A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins

Dynamic nuclear polarization (DNP) magic‐angle spinning (MAS) solid‐state NMR (ssNMR) spectroscopy has the potential to enhance NMR signals by orders of magnitude and to enable NMR characterization of proteins which are inherently dilute, such as membrane proteins. In this work spin‐labeled lipid molecules (SL‐lipids), when used as polarizing agents, lead to large and relatively homogeneous DNP enhancements throughout the lipid bilayer and to an embedded lung surfactant mimetic peptide, KL₄. Specifically, DNP MAS ssNMR experiments at 600 MHz/395 GHz on KL₄ reconstituted in liposomes containing SL‐lipids reveal DNP enhancement values over two times larger for KL₄ compared to liposome suspensions containing the biradical TOTAPOL. These findings suggest an alternative sample preparation strategy for DNP MAS ssNMR studies of lipid membranes and integral membrane proteins.     

Highly Efficient Trityl-Nitroxide Biradicals for Biomolecular High-Field Dynamic Nuclear Polarization

Dynamic nuclear polarization (DNP) is a powerful method to enhance the sensitivity of solid-state magnetic nuclear resonance (ssNMR) spectroscopy. However, its biomolecular applications at high magnetic fields (preferably>14 T) have so far been limited by the intrinsically low efficiency of polarizing agents and sample preparation aspects. Herein, we report a new class of trityl-nitroxide biradicals, dubbed SNAPols that combine high DNP efficiency with greatly enhanced hydrophilicity. SNAPol-1, the best compound in the series, shows DNP enhancement factors at 18.8 T of more than 100 in small molecules and globular proteins and also exhibits strong DNP enhancements in membrane proteins and cellular preparations. By integrating optimal sensitivity and high resolution, we expect widespread applications of this new polarizing agent in high-field DNP/ssNMR spectroscopy, especially for complex biomolecules.     

Analysis of Molecular Orientation in Organic Semiconducting Thin Films Using Static Dynamic Nuclear Polarization Enhanced Solid‐State NMR Spectroscopy

Molecular orientation in amorphous organic semiconducting thin‐film devices is an important issue affecting device performance. However, to date it has not been possible to analyze the “distribution” of the orientations. Although solid‐state NMR (ssNMR) spectroscopy can provide information on the “distribution” of molecular orientations, the technique is limited because of the small amount of sample in the device and the low sensitivity of ssNMR. Here, we report the first application of dynamic nuclear polarization enhanced ssNMR (DNP‐ssNMR) spectroscopy for the orientational analysis of amorphous phenyldi(pyren‐1‐yl)phosphine oxide (POPy₂). The ³¹P DNP‐ssNMR spectra exhibited a sufficient signal‐to‐noise ratio to quantify the distribution of molecular orientations in amorphous films: the P=O axis of the vacuum‐deposited and drop‐cast POPy₂ shows anisotropic and isotropic distribution, respectively. The different molecular orientations reflect the molecular origin of the different charge transport behaviors.     

Towards a native environment: structure and function of membrane proteins in lipid bilayers by NMR

Solid-state NMR (ssNMR) is a versatile technique that can be used for the characterization of various materials, ranging from small molecules to biological samples, including membrane proteins. ssNMR can probe both the structure and dynamics of membrane proteins, revealing protein function in a near-native lipid bilayer environment. The main limitation of the method is spectral resolution and sensitivity, however recent developments in ssNMR hardware, including the commercialization of 28 T magnets (1.2 GHz proton frequency) and ultrafast MAS spinning (<100 kHz) promise to accelerate acquisition, while reducing sample requirement, both of which are critical to membrane protein studies. Here, we review recent advances in ssNMR methodology used for structure determination of membrane proteins in native and mimetic environments, as well as the study of protein functions such as protein dynamics, and interactions with ligands, lipids and cholesterol.

Solid-state NMR (ssNMR) is a versatile technique that can be used for the characterization of various materials, ranging from small molecules to biological samples, including membrane proteins, as reviewed here.     

Studies on the MxiH Protein in T3SS Needles Using DNP‐Enhanced ssNMR Spectroscopy

Bacterial T3SS needles formed by the protein MxiH are studied using DNP‐enhanced ssNMR spectroscopy at 14.1 T (600 MHz). This technique provides spectra of good resolution, allowing us to draw conclusions about the protein dynamics. With the obtained signal enhancement, samples of limited quantity now get within reach of ssNMR studies.     

Electrostatics and Intrinsic Disorder Drive Translocon Binding of the SRP Receptor FtsY

Integral membrane proteins in bacteria are co‐translationally targeted to the SecYEG translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N‐terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C‐terminal membrane targeting sequence. The central A domain binds to the translocon non‐specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome‐bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane.     

Complex Formation of the Tetracycline‐Binding Aptamer Investigated by Specific Cross‐Relaxation under DNP

While dynamic nuclear polarization (DNP) under magic‐angle spinning (MAS) is generally a powerful method capable of greatly enhancing the sensitivity of solid‐state NMR spectroscopy, hyperpolarization also gives rise to peculiar spin dynamics. Here, we elucidate how specific cross‐relaxation enhancement by active motions under DNP (SCREAM‐DNP) can be utilized to selectively obtain MAS‐NMR spectra of an RNA aptamer in a tightly bound complex with a methyl‐bearing ligand (tetracycline) due to the effective CH₃‐reorientation at an optimized sample temperature of approximately 160 K. SCREAM‐DNP can spectrally isolate the complex from non‐bound species in an RNA mixture. This selectivity allows for a competition assay between the aptamer and a mutant with compromised binding affinity. Variations in molecular structure and methyl dynamics, as observed by SCREAM‐DNP, between free tetracycline and RNA‐bound tetracycline are discussed.     

Protein identification with Teflon as matrix-assisted laser desorption/ionization sample support

Protein identification is a critical step in proteomics, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) plays an important role in that identification. Polytetrafluoroethylene (Teflon) was tested as a new MALDI sample support to improve protein identification. The tryptic peptides obtained from a model protein were bound to the surface of a modified MALDI sample holder via the hydrophobic interactions that occur between the Teflon surface and the peptide ion-pairs, and the affinity of alpha-cyano-4-hydroxycinnamic acid for the peptides. During that surface-binding step, the peptide mixture was also desalted and concentrated. A greater number of matched peptides and a larger sequence coverage were obtained for the proteins when Teflon was used as the sample support compared with conventional sample preparation methods and a stainless-steel surface. In addition, the characterization of a small amount of protein was improved with Teflon. Nine silver-stained protein spots obtained from 2-D gel of a human cerebrospinal fluid (CSF) proteome were identified by this method. Among the nine protein spots, peptide 6:c3c fragment and procollagen c-proteinase enhancer were not annotated in any published 2-D map of human CSF. A Teflon MALDI sample support is a low-cost, simple, and effective method that can be used to improve the quality of the MALDI mass spectrum of a complex tryptic peptide mixture, and to achieve a higher level of reliability and success in protein identification.     

Disclosing Interfaces of ZnO Nanocrystals Using Dynamic Nuclear Polarization: Sol‐Gel versus Organometallic Approach

The unambiguous characterization of the coordination chemistry of nanocrystal surfaces produced by wet‐chemical synthesis presently remains highly challenging. Here, zinc oxide nanocrystals (ZnO NCs) coated by monoanionic diphenyl phosphate (DPP) ligands were derived by a sol‐gel process and a one‐pot self‐supporting organometallic (OSSOM) procedure. Atomic‐scale characterization through dynamic nuclear polarization (DNP‐)enhanced solid‐state NMR (ssNMR) spectroscopy has notably enabled resolving their vastly different surface‐ligand interfaces. For the OSSOM‐derived NCs, DPP moieties form stable and strongly‐anchored μ₂‐ and μ₃‐bridging‐ligand pairs that are resistant to competitive ligand exchange. The sol‐gel‐derived NCs contain a wide variety of coordination modes of DPP ligands and a ligand exchange process takes place between DPP and glycerol molecules. This highlights the power of DNP‐enhanced ssNMR for detailed NC surface analysis and of the OSSOM approach for the preparation of ZnO NCs.     

Substrate affinities for membrane transport proteins determined by 13C cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy

We have devised methods in which cross-polarization magic-angle spinning (CP-MAS) solid-state NMR is exploited to measure rigorous parameters for binding of (13)C-labeled substrates to membrane transport proteins. The methods were applied to two proteins from Escherichia coli: a nucleoside transporter, NupC, and a glucuronide transporter, GusB. A substantial signal for the binding of methyl [1-(13)C]-beta-d-glucuronide to GusB overexpressed in native membranes was achieved with a sample that contained as little as 20 nmol of GusB protein. The data were fitted to yield a K(D) value of 4.17 mM for the labeled ligand and 0.42 mM for an unlabeled ligand, p-nitrophenyl beta-d-glucuronide, which displaced the labeled compound. CP-MAS was also used to measure binding of [1'-(13)C]uridine to overexpressed NupC. The spectrum of NupC-enriched membranes containing [1'-(13)C]uridine exhibited a large peak from substrate bound to undefined sites other than the transport site, which obscured the signal from substrate bound to NupC. In a novel application of a cross-polarization/polarization-inversion (CPPI) NMR experiment, the signal from undefined binding was eliminated by use of appropriate inversion pulse lengths. By use of CPPI in a titration experiment, a K(D) value of 2.6 mM was determined for uridine bound to NupC. These approaches are broadly applicable to quantifying binding of substrates, inhibitors, drugs, and antibiotics to numerous membrane proteins.     

Structure of a Protein–RNA Complex by Solid-State NMR Spectroscopy

Solid-state NMR (ssNMR) is applicable to high molecular-weight (MW) protein assemblies in a non-amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low-sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical-shift-perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.     

基于固体核磁共振方法的蛋白质组装体三维结构解析

蛋白质组装体广泛存在于生物体内,具有相关生物学功能或与人类的重要疾病密切相关。蛋白质组装体分子量大,通常难以溶解和结晶,限制了常用的结构研究手段如X射线晶体学和液体NMR等在其高分辨三维结构解析中的应用。固体核磁共振技术(ssNMR)在难溶、非结晶样品的三维结构解析中具有独特的优势,尤其随着固体NMR硬件包括高场磁体和高性能的探头、固体NMR多维脉冲实验技术和样品制备技术特别是同位素标记技术的快速发展,固体NMR已经成为了蛋白组装体三维结构解析的重要手段。在样品制备方法方面,强调了样品制备条件的优化对得到构象均一样品的重要性,以及丰富的同位素标记方法的使用对固体NMR谱图分辨率提高的重要作用。同时多种脉冲序列如质子驱动自旋扩散技术(PDSD),偶极辅助旋转共振技术(DARR),质子辅助重偶技术(PAR)或转移回波双共振技术(TEDOR)等的建立和发展为结构约束条件收集提供了基本的技术方法。此外,固体NMR与其它实验技术如扫描透射电镜(STEM),冷冻电镜(Cryo-EM)等和理论模拟方法的联用能显著地提高固体NMR的能力,从而能解析分子量更大、结构更复杂的蛋白质组装体的三维结构。本文以Aβ纤维和T3SS针状体的三维结构解析为例介绍固体NMR在蛋白质组装体结构研究的最新实验方法,重点介绍最新的距离约束条件获取的实验方法进展,以及固体NMR与其它实验和理论模拟研究手段的联用在蛋白质组装体结构解析上的最新进展,期望有助于读者对固体NMR技术在蛋白质组装体的三维结构解析方面的研究进展有所了解。     

Free energy of nascent-chain folding in the translocon

During their synthesis, many water-soluble proteins and nearly all membrane proteins transit through a protein-conducting channel in the membrane, the Sec translocon, from where they are inserted into the lipid bilayer. Increasing evidence indicates that folding of the nascent protein begins already within the ribosomal exit tunnel in a sequence- and environment-dependent fashion. To examine the effects of the translocon on the nascent-chain folding, we have calculated the potential of mean force for α-helix formation of a 10-alanine oligopeptide as a function of its position within the translocon channel. We find that the predominant conformational states, α-helical and extended, reflect those found for the peptide in water. However, the translocon, via its surface properties and its variable diameter, shifts the equilibrium in favor of the α-helical state. Thus, we suggest that the translocon facilitates not only the insertion of membrane proteins into the bilayer but also their folding.     

Structure of a Protein–RNA Complex by Solid‐State NMR Spectroscopy

Solid‐state NMR (ssNMR) is applicable to high molecular‐weight (MW) protein assemblies in a non‐amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low‐sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical‐shift‐perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.     

A study of Src SH2 domain protein-phosphopeptide binding interactions by electrospray ionization mass spectrometry

The noncovalent binding of various peptide ligands to pp60^src (Src) SH2 (Src homology 2) domain protein (12.9 ku) has been used as a model system for development of electrospray ionization mass spectrometry (ESI-MS) as a tool to study noncovalently bound complexes. SH2 motifs in proteins are critical in the signal transduction pathways of the tyrosine kinase growth factor receptors and recognize phosphotyrosine-containing proteins and peptides. ESI-MS with a magnetic sector instrument and array detection has been used to detect the protein-peptide complex with low-picomole sensitivity. The relative abundances of the multiply charged ions for the complex formed between Src SH2 protein and several nonphosphorylated and phosphorylated peptides have been compared. The mass spectrometry data correlate well to the measured binding constants derived from solution-based methods, indicating that the mass spectrometry-based method can be used to assess the affinity of such interactions. Solution-phase equilibrium constants may be determined by measuring the amount of bound and unbound species as a function of concentration for construction of a Scatchard graph. ESI-MS of a solution containing Src SH2 with a mixture of phosphopeptides showed the expected protein-phosphopeptide complex as the dominant species in the mass spectrum, demonstrating the method’s potential for screening mixtures from peptide libraries.     

Targeted DNP for biomolecular solid-state NMR

High-field dynamic nuclear polarization is revolutionizing the scope of solid-state NMR with new applications in surface chemistry, materials science and structural biology. In this perspective article, we focus on a specific DNP approach, called targeted DNP, in which the paramagnets introduced to polarize are not uniformly distributed in the sample but site-specifically located on the biomolecular system. After reviewing the various targeting strategies reported to date, including a bio-orthogonal chemistry-based approach, we discuss the potential of targeted DNP to improve the overall NMR sensitivity while avoiding the use of glass-forming DNP matrix. This is especially relevant to the study of diluted biomolecular systems such as, for instance, membrane proteins within their lipidic environment. We also discuss routes towards extracting structural information from paramagnetic relaxation enhancement (PRE) induced by targeted DNP at cryogenic temperature, and the possibility to recover site-specific information in the vicinity of the paramagnetic moieties using high-resolution selective DNP spectra. Finally, we review the potential of targeted DNP for in-cell NMR studies and how it can be used to extract a given protein NMR signal from a complex cellular background.

In targeted DNP, localization of polarizing agent at specific sites leads to new NMR approaches to improve sensitivity, background suppression for in-cell NMR, access to long-range constraints, and selective observation of binding sites.     

In Vitro Synthesis of the E. coli Sec Translocon from DNA

Difficulties in constructing complex lipid/protein membranes have severely limited the development of functional artificial cells endowed with vital membrane‐related functions. The Sec translocon membrane channel, which mediates the insertion of membrane proteins into the plasma membrane, was constructed in the membrane of lipid vesicles through in vitro expression of its component proteins. The components of the Sec translocon were synthesized from their respective genes in the presence of liposomes, thereby bringing about a functional complex. The synthesized E. coli Sec translocon mediated the membrane translocation of single‐ and multi‐span membrane proteins. The successful translocation of a functional peptidase into the liposome lumen further confirmed the proper insertion of the translocon complex. Our results demonstrate the feasible construction of artificial cells, the membranes of which can be functionalized by directly decoding genetic information into membrane functions.     

Fast multidimensional localized parallel NMR spectroscopy for the analysis of samples

A parallel localized spectroscopy (PALSY) method is presented to speed up the acquisition of multidimensional NMR (nD) spectra. The sample is virtually divided into a discrete number of nonoverlapping slices that relax independently during consecutive scans of the experiment, affording a substantial reduction in the interscan relaxation delay and the total experiment time. PALSY was tested for the acquisition of three experiments 2D COSY, 2D DQF‐COSY and 2D TQF‐COSY in parallel, affording a time‐saving factor of 3–4. Some unique advantages are that the achievable resolution in any dimension is not compromised in any way: it uses conventional NMR data processing, it is not prone to generate spectral artifacts, and once calibrated, it can be used routinely with these and other combinations of NMR spectra. Copyright © 2010 John Wiley & Sons, Ltd.     

Neurotoxin II bound to acetylcholine receptors in native membranes studied by dynamic nuclear polarization NMR

Methods enabling structural studies of membrane-integrated receptor systems without the necessity of purification provide an attractive perspective in membrane protein structural and molecular biology. This has become feasible in principle since the advent of dynamic nuclear polarization (DNP) magic-angle-spinning NMR spectroscopy, which delivers the required sensitivity. In this pilot study, we observed well-resolved solid-state NMR spectra of extensively (13)C-labeled neurotoxin II bound to the nicotinic acetylcholine receptor (nAChR) in native membranes. We show that TOTAPOL, a biradical required for DNP, is localized at membrane and protein surfaces. The concentration of active, membrane-attached biradical decreases with time, probably because of reactive components of the membrane preparation. An optimal distribution of active biradical has strong effects on the NMR data. The presence of inactive TOTAPOL in membrane-proximal situations but active biradical in the surrounding water/glycerol "glass" leads to well-resolved spectra, yet a considerable enhancement (ε = 12) is observed. The resulting spectra of a protein ligand bound to its receptor are paving the way for further DNP investigations of proteins embedded in native membrane patches.     

Combining isoelectric point-based fractionation, liquid chromatography and mass spectrometry to improve peptide detection and protein identification

The off-line coupling of an isoelectric trapping device termed membrane separated wells for isoelectric focusing and trapping (MSWIFT) to mass spectrometry-based proteomic studies is described. The MSWIFT is a high capacity, high-throughput, mass spectrometry-compatible isoelectric trapping device that provides isoelectric point (pI)-based separations of complex mixtures of peptides. In MSWIFT, separation and analyte trapping are achieved by migrating the peptide ions through membranes having fixed pH values until the peptide pI is bracketed by the pH values of adjacent membranes. The pH values of the membranes can be tuned, thus affording a high degree of experimental flexibility. Specific advantages of using MSWIFT for sample prefractionation include: (1) small sample volumes (～200 μL), (2) customized membranes over a large pH range, (3) flexibility in the number of desired fractions, (4) membrane compatibility with a variety of solvents systems, and (5) resulting fractions do not require sample cleanup before MS analysis. Here, we demonstrate the utility of MSWIFT for mass spectrometry-based detection of peptides in improving dynamic range and the reduction of ion suppression effects for high-throughput separations of tryptic peptides.