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Magic angle spinning (MAS) NMR studies of amyloid and membrane proteins and large macromolecular complexes are an important new approach to structural biology. However, the applicability of these experiments, which are based on (13)C- and (15)N-detected spectra, would be enhanced if the sensitivity were improved. Here we discuss two advances that address this problem: high-frequency dynamic nuclear polarization (DNP) and (1)H-detected MAS techniques. DNP is a sensitivity enhancement technique that transfers the high polarization of exogenous unpaired electrons to nuclear spins via microwave irradiation of electron-nuclear transitions. DNP boosts NMR signal intensities by factors of 10(2) to 10(3), thereby overcoming NMR's inherent low sensitivity. Alternatively, it permits structural investigations at the nanomolar scale. In addition, (1)H detection is feasible primarily because of the development of MAS rotors that spin at frequencies of 40 to 60 kHz or higher and the preparation of extensively (2)H-labeled proteins.
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Resonancia Magnética Nuclear Biomolecular/métodos , Amiloide/química , Bacterias/química , Humanos , Hidrógeno/análisis , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/instrumentaciónRESUMEN
Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy and enable structural studies in biologically complex environments. Here, we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an intrinsically disordered region, the yeast prion protein Sup35. We added an exogenously prepared isotopically labeled protein to deuterated lysates, rendering the biological environment "invisible" and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible, and such contexts can influence protein structure.
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Resonancia Magnética Nuclear Biomolecular/métodos , Factores de Terminación de Péptidos/química , Priones/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Factores de Terminación de Péptidos/metabolismo , Priones/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Several publications describing high-resolution structures of amyloid-ß (Aß) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments-namely, 1H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aß1-42 fibrils by high-field 1H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aß1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently â¼6.5 times more sensitive than 1H detection. These results suggest that 1H detection and DNP may be the spectroscopic approaches of choice for future studies of Aß and other amyloid systems.
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Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Espectroscopía de Protones por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Conformación Proteica , TemperaturaRESUMEN
Collagen-mimetic peptides (CMP) have been invaluable tools for understanding the structure and function of collagen, which is the most abundant protein in animals. CMPs have also been developed as probes that detect damaged collagen because of the specificity required to form a collagen triple helix. These probes are not, however, ratiometric. Here, we used EPR spectroscopy to determine the end-to-end distances of CMPs that do not form stable homotrimeric helices. We found that those distances are shorter than the distances in the context of a collagen triple helix, suggesting their potential utility as a "molecular beacon" and guiding the choice and location of a pendant fluorophore-quencher pair. We then showed that a molecular beacon based on a glycine-(2S,4S)-4-fluoroproline-(2S,4R)-4-hydroxyproline tripeptide repeat and EDANS-DABCYL pair enabled the ratiometric detection of its binding to both other CMPs and natural mammalian collagen. These results provide guidance for the development of a new modality for detecting damaged collagen in physiological settings.
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Amyloid fibrils have been implicated in the pathogenesis of several neurodegenerative diseases, the most prevalent example being Alzheimer's disease (AD). Despite the prevalence of AD, relatively little is known about the structure of the associated amyloid fibrils. This has motivated our studies of fibril structures, extended here to the familial Arctic mutant of Aß1-42, E22G-Aß1-42. We found E22G-AßM0,1-42 is toxic to Escherichia coli, thus we expressed E22G-Aß1-42 fused to the self-cleavable tag NPro in the form of its EDDIE mutant. Since the high surface activity of E22G-Aß1-42 makes it difficult to obtain more than sparse quantities of fibrils, we employed 1H detected magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments to characterize the protein. The 1H detected 13C-13C methods were first validated by application to fully protonated amyloidogenic nanocrystals of GNNQQNY, and then applied to fibrils of the Arctic mutant of Aß, E22G-Aß1-42. The MAS NMR spectra indicate that the biosynthetic samples of E22G-Aß1-42 fibrils comprise a single conformation with 13C chemical shifts extracted from hCH, hNH, and hCCH spectra that are very similar to those of wild type Aß1-42 fibrils. These results suggest that E22G-Aß1-42 fibrils have a structure similar to that of wild type Aß1-42.
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Péptidos beta-Amiloides , Fragmentos de Péptidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Amiloide/química , Amiloide/metabolismo , Resonancia Magnética Nuclear Biomolecular , Escherichia coli/genética , Escherichia coli/metabolismo , Mutación , HumanosRESUMEN
The application of radio frequency (RF) vacuum electronics for the betterment of the human condition began soon after the invention of the first vacuum tubes in the 1920s and has not stopped since. Today, microwave vacuum devices are powering important applications in health treatment, material and biological science, wireless communication-terrestrial and space, Earth environment remote sensing, and the promise of safe, reliable, and inexhaustible energy. This article highlights some of the exciting application frontiers of vacuum electronics.
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Recent advancements in quantum sensing have sparked transformative detection technologies with high sensitivity, precision, and spatial resolution. Owing to their atomic-level tunability, molecular qubits and ensembles thereof are promising candidates for sensing chemical analytes. Here, we show quantum sensing of lithium ions in solution at room temperature with an ensemble of organic radicals integrated in a microporous metal-organic framework (MOF). The organic radicals exhibit electron spin coherence and microwave addressability at room temperature, thus behaving as qubits. The high surface area of the MOF promotes accessibility of the guest analytes to the organic qubits, enabling unambiguous identification of lithium ions and quantitative measurement of their concentration through relaxometric and hyperfine spectroscopic methods based on electron paramagnetic resonance (EPR) spectroscopy. The sensing principle presented in this work is applicable to other metal ions with nonzero nuclear spin.
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Dynamic nuclear polarization (DNP) is an NMR sensitivity enhancement technique that mediates polarization transfer from unpaired electrons to NMR-active nuclei. Despite its success in elucidating important structural information on biological and inorganic materials, the detailed polarization-transfer pathway from the electrons to the nearby and then the bulk solvent nuclei, and finally to the molecules of interest-remains unclear. In particular, the nuclei in the paramagnetic polarizing agent play significant roles in relaying the enhanced NMR polarizations to more remote nuclei. Despite their importance, the direct NMR observation of these nuclei is challenging because of poor sensitivity. Here, we show that a combined DNP and electron decoupling approach can facilitate direct NMR detection of these nuclei. We achieved an â¼80 % improvement in NMR intensity via electron decoupling at 0.35â T and 80â K on trityl radicals. Moreover, we recorded a DNP enhancement factor of ϵ ${\varepsilon{} }$ â¼90 and â¼11 % higher NMR intensity using electron decoupling on paramagnetic metal-organic framework, magnesium hexaoxytriphenylene (MgHOTP MOF).
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ElectronesRESUMEN
The two-spin solid effect (2SSE) is one of the established continuous wave dynamic nuclear polarization mechanisms that enables enhancement of nuclear magnetic resonance signals. It functions via a state-mixing mechanism that mediates the excitation of forbidden transitions in an electron-nuclear spin system. Specifically, microwave irradiation at frequencies ωµw â¼ ω0S ± ω0I, where ω0S and ω0I are electron and nuclear Larmor frequencies, respectively, yields enhanced nuclear spin polarization. Following the recent rediscovery of the three-spin solid effect (3SSE) [Tan et al., Sci. Adv. 5, eaax2743 (2019)], where the matching condition is given by ωµw = ω0S ± 2ω0I, we report here the first direct observation of the four-spin solid effect (4SSE) at ωµw = ω0S ± 3ω0I. The forbidden double- and quadruple-quantum transitions were observed in samples containing trityl radicals dispersed in a glycerol-water mixture at 0.35 T/15 MHz/9.8 GHz and 80 K. We present a derivation of the 4SSE effective Hamiltonian, matching conditions, and transition probabilities. Finally, we show that the experimental observations agree with the results from numerical simulations and analytical theory.
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Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is an abundant innate immune protein that contributes to the host metal-withholding response. Its ability to sequester transition metal nutrients from microbial pathogens depends on a complex interplay of Ca(II) binding and self-association, which converts the αß heterodimeric apo protein into a Ca(II)-bound (αß)2 heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αß heterodimer has hampered molecular understanding of how Ca(II) binding enables CP to exert its metal-sequestering innate immune function. We report solution NMR data that reveal how Ca(II) binding affects the structure and dynamics of the CP αß heterodimer. These studies provide a structural model in which the apo αß heterodimer undergoes conformational exchange and switches between two states, a tetramerization-incompetent or "inactive" state and a tetramerization-competent or "active" state. Ca(II) binding to the EF-hands of the αß heterodimer causes the active state to predominate, resulting in self-association and formation of the (αß)2 heterotetramer. Moreover, Ca(II) binding causes local and allosteric ordering of the His3Asp and His6 metal-binding sites. Ca(II) binding to the noncanonical EF-hand of S100A9 positions (A9)D30 and organizes the His3Asp site. Remarkably, Ca(II) binding causes allosteric effects in the C-terminal region of helix αIV of S100A9, which stabilize the α-helicity at positions H91 and H95 and thereby organize the functionally versatile His6 site. Collectively, this study illuminates the molecular basis for how CP responds to high extracellular Ca(II) concentrations, which enables its metal-sequestering host-defense function.
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Calcio/metabolismo , Complejo de Antígeno L1 de Leucocito/metabolismo , Multimerización de Proteína/efectos de los fármacos , Elementos de Transición/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Histidina/química , Humanos , Complejo de Antígeno L1 de Leucocito/genética , Metales Pesados/metabolismo , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica en Hélice alfa/efectos de los fármacos , Multimerización de Proteína/genéticaRESUMEN
The Overhauser effect (OE), commonly observed in NMR spectra of liquids and conducting solids, was recently discovered in insulating solids doped with the radical 1,3-bisdiphenylene-2-phenylallyl (BDPA). However, the mechanism of polarization transfer in OE-DNP in insulators is yet to be established, but hyperfine coupling of the radical to protons in BDPA has been proposed. In this paper we present a study that addresses the role of hyperfine couplings via the EPR and DNP measurements on some selectively deuterated BDPA radicals synthesized for this purpose. Newly developed synthetic routes enable selective deuteration at orthogonal positions or perdeuteration of the fluorene moieties with 2H incorporation of >93%. The fluorene moieties were subsequently used to synthesize two octadeuterated BDPA radicals, 1,3-[α,γ-d8]-BDPA and 1,3-[ß,δ-d8]-BDPA, and a BDPA radical with perdeuterated fluorene moieties, 1,3-[α,ß,γ,δ-d16]-BDPA. In contrast to the strong positive OE enhancement observed in degassed samples of fully protonated h21-BDPA (ε â¼ +70), perdeuteration of the fluorenes results in a negative enhancement (ε â¼ -13), while selective deuteration of α- and γ-positions (aiso â¼ 5.4 MHz) in BDPA results in a weak negative OE enhancement (ε â¼ -1). Furthermore, deuteration of ß- and δ-positions (aiso â¼ 1.2 MHz) results in a positive OE enhancement (ε â¼ +36), albeit with a reduced magnitude relative to that observed in fully protonated BDPA. Our results clearly show the role of the hyperfine coupled α and γ 1H spins in the BDPA radical in determining the dominance of the zero and double-quantum cross-relaxation pathways and the polarization-transfer mechanism to the bulk matrix.
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Compuestos Alílicos/química , Fluorenos/química , Radicales Libres/química , Compuestos Alílicos/síntesis química , Deuterio/química , Fluorenos/síntesis química , Radicales Libres/síntesis química , Espectroscopía de Resonancia MagnéticaRESUMEN
Recent structural investigation of amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations, which are different from those of in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy patients adopt distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments formed in the presence of a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filaments reveals some similarities to one of the previously reported polymorphs of in vitro α-synuclein filaments in the core region, while illustrating distinct conformations in the N- and C-terminal regions. The structural study highlights the conformational plasticity of α-synuclein filaments and the importance of the co-factors, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors. The comparative structural analyses will help better understand molecular basis of diverse structures of α-synuclein filaments and possible relevance of each structure to the disease phenotype.
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Amiloide/química , Microscopía por Crioelectrón/métodos , Espectroscopía de Resonancia Magnética/métodos , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Química Encefálica , Humanos , Microscopía Inmunoelectrónica/métodos , Conformación Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
All cells obtain 2'-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, ß; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cysâ¢). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys⢠in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPAâ¢) in ß. The three-electron oxidation producing DOPA⢠occurs in Escherichia coli only if the ß is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.
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Dihidroxifenilalanina/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Radicales Libres/química , Ribonucleótido Reductasas/química , Tirosina/química , Dihidroxifenilalanina/metabolismo , Proteínas de Escherichia coli/metabolismo , Radicales Libres/metabolismo , Ribonucleótido Reductasas/metabolismo , Tirosina/metabolismoRESUMEN
Amyloid formation of full-length TTR involves dissociation of the native tetramers into misfolded monomers that self-assemble into amyloid. In addition to the full-length TTR, C-terminal fragments including residues 49-127 were also observed in vivo, implying the presence of additional misfolding pathways. It was previously proposed that a proteolytic cleavage might lead to the formation of the C-terminal fragment TTR amyloid. Here, we report mechanistic studies of misfolding and aggregation of a TTR variant (G53A) in the absence and presence of a serine protease. A proteolytic cleavage of G53A in the CD loop (K48 and T49) with agitation promoted TTR misfolding and aggregation, suggesting that the proteolytic cleavage may lead to the aggregation of the C-terminal fragment (residues 49-127). To gain more detailed insights into TTR misfolding promoted by proteolytic cleavage, we investigated structural changes in G53A TTR in the presence and absence of trypsin. Our combined biophysical analyses revealed that the proteolytic cleavage accelerated the formation of spherical small oligomers, which exhibited cytotoxic activities. However, the truncated TTR appeared to maintain native-like structures, rather than the C-terminal fragment (residues 49-127) being released and unfolded from the native state. In addition, our solid-state nuclear magnetic resonance and Fourier transform infrared structural studies showed that the two aggregates derived from the full-length and cleaved TTR exhibited nearly identical molecular structural features, suggesting that the proteolytic cleavage in the CD loop destabilizes the native tetrameric structure and accelerates oligomer formation through a common TTR misfolding and aggregation mechanism rather than through a distinct molecular mechanism.
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Proteínas Amiloidogénicas/metabolismo , Prealbúmina/metabolismo , Tripsina/química , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/genética , Línea Celular Tumoral , Humanos , Mutación , Prealbúmina/química , Prealbúmina/genética , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , ProteolisisRESUMEN
Structural characterization of misfolded protein aggregates is essential to understanding the molecular mechanism of protein aggregation associated with various protein misfolding disorders. Here, we report structural analyses of ex vivo transthyretin aggregates extracted from human cardiac tissue. Comparative structural analyses of in vitro and ex vivo transthyretin aggregates using various biophysical techniques revealed that cardiac transthyretin amyloid has structural features similar to those of in vitro transthyretin amyloid. Our solid-state nuclear magnetic resonance studies showed that in vitro amyloid contains extensive nativelike ß-sheet structures, while other loop regions including helical structures are disrupted in the amyloid state. These results suggest that transthyretin undergoes a common misfolding and aggregation transition to nativelike aggregation-prone monomers that self-assemble into amyloid precipitates in vitro and in vivo.
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Amiloide/química , Amiloide/metabolismo , Miocitos Cardíacos/química , Prealbúmina/química , Prealbúmina/metabolismo , Agregado de Proteínas , Pliegue de Proteína , Amiloide/aislamiento & purificación , Humanos , Modelos Moleculares , Tamaño de la Partícula , Prealbúmina/aislamiento & purificación , Conformación Proteica , Propiedades de SuperficieRESUMEN
Correction for 'Conformation of bis-nitroxide polarizing agents by multi-frequency EPR spectroscopy' by Janne Soetbeer et al., Phys. Chem. Chem. Phys., 2018, 20, 25506-25517.
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We report the design and experimental demonstration of a frequency tunable terahertz gyrotron at 527 GHz built for an 800 MHz Dynamic Nuclear Polarization enhanced Nuclear Magnetic Resonance (DNP-NMR) spectrometer. The gyrotron is designed at the second harmonic (ω = 2ω c) of the electron cyclotron frequency. It produces up to 9.3 W continuous microwave (CW) power at 527.2 GHz frequency using a diode type electron gun operating at V = 16.65 kV, Ib = 110 mA in a TE11,2,1 mode, corresponding to an efficiency of ~0.5%. The gyrotron is tunable within ~ 0.4 GHz by combining voltage and magnetic field tuning. The gyrotron has an internal mode converter that produces a Gaussian-like beam that couples to the HE11 mode of an internal 12 mm i.d. corrugated waveguide periscope assembly leading up to the output window. An external corrugated waveguide transmission line system is built including a corrugated taper from 12 mm to 16 mm i.d. waveguide followed by 3 m of the 16 mm i.d. waveguide The microwave beam profile is measured using a pyroelectric camera showing ~ 84% HE11 mode content.
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The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a ß-helical arrangement, whereas others suggest a parallel in-register organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13C- and 15N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems.
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Factores de Terminación de Péptidos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Marcaje Isotópico , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Saccharomyces cerevisiae/químicaRESUMEN
The second isoform of the human voltage dependent anion channel (VDAC2) is a mitochondrial porin that translocates calcium and other metabolites across the outer mitochondrial membrane. VDAC2 has been implicated in cardioprotection and plays a critical role in a unique apoptotic pathway in tumor cells. Despite its medical importance, there have been few biophysical studies of VDAC2 in large part due to the difficulty of obtaining homogeneous preparations of the protein for spectroscopic characterization. Here we present high resolution magic angle spinning nuclear magnetic resonance (NMR) data obtained from homogeneous preparation of human VDAC2 in 2D crystalline lipid bilayers. The excellent resolution in the spectra permit several sequence-specific assignments of the signals for a large portion of the VDAC2 N-terminus and several other residues in two- and three-dimensional heteronuclear correlation experiments. The first 12 residues appear to be dynamic, are not visible in cross polarization experiments, and they are not sufficiently mobile on very fast timescales to be visible in 13C INEPT experiments. A comparison of the NMR spectra of VDAC2 and VDAC1 obtained from highly similar preparations demonstrates that the spectral quality, line shapes and peak dispersion exhibited by the two proteins are nearly identical. This suggests an overall similar dynamic behavior and conformational homogeneity, which is in contrast to two earlier reports that suggested an inherent conformational heterogeneity of VDAC2 in membranes. The current data suggest that the sample preparation and spectroscopic methods are likely applicable to studying other human membrane porins, including human VDAC3, which has not yet been structurally characterized.
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Membrana Dobles de Lípidos , Resonancia Magnética Nuclear Biomolecular/métodos , Canal Aniónico 2 Dependiente del Voltaje/química , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Canal Aniónico 1 Dependiente del Voltaje/químicaRESUMEN
Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. 15N(SB)-1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C-13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N-13C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.