RESUMEN
The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments. Vn is a major blood protein that regulates many different physiological and pathological processes. The high sensitivity of the CryoProbe enabled us to acquire three-dimensional solid-state NMR spectra for sequential assignment and characterization of site-specific water-protein interactions that provide initial insights into the organization of the Vn-HAP complex. Vn associates with HAP in various pathological settings, including macular degeneration eyes and Alzheimer's disease brains. The ability to probe these assemblies at atomic detail paves the way for understanding their formation.
Asunto(s)
Durapatita , Vitronectina , Humanos , Espectroscopía de Resonancia Magnética/métodos , Imagen por Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
Ultrafast magic angle spinning (MAS) technology and 1H detection have dramatically enhanced the sensitivity of solid-state NMR (ssNMR) spectroscopy of biopolymers. We previously showed that, when combined with polarization optimized experiments (POE), these advancements enable the simultaneous acquisition of multi-dimensional 1H- or 13C-detected experiments using a single receiver. Here, we propose a new sub-class within the POE family, namely HC-DUMAS, HC-MEIOSIS, and HC-MAeSTOSO, that utilize dual receiver technology for the simultaneous detection of 1H and 13C nuclei. We also expand this approach to record 1H-, 13C-, and 15N-detected homonuclear 2D spectra simultaneously using three independent receivers. The combination of POE and multi-receiver technology will further shorten the total experimental time of ssNMR experiments for biological solids.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono/química , Hidrógeno/química , Resonancia Magnética Nuclear Biomolecular/instrumentación , Factores de TiempoRESUMEN
Oriented sample solid-state NMR (OS-ssNMR) spectroscopy is a powerful technique to determine the topology of membrane proteins in oriented lipid bilayers. Separated local field (SLF) experiments are central to this technique as they provide first-order orientational restraints, i.e., dipolar couplings and anisotropic chemical shifts. Despite the use of low-E (or E-free) probes, the heat generated during the execution of 2D and 3D SLF pulse sequences causes sizeable line-shape distortions. Here, we propose a new heat-compensated SE-SAMPI4 (hcSE-SAMPI4) pulse sequence that holds the temperature constant for the duration of the experiment. This modification of the SE-SAMPI4 results in sharper and more intense resonances without line-shape distortions. The spectral improvements are even more apparent when paramagnetic relaxation agents are used to speed up data collection. We tested the hcSE-SAMPI4 pulse sequence on a single-span membrane protein, sarcolipin (SLN), reconstituted in magnetically aligned lipid bicelles. In addition to eliminating peak distortions, the hcSE-SAMPI4 experiment increased the average signal-to-noise ratio by 20% with respect to the original SE-SAMPI4.
Asunto(s)
Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/métodos , Temperatura , Anisotropía , Proteínas Musculares/química , Proteolípidos/química , Relación Señal-RuidoRESUMEN
Magic angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy is a major technique for the characterization of the structural dynamics of biopolymers at atomic resolution. However, the intrinsic low sensitivity of this technique poses significant limitations to its routine application in structural biology. Here we achieve substantial savings in experimental time using a new subclass of Polarization Optimized Experiments (POEs) that concatenate TEDOR and SPECIFIC-CP transfers into a single pulse sequence. Specifically, we designed new 2D and 3D experiments (2D TEDOR-NCX, 3D TEDOR-NCOCX, and 3D TEDOR-NCACX) to obtain distance measurements and heteronuclear chemical shift correlations for resonance assignments using only one experiment. We successfully tested these experiments on N-Acetyl-Val-Leu dipeptide, microcrystalline U-13C,15N ubiquitin, and single- and multi-span membrane proteins reconstituted in lipid membranes. These pulse sequences can be implemented on any ssNMR spectrometer equipped with standard solid-state hardware using only one receiver. Since these new POEs speed up data acquisition considerably, we anticipate their broad application to fibrillar, microcrystalline, and membrane-bound proteins.
Asunto(s)
Espectroscopía de Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Algoritmos , Espectroscopía de Resonancia Magnética/métodos , Proteínas de la Membrana/química , Modelos Teóricos , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
The intrinsic conformational plasticity of membrane proteins directly influences the magnitude of the orientational-dependent NMR interactions such as dipolar couplings (DC) and chemical shift anisotropy (CSA). As a result, the conventional cross-polarization (CP)-based techniques mainly capture the more rigid regions of membrane proteins, while the most dynamic regions are essentially invisible. Nonetheless, dynamic regions can be detected using experiments in which polarization transfer takes place via J-coupling interactions. Here, we review our recent efforts to develop single and dual acquisition pulse sequences with either 1H or 13C detection that utilize both DC and J-coupling mediated transfer to detect both rigid and mobile regions of membrane proteins in native-like lipid environments. We show the application of these new methods for studying the conformational equilibrium of a single-pass membrane protein, phospholamban, which regulates the calcium transport across the sarcoplasmic reticulum (SR) membrane by interacting with the SR Ca2+-ATPase. We anticipate that these methods will be ideal to portray the complex dynamics of membrane proteins in their native environments.
Asunto(s)
Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación ProteicaRESUMEN
Oriented sample solid-state NMR (OS-ssNMR) spectroscopy is uniquely suited to determine membrane protein topology at the atomic resolution in liquid crystalline bilayers under physiological temperature. However, the inherent low sensitivity of this technique has hindered the throughput of multidimensional experiments necessary for resonance assignments and structure determination. In this work, we show that doping membrane protein bicelle preparations with paramagnetic ion chelated lipids and exploiting paramagnetic relaxation effects it is possible to accelerate the acquisition of both 2D and 3D multidimensional experiments with significant saving in time. We demonstrate the efficacy of this method for a small membrane protein, sarcolipin, reconstituted in DMPC/POPC/DHPC oriented bicelles. In particular, using Cu2+-DMPE-DTPA as a dopant, we observed a decrease of 1H T1 of sarcolipin by 2/3, allowing us to reduce the recycle delay up to 3 times. We anticipate that these new developments will enable the routine acquisition of multidimensional OS-ssNMR experiments.
Asunto(s)
Proteínas de la Membrana/química , Proteínas Musculares/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteolípidos/química , Animales , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Proteínas Musculares/análisis , Proteínas Musculares/metabolismo , Conformación Proteica , Proteolípidos/análisis , Proteolípidos/metabolismoRESUMEN
Phospholamban (PLN) is a single-pass membrane protein that regulates the sarco(endo)plasmic reticulum Ca²âº-ATPase (SERCA). Phosphorylation of PLN at Ser16 reverses its inhibitory function under ß-adrenergic stimulation, augmenting Ca²âº uptake in the sarcoplasmic reticulum and muscle contractility. PLN exists in two conformations; a T state, where the cytoplasmic domain is helical and adsorbed on the membrane surface, and an R state, where the cytoplasmic domain is unfolded and membrane detached. Previous studies have shown that the PLN conformational equilibrium is crucial to SERCA regulation. Here, we used a combination of solution and solid-state NMR to compare the structural topology and conformational dynamics of monomeric PLN (PLN(AFA)) with that of the PLN(R14del), a naturally occurring deletion mutant that is linked to the progression of dilated cardiomyopathy. We found that the behavior of the inhibitory transmembrane domain of PLN(R14del) is similar to that of the native sequence. Conversely, the conformational dynamics of R14del both in micelles and lipid membranes are enhanced. We conclude that the deletion of Arg14 in the cytoplasmic region weakens the interactions with the membrane and shifts the conformational equilibrium of PLN toward the disordered R state. This conformational transition is correlated with the loss-of-function character of this mutant and is corroborated by SERCA's activity assays. These findings support our hypothesis that SERCA function is fine-tuned by PLN conformational dynamics and begin to explain the aberrant regulation of SERCA by the R14del mutant.
Asunto(s)
Arginina/química , Proteínas de Unión al Calcio/química , Proteínas de la Membrana/química , Membrana Dobles de Lípidos/química , Micelas , Conformación Proteica , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
The membrane protein complex between the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLN) controls Ca(2+) transport in cardiomyocytes, thereby modulating cardiac contractility. ß-Adrenergic-stimulated phosphorylation of PLN at Ser-16 enhances SERCA activity via an unknown mechanism. Using solid-state nuclear magnetic resonance spectroscopy, we mapped the physical interactions between SERCA and both unphosphorylated and phosphorylated PLN in membrane bilayers. We found that the allosteric regulation of SERCA depends on the conformational equilibrium of PLN, whose cytoplasmic regulatory domain interconverts between three different states: a ground T state (helical and membrane associated), an excited R state (unfolded and membrane detached), and a B state (extended and enzyme-bound), which is noninhibitory. Phosphorylation at Ser-16 of PLN shifts the populations toward the B state, increasing SERCA activity. We conclude that PLN's conformational equilibrium is central to maintain SERCA's apparent Ca(2+) affinity within a physiological window. This model represents a paradigm shift in our understanding of SERCA regulation by posttranslational phosphorylation and suggests strategies for designing innovative therapeutic approaches to enhance cardiac muscle contractility.
Asunto(s)
Proteínas de Unión al Calcio/química , Conformación Proteica , Mapeo de Interacción de Proteínas/métodos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Calcio/química , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Cinética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Espectroscopía de Resonancia Magnética , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Fosforilación , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Conejos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
We present a new method called DAISY (Dual Acquisition orIented ssNMR spectroScopY) for the simultaneous acquisition of 2D and 3D oriented solid-state NMR experiments for membrane proteins reconstituted in mechanically or magnetically aligned lipid bilayers. DAISY utilizes dual acquisition of sine and cosine dipolar or chemical shift coherences and long living (15)N longitudinal polarization to obtain two multi-dimensional spectra, simultaneously. In these new experiments, the first acquisition gives the polarization inversion spin exchange at the magic angle (PISEMA) or heteronuclear correlation (HETCOR) spectra, the second acquisition gives PISEMA-mixing or HETCOR-mixing spectra, where the mixing element enables inter-residue correlations through (15)N-(15)N homonuclear polarization transfer. The analysis of the two 2D spectra (first and second acquisitions) enables one to distinguish (15)N-(15)N inter-residue correlations for sequential assignment of membrane proteins. DAISY can be implemented in 3D experiments that include the polarization inversion spin exchange at magic angle via I spin coherence (PISEMAI) sequence, as we show for the simultaneous acquisition of 3D PISEMAI-HETCOR and 3D PISEMAI-HETCOR-mixing experiments.
Asunto(s)
Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/métodos , Biología Computacional , Escherichia coli/genética , Expresión Génica , Modelos Moleculares , Proteínas Musculares/química , Isótopos de Nitrógeno/química , Proteolípidos/químicaRESUMEN
The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD ~0.44 Å, a tilt angle of 24° ± 1°, and an azimuthal angle of 55° ± 6°. This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-state NMR and magic angle spinning solid-state NMR.
Asunto(s)
Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Proteínas Musculares/química , Resonancia Magnética Nuclear Biomolecular , Proteolípidos/química , Modelos MolecularesRESUMEN
We propose a general method that enables the acquisition of multiple 2D and 3D solid-state NMR spectra for U-(13)C, (15)N-labeled proteins. This method, called MEIOSIS (Multiple ExperIments via Orphan SpIn operatorS), makes it possible to detect four coherence transfer pathways simultaneously, utilizing orphan (i.e., neglected) spin operators of nuclear spin polarization generated during (15)N-(13)C cross polarization (CP). In the MEIOSIS experiments, two phase-encoded free-induction decays are decoded into independent nuclear polarization pathways using Hadamard transformations. As a proof of principle, we show the acquisition of multiple 2D and 3D spectra of U-(13)C, (15)N-labeled microcrystalline ubiquitin. Hadamard decoding of CP coherences into multiple independent spin operators is a new concept in solid-state NMR and is extendable to many other multidimensional experiments. The MEIOSIS method will increase the throughput of solid-state NMR techniques for microcrystalline proteins, membrane proteins, and protein fibrils.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Ubiquitina/química , Isótopos de Carbono , Isótopos de NitrógenoRESUMEN
Fast data collection: a general method for dual data acquisition of multidimensional magic-angle spinning solid-state NMR experiments is presented. The method uses a simultaneous Hartmann-Hahn cross-polarization from (1)H to (13)C and (15)N nuclei and exploits the long-living (15)N polarization for parallel acquisition of two multidimensional experiments.
Asunto(s)
Sustancias Macromoleculares/química , Resonancia Magnética Nuclear Biomolecular , Isótopos de Carbono/química , Hidrógeno/química , Isótopos de Nitrógeno/química , Ubiquitina/químicaRESUMEN
Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring (1)H-(15)N dipolar couplings (DC) and (15)N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles' heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([(1)H,(15)N]-SE-PISEMA-PDSD). The incorporation of 2D (15)N/(15)N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the (15)N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.
Asunto(s)
Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/métodos , Anisotropía , Modelos Moleculares , Isótopos de NitrógenoRESUMEN
NMR anisotropic parameters such as dipolar couplings and chemical shifts are central to structure and orientation determination of aligned membrane proteins and liquid crystals. Among the separated local field experiments, the proton evolved local field (PELF) scheme is particularly suitable to measure dynamically averaged dipolar couplings and give information on local molecular motions. However, the PELF experiment requires the acquisition of several 2D datasets at different mixing times to optimize the sensitivity for the complete range of dipolar couplings of the resonances in the spectrum. Here, we propose a new PELF experiment that takes the advantage of the Hadamard encoding (HE) to obtain higher sensitivity for a broad range of dipolar couplings using a single 2D experiment. The HE scheme is obtained by selecting the spin operators with phase switching of hard pulses. This approach enables one to detect four spin operators, simultaneously, which can be processed into two 2D spectra covering a broader range of dipolar couplings. The advantages of the new approach are illustrated for a U-(15)N NAL single crystal and the U-(15)N labeled single-pass membrane protein sarcolipin reconstituted in oriented lipid bicelles. The HE-PELF scheme can be implemented in other multidimensional experiments to speed up the characterization of the structure and dynamics of oriented membrane proteins and liquid crystalline samples.
Asunto(s)
Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/métodos , Protones , Escherichia coli/genética , Proteínas de la Membrana/genética , Proteínas Musculares/química , Proteínas Musculares/genética , Proteolípidos/química , Proteolípidos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
CONTEXT: Electronic devices, laptops, tablets, ipad and smart phones are an integral part of one's life both in work and personal space. Excessive usage of these devices had led to health-related problems of which computer vision syndrome (CVS) is at risk of becoming a major public health issue. AIMS: Our study aimed to assess the pre-test and post-test level of knowledge regarding CVS among the medical coding trainees and also to determine the effectiveness (Video Teaching Programme) of knowledge about the CVS among them. METHODS AND MATERIAL: After obtaining ethical committee clearance using universal sampling method 480 medical coding trainees working in a firm was included in this study. A structured questionnaire was used to interview and a video teaching programme was given between the pre and post-test sessions for about 45 minutes regarding the effects of Computer Vision Syndrome. RESULTS: The level of knowledge about CVS among medical coding trainees in the pre-test and post-test was 280 (58.3%) and 8 (1.7%) had inadequate knowledge, respectively. With regards to adequate level of knowledge 24 (3%) and 328 (68.3%) had in pre-test and post-test phase, respectively. The comparison of knowledge shows a very good improvement among the medical coding trainees which was significant (p < 0.001). CONCLUSIONS: The study highlights health education as an important factor in increasing the knowledge. From a primary care point of view, knowledge regarding CVS helps in differentiating and addressing the ocular symptoms, headache and shoulder pain symptoms of other aetiology if history is elicited properly.
RESUMEN
We present new sensitivity enhanced schemes for heteronuclear correlation spectroscopy (HETCOR) in solid-state NMR of oriented systems. These schemes will enhance the sensitivity of the HETCOR by 40% for the two-dimensional experiments (SE-HETCOR) and up to 180% for the 3D HETCOR-separated local field version (SE-PISEMAI-HETCOR). The signal enhancement is demonstrated for a single crystal of ((15)N)N-acetylleucine and the integral membrane protein sarcolipin oriented in lipid bicelles. These methods will significantly reduce the time needed to acquire multidimensional experiments for membrane proteins oriented in magnetically or mechanically aligned lipid bilayers as well as liquid crystalline materials.
Asunto(s)
Membrana Dobles de Lípidos/química , Proteínas Musculares/química , Resonancia Magnética Nuclear Biomolecular/métodos , Proteolípidos/química , Leucina/análogos & derivados , Leucina/química , Modelos QuímicosRESUMEN
The atomic structure of small molecules and polypeptides can be attained from anisotropic NMR parameters such as dipolar couplings (DC) and chemical shifts (CS). Separated local field experiments resolve DC and CS correlations into two dimensions. However, crowded NMR spectra represent a significant obstacle for the complete resolution of these anisotropic parameters. Using the PISEMA (Polarization Inversion Spin Exchange at the Magic Angle) experiment as a foundation, we designed new pulse schemes that use a constant time evolution in the dipolar (indirect) dimension to measure DC and CS correlations at high resolution. We demonstrated this approach on a 4-pentyl-4'-cyanobiphenyl (5CB) liquid crystal sample, achieving a resolution enhancement ranging from 30 to 60 % for the resonances in the dipolar dimension. These new experiments open the possibility of obtaining significant resolution enhancement for multidimensional NMR experiments carried out on oriented liquid crystalline samples as well as oriented membrane proteins.
RESUMEN
Proton-detected solid-state NMR (ssNMR) spectroscopy has dramatically improved the sensitivity and resolution of fast magic angle spinning (MAS) methods. While relatively straightforward for fibers and crystalline samples, the routine application of these techniques to membrane protein samples is still challenging. This is due to the low sensitivity of these samples, which require high lipid:protein ratios to maintain the structural and functional integrity of membrane proteins. We previously introduced a family of novel polarization optimized experiments (POE) that enable to make the best of nuclear polarization and obtain multiple-acquisitions from a single pulse sequence and one receiver. Here, we present the 1H-detected versions of POE using ultrafast MAS ssNMR. Specifically, we implemented proton detection into our three main POE strategies, H-DUMAS, H-MEIOSIS, and H-MAeSTOSO, achieving the acquisition of up to ten different experiments using a single pulse sequence. We tested these experiments on a model compound N-Acetyl-Val-Leu dipeptide and applied to a six transmembrane acetate transporter, SatP, reconstituted in lipid membranes. These new methods will speed up the spectroscopy of challenging biomacromolecules such as membrane proteins.
Asunto(s)
Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/métodos , Algoritmos , Isótopos de Carbono , Proteínas de Escherichia coli/química , Membrana Dobles de Lípidos , Lípidos/química , Isótopos de Nitrógeno , Oligopéptidos/química , Transportadores de Anión Orgánico/química , ProtonesRESUMEN
We present a new method for enhancing the sensitivity in static solid-state NMR experiments for a gain in signal-to-noise ratio of up to 40%. This sensitivity enhancement is different from the corresponding solution NMR sensitivity enhancement schemes and is achieved by combining single- and multiple-quantum dipolar coherences. While this new approach is demonstrated for the polarization inversion spin exchange at magic angle (PISEMA) experiment, it can be generalized to the other separated local field experiments for solid-state NMR spectroscopy. This method will have a direct impact on solid-state NMR spectroscopy of liquid crystals as well as of membrane proteins aligned in lipid membranes.