RESUMEN
ATP-binding cassette (ABC) exporters actively move chemically diverse substrates across biological membranes. Their malfunction leads to human diseases. Many ABC exporters encompass asymmetric nucleotide-binding sites (NBSs), and some of them are inhibited by the transported substrate. The functional relevance of the catalytic asymmetry or the mechanism for trans-inhibition remains elusive. Here, we investigated TmrAB, a functional homologue of the human antigen translocation complex TAP using advanced electron-electron double resonance spectroscopy. In the presence of ATP, the heterodimeric ABC exporter exists in a tunable equilibrium between inward- and outward-facing conformations. The two NBSs exhibit pronounced asymmetry in the open-to-close equilibrium. The closed conformation is more favored at the degenerate NBS, and closure of either of the NBS is sufficient to open the extracellular gate. We define the mechanistic basis for trans-inhibition, which operates by a reverse transition from the outward-facing state through an occluded conformation. These novel findings uncover the central role of reversible conformational equilibrium in the function and regulation of an ABC exporter and establish a mechanistic framework for future investigations on other medically important transporters with imprinted asymmetry. Also, this study demonstrates for the first-time the feasibility to resolve equilibrium populations at multiple domains and their interdependence for global conformational changes in a large membrane protein complex.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Modelos Biológicos , Conformación Proteica , Dominios ProteicosRESUMEN
Ubiquitination is the most versatile posttranslational modification. The information is encoded by linkage type as well as chain length, which are translated by ubiquitin binding domains into specific signaling events. Chain topology determines the conformational space of a ubiquitin chain and adds an additional regulatory layer to this ubiquitin code. In particular, processes that modify chain length will be affected by chain conformations as they require access to the elongation or cleavage sites. We investigated conformational distributions in the context of chain elongation and disassembly using pulsed electron-electron double resonance spectroscopy in combination with molecular modeling. Analysis of the conformational space of diubiquitin revealed conformational selection or remodeling as mechanisms for chain recognition during elongation or hydrolysis, respectively. Chain elongation to tetraubiquitin increases the sampled conformational space, suggesting that a high intrinsic flexibility of K48-linked chains may contribute to efficient proteasomal degradation.
Asunto(s)
Ubiquitina/metabolismo , Ubiquitinación/fisiología , Humanos , Modelos Moleculares , Conformación Molecular , Unión ProteicaRESUMEN
This article describes current uses of shaped pulses, generated by an arbitrary waveform generator, in the field of EPR spectroscopy. We show applications of sech/tanh and WURST pulses to dipolar spectroscopy, including new pulse schemes and procedures, and discuss the more general concept of optimum-control-based pulses for applications in EPR spectroscopy. The article also describes a procedure to correct for experimental imperfections, mostly introduced by the microwave resonator, and discusses further potential applications and limitations of such pulses.
RESUMEN
Applications of broadband pulses for EPR have been reported for FID, echo detection and inversion pulses recently. Here we present a broadband Hahn, stimulated and refocused echo sequence derived from adiabatic pulses. The formation of echoes is accomplished by using variable chirp rates and pulse lengths. In all three broadband echo experiments the complete spectral shape of a nitroxide (about 70 Gauss at X-band frequency) could be recovered by Fourier transformation of the quadrature detected echo signals. Such broadband echoes provide an exciting opportunity to optimize pulse sequences where a full excitation of the spectrum is mandatory for an optimum performance. We applied our pulses to the SIFTER (single frequency technique for refocusing dipolar couplings) experiment, a solid echo based pulse sequence to measure the dipolar coupling between two unpaired electron spins. By employing our broadband Hahn echo sequence on a nitroxide biradical we could achieve an artifact free dipolar evolution time trace in the SIFTER experiment with 95% modulation depth at X-band frequency and of 10% modulation depth at Q-band frequency.
RESUMEN
Pulsed electron paramagnetic resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5-8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized membrane proteins, the maximum distance accessible is much lower, because of a strongly reduced coherence time of the electron spins. Here we introduce a pulse sequence, based on a Carr-Purcell decoupling scheme on the observer spin, where each π-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome this limitation. This pump/probe excitation scheme efficiently recouples the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric betaine transporter BetP. Interprotomer distances as long as 6 nm could be reliably determined, which is impossible with the existing methods.
Asunto(s)
Proteínas Portadoras/química , Electrones , Óxidos de Nitrógeno/química , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Transportadoras de GABA en la Membrana Plasmática , Modelos MolecularesRESUMEN
A 1 ns resolution pulse shaping unit has been developed for pulsed EPR spectroscopy to enable 14-bit amplitude and phase modulation. Shaped broadband excitation pulses designed using optimal control theory (OCT) have been tested with this device at X-band frequency (9 GHz). FT-EPR experiments on organic radicals in solution have been performed with the new pulses, designed for uniform excitation over a significantly increased bandwidth compared to a classical rectangular π/2 pulse of the same B(1) amplitude. The concept of a dead-time compensated prefocused pulse has been introduced to EPR with a self-refocusing of 200 ns after the end of the pulse. Echo-like refocused signals have been recorded and compared to the performance of a classical Hahn-echo sequence. The impulse response function of the microwave setup has been measured and incorporated into the algorithm for designing OCT pulses, resulting in further significant improvements in performance. Experimental limitations and potential new applications of OCT pulses in EPR spectroscopy will be discussed.