A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes

A method for measuring intermolecular NOEs in protein complexes based on asymmetric sample deuteration is described. ¹³C/¹H-I,L,V-methyl, U-²H labeled protein is produced using the biosynthetic precursors [-¹³C]--ketobutyrate and [,-¹³C₂]--ketoisovalerate. The labeled protein is mixed with its unlabeled binding partner and a 3D ¹³C-HMQC-NOESY is recorded, yielding unambiguous intermolecular aromatic/methyl NOEs. A simple synthesis of the biosynthetic precursors via reaction of diethyl oxalate with alkyl Grignard compounds is reported. The method is demonstrated for a 35 kDa heterodimeric protein complex dissolved in a CHAPS micelle. This approach will facilitate the solution structure determination of protein/protein, protein/ligand or protein/nucleic acid complexes.These authors contributed equallyThese authors contributed equally     

Simultaneous measurement of intra- and intermolecular NOEs in differentially labeled protein-ligand complexes*

A new NOE strategy is presented that allows the simultaneous observation of intermolecular and intramolecular NOEs between an unlabeled ligand and a ¹³C,¹⁵N-labeled protein. The method uses an adiabatic ¹³C inversion pulse optimized to an empirically observed relationship between ¹ J _CH and carbon chemical shift to selectively invert the protein protons (attached to ¹³C). Two NOESY data sets are recorded where the intermolecular and intramolecular NOESY cross peaks have either equal or opposite signs, respectively. Addition and subtraction yield two NOESY spectra which contain either NOEs within the labeled protein (or unlabeled ligand) or along the binding interface. The method is demonstrated with an application to the B₁₂-binding subunit of Glutamate Mutase from Clostridium tetanomorphum complexed with the B₁₂-nucleotide loop moiety of the natural cofactor adenosylcobalamin (Coenzyme B₁₂).     

Tools for the automated assignment of high-resolution three-dimensional protein NMR spectra based on pattern recognition techniques

One of the major bottlenecks in the determination of proteinstructures by NMR is in the evaluation of the data produced by theexperiments. An important step in this process is assignment, where thepeaks in the spectra are assigned to specific spins within specificresidues. In this paper, we discuss a spin system assignment tool based onpattern recognition techniques. This tool employs user-specified templatesto search for patterns of peaks in the original spectra; these patterns maycorrespond to side-chain or backbone fragments. Multiple spectra willnormally be searched simultaneously to reduce the impact of noise. Thesearch generates a preliminary list of putative assignments, which arefiltered by a set of heuristic algorithms to produce the final results list.Each result contains a set of chemical shift values plus information aboutthe peaks found. The results may be used as input for combinatorialroutines, such as sequential assignment procedures, in place of peak lists.Two examples are presented, in which (i) HCCH-COSY and -TOCSY spectra arescanned for side-chain spin systems; and (ii) backbone spin systems aredetected in a set of spectra comprising HNCA, HN(CO)CA, HNCO, HN(CA)CO,CBCANH and CBCA(CO)NH.     

Solvation study of the non-specific lipid transfer protein from wheat by intermolecular NOEs with water and small organic molecules

Intermolecular nuclear Overhauser effects (NOEs) were measured between the protons of various small solvent or gas molecules and the non-specific lipid transfer protein (ns-LTP) from wheat. Intermolecular NOEs were observed with the hydrophobic pocket in the interior of wheat ns-LTP, which grew in intensity in the order cyclopropane (saturated solution) < methane (140 bar) < ethane (40 bar) < acetonitrile (5% in water) < cyclohexane (saturated solution) < benzene (saturated solution). No intermolecular NOEs were observed with dioxane (5% in water). The intermolecular NOEs were negative for all of the organic molecules tested. Intermolecular NOEs between wheat ns-LTP and water were weak or could not be distinguished from exchange-relayed NOEs. As illustrated by the NOEs with cyclohexane versus dioxane, the hydrophobic pocket in wheat ns-LTP preferably binds non-polar molecules. Yet, polar molecules like acetonitrile can also be accommodated. The pressure dependence of the NOEs between methane and wheat ns-LTP indicated incomplete occupancy, even at 190 bar methane pressure. In general, NOE intensities increased with the size of the ligand molecule and its vapor pressure. NMR of the vapor phase showed excellent resolution between the signals from the gas phase and those from the liquid phase. The vapor concentration of cyclohexane was fivefold higher than that of the dioxane solution, supporting the binding of cyclohexane versus uptake of dioxane.     

TSAR: a program for automatic resonance assignment using 2D cross-sections of high dimensionality, high-resolution spectra

While NMR studies of proteins typically aim at structure, dynamics or interactions, resonance assignments represent in almost all cases the initial step of the analysis. With increasing complexity of the NMR spectra, for example due to decreasing extent of ordered structure, this task often becomes both difficult and time-consuming, and the recording of high-dimensional data with high-resolution may be essential. Random sampling of the evolution time space, combined with sparse multidimensional Fourier transform (SMFT), allows for efficient recording of very high dimensional spectra (≥4 dimensions) while maintaining high resolution. However, the nature of this data demands for automation of the assignment process. Here we present the program TSAR (Tool for SMFT-based Assignment of Resonances), which exploits all advantages of SMFT input. Moreover, its flexibility allows to process data from any type of experiments that provide sequential connectivities. The algorithm was tested on several protein samples, including a disordered 81-residue fragment of the δ?subunit of RNA polymerase from Bacillus subtilis containing various repetitive sequences. For our test examples, TSAR achieves a high percentage of assigned residues without any erroneous assignments.     

Solvation study of the non-specific lipid transfer protein from wheat by intermolecular NOEs with water and small organic molecules

Intermolecular nuclear Overhauser effects (NOEs) were measured between the protons of various small solvent or gas molecules and the non-specific lipid transfer protein (ns-LTP) from wheat. Intermolecular NOEs were observed with the hydrophobic pocket in the interior of wheat ns-LTP, which grew in intensity in the order cyclopropane (saturated solution) < methane (140 bar) < ethane (40 bar) < acetonitrile (5% in water) < cyclohexane (saturated solution) < benzene (saturated solution). No intermolecular NOEs were observed with dioxane (5% in water). The intermolecular NOEs were negative for all of the organic molecules tested. Intermolecular NOEs between wheat ns-LTP and water were weak or could not be distinguished from exchange-relayed NOEs. As illustrated by the NOEs with cyclohexane versus dioxane, the hydrophobic pocket in wheat ns-LTP preferably binds non-polar molecules. Yet, polar molecules like acetonitrile can also be accommodated. The pressure dependence of the NOEs between methane and wheat ns-LTP indicated incomplete occupancy, even at 190 bar methane pressure. In general, NOE intensities increased with the size of the ligand molecule and its vapor pressure. NMR of the vapor phase showed excellent resolution between the signals from the gas phase and those from the liquid phase. The vapor concentration of cyclohexane was fivefold higher than that of the dioxane solution, supporting the binding of cyclohexane versus uptake of dioxane.     

Automated combined assignment of NOESY spectra and three-dimensional protein structure determination

A procedure for automated protein structure determination is presented that is based on an iterative procedure during which the NOESY peak list assignment and the structure calculation are performed simultaneously. The input consists of a list of NOESY peak positions and a list of chemical shifts as obtained from sequence-specific resonance assignment. For the present applications of this approach the previously introduced NOAH routine was implemented in the distance geometry program DIANA. As an illustration, experimental 2D and 3D NOESY cross-peak lists of six proteins have been analyzed, for which complete sequence-specific ¹H assignments are available for the polypeptide backbone and the amino acid side chains. The automated method assigned 70–90% of all NOESY cross peaks, which is on average 10% less than with the interactive approach, and only between 0.8% and 2.4% of the automatically assigned peaks had a different assignment than in the corresponding manually assigned peak lists. The structures obtained with NOAH/DIANA are in close agreement with those from manually assigned peak lists, and with both approaches the residual constraint violations correspond to high-quality NMR structure determinations. Systematic comparisons of the bundles of conformers that represent corresponding automatically and interactively determined structures document the absence of significant bias in either approach, indicating that an important step has been made towards automation of structure determination from NMR spectra.     

IBIS--a tool for automated sequential assignment of protein spectra from triple resonance experiments

We have developed a tool for computer-assisted assignments of protein NMR spectra from triple resonance data. The program is designed to resemble established manual assignment procedures as closely as possible. IBIS exports its results in XEASY format. Thus, using IBIS the operator has continuous visual and accounting control over the progress of the assignment procedure. IBIS achieves complete assignments for those residues that exhibit sequential triple resonance connectivities within a few hours or days.     

Spectral editing of two-dimensional magic-angle-spinning solid-state NMR spectra for protein resonance assignment and structure determination

Several techniques for spectral editing of 2D ¹³C?C¹³C correlation NMR of proteins are introduced. They greatly reduce the spectral overlap for five common amino acid types, thus simplifying spectral assignment and conformational analysis. The carboxyl (COO) signals of glutamate and aspartate are selected by suppressing the overlapping amide N?CCO peaks through ¹³C?C¹⁵N dipolar dephasing. The sidechain methine (CH) signals of valine, lecuine, and isoleucine are separated from the overlapping methylene (CH₂) signals of long-chain amino acids using a multiple-quantum dipolar transfer technique. Both the COO and CH selection methods take advantage of improved dipolar dephasing by asymmetric rotational-echo double resonance (REDOR), where every other ??-pulse is shifted from the center of a rotor period t_r by about 0.15 t_r. This asymmetry produces a deeper minimum in the REDOR dephasing curve and enables complete suppression of the undesired signals of immobile segments. Residual signals of mobile sidechains are positively identified by dynamics editing using recoupled ¹³C?C¹H dipolar dephasing. In all three experiments, the signals of carbons within a three-bond distance from the selected carbons are detected in the second spectral dimension via ¹³C spin exchange. The efficiencies of these spectral editing techniques range from 60?% for the COO and dynamic selection experiments to 25?% for the CH selection experiment, and are demonstrated on well-characterized model proteins GB1 and ubiquitin.     

Ansig for Windows: An interactive computer program for semiautomatic assignment of protein NMR spectra

Assignment of NMR spectra is a prerequisite for structure determination of proteins using NMR. The time spent on the assignment is comparatively long compared to that spent on other parts in the protein structure determination process, but it can be shortened by using either interactive or fully automated computer programs. To benefit from the advantages of both types of program we have developed a version of the interactive assignment program ANSIG to include automatized, yet user-supervised, routines. The new program includes tools for (i) semiautomatic sequential assignment, (ii) plotting of distances from PDB structure files directly in NMR spectra and (iii) statistical analysis of distance restraint violations with the possibility to directly zoom to violated NOEs in NOESY spectra.     

Distinguishing crystallographic from biological interfaces in protein complexes: role of intermolecular contacts and energetics for classification

Background

Study of macromolecular assemblies is fundamental to understand functions in cells. X-ray crystallography is the most common technique to solve their 3D structure at atomic resolution. In a crystal, however, both biologically-relevant interfaces and non-specific interfaces resulting from crystallographic packing are observed. Due to the complexity of the biological assemblies currently tackled, classifying those interfaces, i.e. distinguishing biological from crystal lattice interfaces, is not trivial and often prone to errors. In this context, analyzing the physico-chemical characteristics of biological/crystal interfaces can help researchers identify possible features that distinguish them and gain a better understanding of the systems.

Results

In this work, we are providing new insights into the differences between biological and crystallographic complexes by focusing on “pair-properties” of interfaces that have not yet been fully investigated. We investigated properties such intermolecular residue-residue contacts (already successfully applied to the prediction of binding affinities) and interaction energies (electrostatic, Van der Waals and desolvation). By using the XtalMany and BioMany interface datasets, we show that interfacial residue contacts, classified as a function of their physico-chemical properties, can distinguish between biological and crystallographic interfaces. The energetic terms show, on average, higher values for crystal interfaces, reflecting a less stable interface due to crystal packing compared to biological interfaces. By using a variety of machine learning approaches, we trained a new interface classification predictor based on contacts and interaction energetic features. Our predictor reaches an accuracy in classifying biological vs crystal interfaces of 0.92, compared to 0.88 for EPPIC (one of the main state-of-the-art classifiers reporting same performance as PISA).

Conclusion

In this work we have gained insights into the nature of intermolecular contacts and energetics terms distinguishing biological from crystallographic interfaces. Our findings might have a broader applicability in structural biology, for example for the identification of near native poses in docking. We implemented our classification approach into an easy-to-use and fast software, freely available to the scientific community from http://github.com/haddocking/interface-classifier.

     

A geometric arrangement algorithm for structure determination of symmetric protein homo-oligomers from NOEs and RDCs

Nuclear magnetic resonance (NMR) spectroscopy is a primary tool to perform structural studies of proteins in physiologically-relevant solution conditions. Restraints on distances between pairs of nuclei in the protein, derived from the nuclear Overhauser effect (NOE), provide information about the structure of the protein in its folded state. NMR studies of symmetric protein homo-oligomers present a unique challenge. Using X-filtered NOESY experiments, it is possible to determine whether an NOE restrains a pair of protons across different subunits or within a single subunit, but current experimental techniques are unable to determine in which subunits the restrained protons lie. Consequently, it is difficult to assign NOEs to particular pairs of subunits with certainty, thus hindering the structural analysis of the oligomeric state. Computational approaches are needed to address this subunit ambiguity, but traditional solutions often rely on stochastic search coupled with simulated annealing and simulations of simplified molecular dynamics, which have many tunable parameters that must be chosen carefully and can also fail to report structures consistent with the experimental restraints. In addition, these traditional approaches rarely provide guarantees on running time or solution quality. We reduce the structure determination of homo-oligomers with cyclic symmetry to computing geometric arrangements of unions of annuli in a plane. Our algorithm, disco, runs in expected O(n2) time, where n is the number of distance restraints, potentially assigned ambiguously. disco is guaranteed to report the exact set of oligomer structures consistent with the distance restraints and also with orientational restraints from residual dipolar couplings (RDCs). We demonstrate our method using two symmetric protein complexes: the trimeric E. coli diacylglycerol kinase (DAGK) and a dimeric mutant of the immunoglobulin-binding domain B1 of streptococcal protein G (GB1). In both cases, disco computes oligomer structures with high precision and also finds distance restraints that are either mutually inconsistent or inconsistent with the RDCs. The entire protocol DISCO has been completely automated in a software package that is freely available and open-source at www.cs.duke.edu/donaldlab/software.php.     

Computational detection of protein complexes in AP-MS experiments

Protein complex identification is an important goal of protein-protein interaction analysis. To date, development of computational methods for detecting protein complexes has been largely motivated by genome-scale interaction data sets from high-throughput assays such as yeast two-hybrid or tandem affinity purification coupled with mass spectrometry (TAP-MS). However, due to the popularity of small to intermediate-scale affinity purification-mass spectrometry (AP-MS) experiments, protein complex detection is increasingly discussed in local network analysis. In such data sets, protein complexes cannot be detected using binary interaction data alone because the data contain interactions with tagged proteins only and, as a result, interactions between all other proteins remain unobserved, limiting the scope of existing algorithms. In this article, we provide a pragmatic review of network graph-based computational algorithms for protein complex analysis in global interactome data, without requiring any computational background. We discuss the practical gap in applying these algorithms to recently surging small to intermediate-scale AP-MS data sets, and review alternative clustering algorithms using quantitative proteomics data and their limitations.     

Specific, sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy

More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell's interior are lagging behind. We describe an approach, metal-tagging TEM (METTEM), that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity, and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before.     

Base-type-selective high-resolution 13C edited NOESY for sequential assignment of large RNAs

Extensive spectral overlap presents a major problem for the NMR study of large RNAs. Here we present NMR techniques for resolution enhancement and spectral simplification of fully ¹³C labelled RNA. High-resolution ¹H-¹³C correlation spectra are obtained by combining TROSY-type experiments with multiple-band-selective homonuclear ¹³C decoupling. An additional C-C filter sequence performs base-type-selective spectral editing. Signal loss during the filter is significantly reduced because of TROSY-type spin evolution. These tools can be inserted in any ¹³C-edited multidimensional NMR experiment. As an example we have chosen the ¹³C-edited NOESY which is a crucial experiment for sequential resonance assignment of RNA. Application to a 33-nucleotide RNA aptamer and a 76-nucleotide tRNA illustrates the potential of this new methodology.     

Detection of protein–ligand NOEs with small, weakly binding ligands by combined relaxation and diffusion filtering

The use of a diffusion filter is proposed to suppress the NMRsignals of small organic compounds in the presence of macromolecules.Combined with a spin-echo relaxation filter, the diffusion filter enablesthe selective and simultaneous detection of intermolecularsolvent–protein NOEs in a straightforward two-dimensional NOESYexperiment. Using the intermolecular NOEs observed betweenN,N-dimethylformamide (DMF) and hen egg-white lysozyme in an aqueoussolution containing 2 M DMF, the binding of DMF at thespecificity-determining substrate binding site C of the enzyme was modelled.     

A modified strategy for sequence specific assignment of protein NMR spectra based on amino acid type selective experiments

The determination of the three-dimensional structure of a protein or the study of protein–ligand interactions requires the assignment of all relevant nuclei as an initial step. This is nowadays almost exclusively performed using triple-resonance experiments. The conventional strategy utilizes one or more pairs of three dimensional spectra to obtain redundant information and thus reliable assignments. Here, a modified strategy for obtaining sequence specific assignments based on two dimensional amino acid type selective triple-resonance experiments is proposed. These experiments can be recorded with good resolution in a relatively short time. They provide very specific and redundant information, in particular on sequential connectivities, that drastically increases the ease and reliability of the assignment procedure, done either manually or in an automated fashion. The new strategy is demonstrated with the protein domain PB1 from yeast CDC24p. Dedicated to Rüdiger Winter ( 06.04.2004)     

细胞骨架蛋白4荧光免疫层析检测方法的建立

采用基于免疫层析技术与荧光微球标记技术相结合的方式,建立一种快速、简单的定量检测肝癌肿瘤标记物的方法。依据双抗体夹心原理,将细胞骨架蛋白4(CKAP4)配对抗体分别作为标记与包被抗体,羊抗兔多抗作为质控线包被抗体,制备CKAP4荧光免疫层析试纸条,并对试纸条的线性、精密性、稳定性等各项性能指标进行评价。结果表明,采用时间分辨荧光微球所制备的CKAP4免疫层析试纸条灵敏度高,特异性好,精密性在15%以内,回收率在85%–115%之间,线性范围为25–1 000 pg/mL,可在37℃稳定保存20 d,与商业化的ELISA试剂盒的相关性良好。结论:初步建立了CKAP4荧光免疫层析方法,能够定量检测血清中CKAP4的含量,且具有快速、灵敏、简便、经济、可单人份操作等优点,有望成为肝癌辅助诊疗的新方法。     

Preparation of single molecules and supramolecular complexes for high-resolution metal shadowing

We have compared the appearance and preservation of molecular and supramolecular structures in preparations that were dried in vacuo at room temperature or freeze-dried. Fibrinogen and brain spectrin molecules appear similar in both types of preparation provided that drying at room temperature is performed in the presence of glycerol, which results in an even and reproducible distribution of such molecules (Shotton et al., 1979, J. Mol. Biol. 131, 303-329; Fowler and Erickson, 1979, J. Mol. Biol. 134, 241-249). In the case of crystalline actin sheets, actin filaments, and keratin filaments, freeze-drying preserves structural details that are often completely lost during drying at room temperature, whether or not glycerol is used. On the other hand, keratin filaments prepared by drying in the presence of glycerol display a beaded axial repeat that is probably due to "glycerol decoration." We conclude that although freeze-drying has no clear advantage over glycerol spraying/vacuum-drying in the case of single extended molecules, it may provide insight into the multiple effects of glycerol in specimen preparation. In the case of supramolecular assemblies such as filaments or crystalline sheets, freeze-drying preserves significantly more substructure and surface detail. The loss of such detail during drying at room temperature, probably through collapse phenomena such as distortion and flattening, cannot be prevented by glycerol.