蛋白质溶液NMR结构测定的一些新进展

新的标记技术的进展和采用稀释的液晶作为溶剂以提供额外的结构信息,提高了核磁共振技术测定蛋白质溶液三维结构的精度,扩大了分子质量测定范围.目前已经利用多维 ¹⁵N,¹³C,²H标记NMR测定了许多分子质量为30 ku左右的蛋白质溶液结构,这一上限可能还会被进一步提高.     

A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

Structural information on RNA, emerging more and more as a major regulator in gene expression, dramatically lags behind compared with information on proteins. Although NMR spectroscopy has proven to be an excellent tool to solve RNA structures, it is hampered by the severe spectral resonances overlap found in RNA, limiting its use for large RNA molecules. Segmental isotope labeling of RNA or ligation of a chemically synthesized RNA containing modified nucleotides with an unmodified RNA fragment have proven to have high potential in overcoming current limitations in obtaining structural information on RNA. However, low yields, cumbersome preparations and sequence requirements have limited its broader application in structural biology. Here we present a fast and efficient approach to generate multiple segmentally labeled RNAs with virtually no sequence requirements with very high yields (up to 10-fold higher than previously reported). We expect this approach to open new avenues in structural biology of RNA.     

Conjoined Use of EM and NMR in RNA Structure Refinement

More than 40% of the RNA structures have been determined using nuclear magnetic resonance (NMR) technique. NMR mainly provides local structural information of protons and works most effectively on relatively small biomacromolecules. Hence structural characterization of large RNAs can be difficult for NMR alone. Electron microscopy (EM) provides global shape information of macromolecules at nanometer resolution, which should be complementary to NMR for RNA structure determination. Here we developed a new energy term in Xplor-NIH against the density map obtained by EM. We conjointly used NMR and map restraints for the structure refinement of three RNA systems — U2/U6 small-nuclear RNA, genome-packing motif (Ψ^CD)₂ from Moloney murine leukemia virus, and ribosome-binding element from turnip crinkle virus. In all three systems, we showed that the incorporation of a map restraint, either experimental or generated from known PDB structure, greatly improves structural precision and accuracy. Importantly, our method does not rely on an initial model assembled from RNA duplexes, and allows full torsional freedom for each nucleotide in the torsion angle simulated annealing refinement. As increasing number of macromolecules can be characterized by both NMR and EM, the marriage between the two techniques would enable better characterization of RNA three-dimensional structures.     

Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of ¹³C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-¹³C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-¹³C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.     

RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation (¹H–¹⁵N 2D HMQC) and proton–proton nuclear Overhauser enhancement spectroscopy (¹H–¹H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino resonances for a majority of the NMR resonances, even when the initial predictions are only modestly accurate. RNA-PAIRS is available as a public web-server at .     

Asymmetry of <Superscript>13</Superscript>C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of ¹³C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-¹³C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-¹³C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.     

Site-selective <Superscript>13</Superscript>C labeling of histidine and tryptophan using ribose

Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific ¹³C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct ¹³C chemical shifts and multiple magnetically equivalent ¹H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with ¹³C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated ¹H-¹³C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.     

An integrative NMR-SAXS approach for structural determination of large RNAs defines the substrate-free state of a trans-cleaving Neurospora Varkud Satellite ribozyme

The divide-and-conquer strategy is commonly used for protein structure determination, but its applications to high-resolution structure determination of RNAs have been limited. Here, we introduce an integrative approach based on the divide-and-conquer strategy that was undertaken to determine the solution structure of an RNA model system, the Neurospora VS ribozyme. NMR and SAXS studies were conducted on a minimal trans VS ribozyme as well as several isolated subdomains. A multi-step procedure was used for structure determination that first involved pairing refined NMR structures with SAXS data to obtain structural subensembles of the various subdomains. These subdomain structures were then assembled to build a large set of structural models of the ribozyme, which was subsequently filtered using SAXS data. The resulting NMR-SAXS structural ensemble shares several similarities with the reported crystal structures of the VS ribozyme. However, a local structural difference is observed that affects the global fold by shifting the relative orientation of the two three-way junctions. Thus, this finding highlights a global conformational change associated with substrate binding in the VS ribozyme that is likely critical for its enzymatic activity. Structural studies of other large RNAs should benefit from similar integrative approaches that allow conformational sampling of assembled fragments.     

Automated and assisted RNA resonance assignment using NMR chemical shift statistics

The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and ¹H and ¹³C chemical shifts. Statistics of resonances from regularly Watson–Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1′/C1′ chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92–100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding ¹³C resonances, which provides an important step toward automated structure determination of RNAs.     

RNA metabolism during light-induced chloroplast development in euglena

Methods are described which provide good recoveries of non-degraded chloroplast and non-chloroplast RNAs from Euglena gracilis var. bacillaris. These have been characterized by comparing the RNA from W₃BUL (an aplastidic mutant of Euglena), with that of wild-type cells which have been resolved into chloroplast and non-chloroplast fractions. Using E. coli RNA as a standard, the RNAs from W₃BUL and from the non-chloroplast fraction of green cells exhibit optical density peaks, upon sucrose gradient centrifugation, at 4S, 10S, and 19S. The chloroplast fraction exhibits optical density peaks at 19S and 14S with the 19S component predominating. Application of various techniques for the separation of RNAs to the problem of separating the chloroplast and non-chloroplast RNAs, without prior separation of the organelle, have not proven successful.

³²P_i is readily incorporated into RNA by cells undergoing light-induced chloroplast development, and fractionation at the end of development reveals that although chloroplast RNAs have a higher specific activity, the other RNAs of the cells are significantly labeled as well. The succession of labeling patterns of total cellular RNA as light-induced chloroplast development proceeds are displayed and reveal that all RNA species mentioned above eventually become labeled. In contrast, cells kept in darkness during this period incorporate little ³²P_i into any RNA fraction. In addition, a heavy RNA component, designated as 28S, while representing a negligible fraction of the total RNA, becomes significantly labeled during the first 24 hours of illumination. While there is light stimulated uptake of ³²P_i into the cells, this uptake is never limiting in the light or dark, for RNA labeling.

On the basis of these findings, we suggest that extensive activation of non-chloroplast RNA labeling during chloroplast development is the result of the activation of the cellular synthetic machinery external to the chloroplast necessary to provide metabolic precursors for plastid development. Thus the plastid is viewed as an auxotrophic resident within the cell during development. Other possibilities for interaction at this and other levels are also discussed.

     

Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote <Emphasis Type="Italic">Pichia pastoris</Emphasis>

¹³C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific ¹³C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient ¹³C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.     

An Economical Method for Production of 2H,13CH3-Threonine for Solution NMR Studies of Large Protein Complexes: Application to the 670 kDa Proteasome

NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby ¹³CH₃ methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-²H; β−²H;γ-¹³C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa proteasome core particle, where high quality Thr ¹³C,¹H correlation spectra are obtained that could not be generated from samples prepared with commercially available U-[¹³C,¹H]-Thr.     

Structural representative of the protein family PF14466 has a new fold and establishes links with the C2 and PLAT domains from the widely distant Pfams PF00168 and PF01477

The domain of unknown function (DUF) YP_001302112.1, a protein secreted by the human intestinal microbita, has been determined by NMR and represents the first structure for the Pfam PF14466. Its NMR structure is classified as a new fold, which, nonetheless, shows limited similarities with representatives of the PLAT/LH2 domains from PF01477 and the C2 domains from PF00168, both of which bind Ca²⁺ for their physiological functions. Further experiments revealed affinity of YP_001302112.1 for Ca²⁺, and the NMR structure in the presence of CaCl₂ was better defined than that of the apo‐protein. Overall, these NMR structures establish a new connection between structural representatives from two widely different Pfams that include the calcium‐binding domain of a sialidase from Vibrio cholerae and the α‐toxin from Clostridium perfrigens, whereby these two proteins have only 7% sequence identity. Furthermore, it provides information toward the functional annotation of YP_001302112.1, based on its capacity to bind Ca²⁺, and thus adds to the structural and functional coverage of the protein sequence universe. © 2013 The Protein Society     

One simple step in the identification of the cofactors signals, one giant leap for the solution structure determination of multiheme proteins

Multiheme proteins play major roles in various biological systems. Structural information on these systems in solution is crucial to understand their functional mechanisms. However, the presence of numerous proton-containing groups in the heme cofactors and the magnetic properties of the heme iron, in particular in the oxidised state, complicates significantly the assignment of the NMR signals. Consequently, the multiheme proteins superfamily is extremely under-represented in structural databases, which constitutes a severe bottleneck in the elucidation of their structural-functional relationships. In this work, we present a strategy that simplifies the assignment of the NMR signals in multiheme proteins and, concomitantly, their solution structure determination, using the triheme cytochrome PpcA from the bacterium Geobacter sulfurreducens as a model. Cost-effective isotopic labeling was used to double label (¹³C/¹⁵N) the protein in its polypeptide chain, with the correct folding and heme post-translational modifications. The combined analysis of ¹H-¹³C HSQC NMR spectra obtained for labeled and unlabeled samples of PpcA allowed a straight discrimination between the heme cofactors and the polypeptide chain signals and their confident assignment. The results presented here will be the foundations to assist solution structure determination of multiheme proteins, which are still very scarce in the literature.     

Expression of human chorionic gonadotropin uniformly labeled with NMR isotpes in Chinese hamster ovary cells: An advance toward rapid determination of glycoprotein structures

Summary Most secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure requires confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein back bone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with ¹³C and ¹⁵N isotopes. We report the first such uniform labeling of a glycoprotein using a universal ¹³C-and ¹⁵N-labeling medium to express ¹³C, ¹⁵N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The ¹³C, ¹⁵N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.To whom correspondence should be addressed.     

A procedure to validate and correct the 13C chemical shift calibration of RNA datasets

Chemical shifts reflect the structural environment of a certain nucleus and can be used to extract structural and dynamic information. Proper calibration is indispensable to extract such information from chemical shifts. Whereas a variety of procedures exist to verify the chemical shift calibration for proteins, no such procedure is available for RNAs to date. We present here a procedure to analyze and correct the calibration of ¹³C NMR data of RNAs. Our procedure uses five ¹³C chemical shifts as a reference, each of them found in a narrow shift range in most datasets deposited in the Biological Magnetic Resonance Bank. In 49 datasets we could evaluate the ¹³C calibration and detect errors or inconsistencies in RNA ¹³C chemical shifts based on these chemical shift reference values. More than half of the datasets (27 out of those 49) were found to be improperly referenced or contained inconsistencies. This large inconsistency rate possibly explains that no clear structure–¹³C chemical shift relationship has emerged for RNA so far. We were able to recalibrate or correct 17 datasets resulting in 39 usable ¹³C datasets. 6 new datasets from our lab were used to verify our method increasing the database to 45 usable datasets. We can now search for structure–chemical shift relationships with this improved list of ¹³C chemical shift data. This is demonstrated by a clear relationship between ribose ¹³C shifts and the sugar pucker, which can be used to predict a C2′- or C3′-endo conformation of the ribose with high accuracy. The improved quality of the chemical shift data allows statistical analysis with the potential to facilitate assignment procedures, and the extraction of restraints for structure calculations of RNA.     

Structural Studies of Bcl-xL/ligand Complexes using 19F NMR

Fluorine atoms are often incorporated into drug molecules as part of the lead optimization process in order to improve affinity or modify undesirable metabolic and pharmacokinetic profiles. From an NMR perspective, the abundance of fluorinated drug leads provides an exploitable niche for structural studies using ¹⁹F NMR in the drug discovery process. As ¹⁹F has no interfering background signal from biological sources, ¹⁹F NMR studies of fluorinated drugs bound to their protein receptors can yield easily interpretable and unambiguous structural constraints. ¹⁹F can also be selectively incorporated into proteins to obtain additional constraints for structural studies. Despite these advantages, ¹⁹F NMR has rarely been exploited for structural studies due to its broad lines in macromolecules and their ligand complexes, leading to weak signals in ¹H/¹⁹F heteronuclear NOE experiments. Here we demonstrate several different experimental strategies that use ¹⁹F NMR to obtain ligand–protein structural constraints for ligands bound to the anti-apoptotic protein Bcl-xL, a drug target for anti-cancer therapy. These examples indicate the applicability of these methods to typical structural problems encountered in the drug development process.     

Database proton NMR chemical shifts for RNA signal assignment and validation

The Biological Magnetic Resonance Data Bank contains NMR chemical shift depositions for 132 RNAs and RNA-containing complexes. We have analyzed the ¹H NMR chemical shifts reported for non-exchangeable protons of residues that reside within A-form helical regions of these RNAs. The analysis focused on the central base pair within a stretch of three adjacent base pairs (BP triplets), and included both Watson–Crick (WC; G:C, A:U) and G:U wobble pairs. Chemical shift values were included for all 4³ possible WC-BP triplets, as well as 137 additional triplets that contain one or more G:U wobbles. Sequence-dependent chemical shift correlations were identified, including correlations involving terminating base pairs within the triplets and canonical and non-canonical structures adjacent to the BP triplets (i.e. bulges, loops, WC and non-WC BPs), despite the fact that the NMR data were obtained under different conditions of pH, buffer, ionic strength, and temperature. A computer program (RNAShifts) was developed that enables convenient comparison of RNA ¹H NMR assignments with database predictions, which should facilitate future signal assignment/validation efforts and enable rapid identification of non-canonical RNA structures and RNA-ligand/protein interaction sites.