cl₃SiSiCl₃·TMEDA was formed quantitatively by treatment of Cl₃SiSiCl₃ with tetramethylethylenediamine (TMEDA) in pentane at room temperature. The crystal structure of Cl₃SiSiCl₃·TMEDA displays one tetrahedrally and one octahedrally bonded Si atom (monoclinic, <i>P</i>2₁/<i>n</i>). ²⁹Si CP/MAS NMR spectroscopy confirms this structure. Density functional theory (DFT) calculations have shown that the structure of the <i>meridional</i> isomer of Cl₃SiSiCl₃·TMEDA is 6.3 kcal lower in energy than that of <i>facial</i> coordinate species. Dissolving of Cl₃SiSiCl₃·TMEDA in CH₂Cl₂ resulted in an immediate reaction by which oligochlorosilanes Si_<i>n</i>Cl_2<i>n</i> (<i>n</i> = 4, 6, 8, 10; precipitate) and the Cl^–-complexed dianions [Si_<i>n</i>Cl_2<i>n</i>+2]^2– (<i>n</i> = 6, 8, 10, 12; CH₂Cl₂ extract) were formed. The constitutions of these compounds were confirmed by MALDI mass spectrometry. Additionally, single crystals of [Me₃NCH₂CH₂NMe₂]₂[Si₆Cl₁₄] and [Me₃NCH₂CH₂NMe₂]₂[Si₈Cl₁₈] were obtained from the CH₂Cl₂ extract. We found that Cl₃SiSiCl₃·TMEDA reacts with MeCl, forming MeSiCl₃ and the products that had been formed in the reaction of Cl₃SiSiCl₃·TMEDA with CH₂Cl₂. X-ray structure analysis indicates that the structures of [Me₃NCH₂CH₂NMe₂]₂[Si₆Cl₁₄] (monoclinic, <i>P</i>2₁/<i>n</i>) and [Me₃NCH₂CH₂NMe₂]₂[Si₈Cl₁₈] (monoclinic, <i>P</i>2₁/<i>n</i>) contain dianions adopting an “inverse sandwich” structure with inverse polarity and [Me₃NCH₂CH₂NMe₂]⁺ as countercations. Single crystals of SiCl₄·TMEDA (monoclinic, <i>Cc</i>) could be isolated by thermolysis reaction of Cl₃SiSiCl₃·TMEDA (50 °C) in tetrahydrofuran (THF)