Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (¹H-MRS) is a novel non-destructive technique that improves spectral line-widths and allows high-resolution spectra to be obtained from extracts, intact cells, cell cultures, and more importantly intact tissue to investigate relationships between metabolites and cellular processes. In vivo HRMAS ¹H-MRS studies have yet to be reported in the live fruit fly Drosophila melanogaster.Drosophila, as a simpler genetic organism, allows the multiple biological functions and various evolutionarily conserved signaling pathways to be examined at the whole organism level and it is a useful model for investigating genetics and physiology. To this end, we developed and implemented an in vivo HRMAS ¹H-MRS method to investigate live Drosophila at 14.1 T. Here, we outline an HRMAS ¹H-MRS protocol for the molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. This technique is a novel, in vivo, non-destructive Drosophila metabolite measurement approach, which enables the identification of disease biomarkers and thus may contribute to novel therapeutic development.Download video file.^{(85M, mp4)}     

Discrimination of Basal Cell Carcinoma from Normal Skin Tissue Using High-Resolution Magic Angle Spinning 1H NMR Spectroscopy

High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy is a useful tool for investigating the metabolism of various cancers. Basal cell carcinoma (BCC) is the most common skin cancer. However, to our knowledge, data on metabolic profiling of BCC have not been reported in the literature. The objective of the present study was to investigate the metabolic profiling of cutaneous BCC using HR-MAS ¹H NMR spectroscopy. HR-MAS ¹H NMR spectroscopy was used to analyze the metabolite profile and metabolite intensity of histopathologically confirmed BCC tissues and normal skin tissue (NST) samples. The metabolic intensity normalized to the total spectral intensities in BCC and NST was compared, and multivariate analysis was performed with orthogonal partial least-squares discriminant analysis (OPLS-DA). P values < 0.05 were considered statistically significant. Univariate analysis revealed 9 metabolites that showed statistically significant difference between BCC and NST. In multivariate analysis, the OPLS-DA models built with the HR-MAS NMR metabolic profiles revealed a clear separation of BCC from NST. The receiver operating characteristic curve generated from the results revealed an excellent discrimination of BCC from NST with an area under the curve (AUC) value of 0.961. The present study demonstrated that the metabolite profile and metabolite intensity differ between BCC and NST, and that HR-MAS ¹H NMR spectroscopy can be a valuable tool in the diagnosis of BCC.     

Three-Dimensional Structure of CAP-Gly Domain of Mammalian Dynactin Determined by Magic Angle Spinning NMR Spectroscopy: Conformational Plasticity and Interactions with End-Binding Protein EB1

Microtubules and their associated proteins play important roles in vesicle and organelle transport, cell motility and cell division. Perturbation of these processes by mutation typically gives rise to severe pathological conditions. In our efforts to obtain atomic information on microtubule-associated protein/microtubule interactions with the goal to understand mechanisms that might potentially assist in the development of treatments for these diseases, we have determined the three-dimensional structure of CAP-Gly (cytoskeleton-associated protein, glycine-rich) domain of mammalian dynactin by magic angle spinning NMR spectroscopy. We observe two conformations in the β2 strand encompassing residues T43-V44-A45, residues that are adjacent to the disease-associated mutation, G59S. Upon binding of CAP-Gly to microtubule plus-end tracking protein EB1, the CAP-Gly shifts to a single conformer. We find extensive chemical shift perturbations in several stretches of residues of CAP-Gly upon binding to EB1, from which we define accurately the CAP-Gly/EB1 binding interface. We also observe that the loop regions may exhibit unique flexibility, especially in the GKNDG motif, which participates in the microtubule binding. This study in conjunction with our previous reports suggests that conformational plasticity is an intrinsic property of CAP-Gly likely due to its unusually high loop content and may be required for its biological functions.     

Model-based Analysis of ChIP-Seq (MACS)

We present Model-based Analysis of ChIP-Seq data, MACS, which analyzes data generated by short read sequencers such as Solexa's Genome Analyzer. MACS empirically models the shift size of ChIP-Seq tags, and uses it to improve the spatial resolution of predicted binding sites. MACS also uses a dynamic Poisson distribution to effectively capture local biases in the genome, allowing for more robust predictions. MACS compares favorably to existing ChIP-Seq peak-finding algorithms, and is freely available.     

Magic angle spinning NMR study of interaction of N-terminal sequence of dermorphin (Tyr-d-Ala-Phe-Gly) with phospholipids

Two modifications of the Tyr-d-Ala-Phe-Gly tetrapeptide with different C-terminal groups (Tyr-d-Ala-Phe-Gly-OH 1 and Tyr-d-Ala-Phe-Gly-NH(2)2) were investigated by various nuclear magnetic resonance sequences under magic angle spinning. The structural constraints obtained from the magic angle spinning nuclear magnetic resonance measurements suggest that both peptides are aligned on the surface of the membrane and that the sandwich-like π-CH(3)-π arrangement of the pharmacophore is preserved. The influence of the chemical modification of the C-terminal residue of 1 and 2 on their interaction with phosphate group of the phospholipid in the subgel phase L(c) and the conformation of the peptides in the liquid crystalline phase L(α) are discussed. The correlation between the X-ray structure of 1 in the solid state and 1 embedded into a membrane in the L(c) phase is presented on the basis of the comparative analysis of the two-dimensional (13)C-(13)C dipolar-assisted rotational resonance cross-peaks and the (13)C isotropic chemical shifts.     

Multiple alternate channel selection (MACS): new versatility for zonal centrifuge rotors

We describe a system of Multiple Alternate Channel Selection (MACS) for zonal rotors in which conical adapters, which attach to the dynamic seal, connect at different vertical heights to any pair of channels in the core. In this way one can pump fluids to and from different positions or compartments in the same rotor during a single run.The specific applications described in this paper include edge-unloading of sensitive particles through the center channel of the dynamic seal and direct insertion of samples at a predetermined radius. Other applications are outlined.     

High-Resolution Two-Dimensional J-Resolved NMR Spectroscopy for Biological Systems

NMR spectroscopy is a principal tool in metabolomic studies and can, in theory, yield atom-level information critical for understanding biological systems. Nevertheless, NMR investigations on biological tissues generally have to contend with field inhomogeneities originating from variations in macroscopic magnetic susceptibility; these field inhomogeneities broaden spectral lines and thereby obscure metabolite signals. The congestion in one-dimensional NMR spectra of biological tissues often leads to ambiguities in metabolite identification and quantification. We propose an NMR approach based on intermolecular double-quantum coherences to recover high-resolution two-dimensional (2D) J-resolved spectra from inhomogeneous magnetic fields, such as those created by susceptibility variations in intact biological tissues. The proposed method makes it possible to acquire high-resolution 2D J-resolved spectra on intact biological samples without recourse to time-consuming shimming procedures or the use of specialized hardware, such as magic-angle-spinning probes. Separation of chemical shifts and J couplings along two distinct dimensions is achieved, which reduces spectral crowding and increases metabolite specificity. Moreover, the apparent J coupling constants observed are magnified by a factor of 3, facilitating the accurate measurement of small J couplings, which is useful in metabolic analyses. Dramatically improved spectral resolution is demonstrated in our applications of the technique on pig brain tissues. The resulting spectra contain a wealth of chemical shift and J-coupling information that is invaluable for metabolite analyses. A spatially localized experiment applied on an intact fish (Crossocheilus siamensis) reveals the promise of the proposed method in in vivo metabolite studies. Moreover, the proposed method makes few demands on spectrometer hardware and therefore constitutes a convenient and effective manner for metabonomics study of biological systems.     

Magnetic activated cell sorting (MACS): utility in assisted reproduction

Assisted reproductive techniques (ART) have now been extensively incorporated in the management of infertile couples. But even after rapid methodological and technological advances the success rates of these procedures have been below expectations. This has led to development of many sperm preparation protocols to obtain an ideal semen sample for artificial reproduction. Sperm apoptosis has been heavily linked to failures in reproductive techniques. One of the earliest changes shown by apoptotic spermatozoa is externalization of phosphatidyl serine. Magnetic activated cell sorting (MACS) is a novel sperm preparation technique that separates apoptotic and non-apoptotic spermatozoa based on the expression of phosphatidylserine. This has led to the incorporation of MACS as a sperm preparation technique. The review highlights the principle and mechanism of this novel technique and enumerates its advantages as a sperm preparation technique. Its utility in ART as an efficient tool for sperm recovery and its application in cryopreservation of semen samples is also explained.     

Multiple alignment of complete sequences (MACS) in the post-genomic era

Multiple alignment, since its introduction in the early seventies, has become a cornerstone of modern molecular biology. It has traditionally been used to deduce structure / function by homology, to detect conserved motifs and in phylogenetic studies. There has recently been some renewed interest in the development of multiple alignment techniques, with current opinion moving away from a single all-encompassing algorithm to iterative and / or co-operative strategies. The exploitation of multiple alignments in genome annotation projects represents a qualitative leap in the functional analysis process, opening the way to the study of the co-evolution of validated sets of proteins and to reliable phylogenomic analysis. However, the alignment of the highly complex proteins detected by today's advanced database search methods is a daunting task. In addition, with the explosion of the sequence databases and with the establishment of numerous specialized biological databases, multiple alignment programs must evolve if they are to successfully rise to the new challenges of the post-genomic era. The way forward is clearly an integrated system bringing together sequence data, knowledge-based systems and prediction methods with their inherent unreliability. The incorporation of such heterogeneous, often non-consistent, data will require major changes to the fundamental alignment algorithms used to date. Such an integrated multiple alignment system will provide an ideal workbench for the validation, propagation and presentation of this information in a format that is concise, clear and intuitive.     

Triosephosphate Isomerase: N and C Chemical Shift Assignments and Conformational Change upon Ligand Binding by Magic-Angle Spinning Solid-State NMR Spectroscopy

Microcrystalline uniformly ¹³C,¹⁵N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.     

Microfabricated post-array-detectors (mPADs): an approach to isolate mechanical forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts. Traction forces are generated through myosin-actin interactions and play an important role in our physiology. During development, they enable cells to move from one location to the next in order to form the early structures of tissue. Traction forces help in the healing processes. They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body. These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor. The most common method by which to study cells in vitro has been to use a glass or polystyrene dish. However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces. Our lab has developed a technique to overcome these limitations. The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process. The pillars we use are 3 microm in diameter, 10 microm tall, and are configured in a regular array with 9 microm center-to-center spacing. But these physical dimensions can be readily varied to accommodate a variety of studies. We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS. We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections. We call these substrates microfabricated post-array-detectors, or mPADs. Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.     

Comparison of Different Torsion Angle Approaches for NMR Structure Determination

A new procedure for NMR structure determination, based on the Internal Coordinate Molecular Dynamics (ICMD) approach, is presented. The method finds biopolymer conformations that satisfy usual NMR-derived restraints by using high temperature dynamics in torsion angle space. A variable target function algorithm gradually increases the number of NOE-based restraints applied, with the treatment of ambiguous and floating restraints included. This soft procedure allows combining artificially high temperature with a general purpose force-field including Coulombic and Lennard-Jones non-bonded interactions, which improves the quality of the ensemble of conformations obtained in the gas-phase. The new method is compared to existing algorithms by using the structures of eight ribosomal proteins earlier obtained with state-of-the-art procedures and included into the RECOORD database [Nederveen, A., Doreleijers, J., Vranken, W., Miller, Z., Spronk, C., Nabuurs, S., Guntert, P., Livny, M., Markley, M., Nilges, M., Ulrich, E., Kaptein, R. and Bonvin, A.M. (2005) Proteins, 59, 662–672]. For the majority of tested proteins, the ICMD algorithm shows similar convergence and somewhat better quality Z scores for the ϕ, ψ distributions. The new method is more computationally demanding although the overall load is reasonable.     

High-Resolution Two-Dimensional J-Resolved NMR Spectroscopy for Biological Systems

NMR spectroscopy is a principal tool in metabolomic studies and can, in theory, yield atom-level information critical for understanding biological systems. Nevertheless, NMR investigations on biological tissues generally have to contend with field inhomogeneities originating from variations in macroscopic magnetic susceptibility; these field inhomogeneities broaden spectral lines and thereby obscure metabolite signals. The congestion in one-dimensional NMR spectra of biological tissues often leads to ambiguities in metabolite identification and quantification. We propose an NMR approach based on intermolecular double-quantum coherences to recover high-resolution two-dimensional (2D) J-resolved spectra from inhomogeneous magnetic fields, such as those created by susceptibility variations in intact biological tissues. The proposed method makes it possible to acquire high-resolution 2D J-resolved spectra on intact biological samples without recourse to time-consuming shimming procedures or the use of specialized hardware, such as magic-angle-spinning probes. Separation of chemical shifts and J couplings along two distinct dimensions is achieved, which reduces spectral crowding and increases metabolite specificity. Moreover, the apparent J coupling constants observed are magnified by a factor of 3, facilitating the accurate measurement of small J couplings, which is useful in metabolic analyses. Dramatically improved spectral resolution is demonstrated in our applications of the technique on pig brain tissues. The resulting spectra contain a wealth of chemical shift and J-coupling information that is invaluable for metabolite analyses. A spatially localized experiment applied on an intact fish (Crossocheilus siamensis) reveals the promise of the proposed method in in vivo metabolite studies. Moreover, the proposed method makes few demands on spectrometer hardware and therefore constitutes a convenient and effective manner for metabonomics study of biological systems.     

Zwitterionic Phosphorylated Quinines as Chiral Solvating Agents for NMR Spectroscopy

Because of their unique 3D arrangement, naturally occurring Cinchona alkaloids and their synthetic derivatives have found wide‐ranging applications in chiral recognition. Recently, we determined the enantioselective properties of C‐9‐phosphate mixed triesters of quinine as versatile chiral solvating agents in nuclear magnetic resonance (NMR) spectroscopy. In the current study, we introduce new zwitterionic members of this class of molecules containing a negatively charged phosphate moiety (i.e., ethyl, n‐butyl and phenyl hydrogen quininyl phosphate). An efficient approach for synthesizing these compounds is elaborated, and full characterization, including conformational and autoaggregation phenomena studies, was performed. Therefore, their ability to induce NMR anisochrony of selected enantiomeric substrates (i.e., primarily N‐DNB‐protected amino acids and their methyl esters) was analyzed compared to uncharged diphenyl quininyl phosphate and its positively charged quaternary ammonium hydrochloride salt. In addition, ¹H and ¹³C NMR experiments revealed their enantiodiscrimination potential toward novel analytes, such as secondary amines and nonprotected amino acids. Chirality 27:752–760, 2015. © 2015 Wiley Periodicals, Inc.     

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts. Traction forces are generated through myosin-actin interactions and play an important role in our physiology. During development, they enable cells to move from one location to the next in order to form the early structures of tissue. Traction forces help in the healing processes. They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body. These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor. The most common method by which to study cells in vitro has been to use a glass or polystyrene dish. However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces. Our lab has developed a technique to overcome these limitations. The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process. The pillars we use are 3 μm in diameter, 10 μm tall, and are configured in a regular array with 9 μm center-to-center spacing. But these physical dimensions can be readily varied to accommodate a variety of studies. We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS. We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections. We call these substrates microfabricated post-array-detectors, or mPADs. Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.     

Application of Photoacoustic Phase Angle Spectroscopy (φAS) to Eumelanins and Pheomelanins

A new technique, based on the measurement of the phase angle of the photoacoustic signal, was used for investigating the absorption spectra of melanins in the dry state. The main advantages of such a method are the insensitivity to scattering of the light and the applicability to nontransparent or highly adsorbing and thermally thick samples. The spectra obtained on different kinds of natural and synthetic melanins show much more details than the corresponding ones obtained spectrophotometrically on aqueous suspensions of the pigment. Some relevant features of the spectra still cannot be interpreted, yet we were able to show the presence in eumelanins of dopachrome at 475 nm and melanochrome at 540 nm, and in pheomelanins of compounds previously isolated from red chicken feathers. Moreover, the well known trend of the optical absorption, i.e., its decrease with increasing wavelength, was unambiguously confirmed.     

Magic angle spinning (MAS) NMR: a new tool to study the spatial and electronic structure of photosynthetic complexes

Photosynthesis Research - In the last two decades, Magic Angle Spinning (MAS) NMR has created its own niche in studies involving photosynthetic membrane protein complexes, owing to its ability to...     

In-cell NMR for protein-protein interactions (STINT-NMR)

We describe an in-cell NMR-based method for mapping the structural interactions (STINT-NMR) that underlie protein-protein complex formation. This method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring their interactions using in-cell NMR spectroscopy. The resulting NMR data provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution. Unlike the case where interacting proteins are simultaneously overexpressed in the labeled medium, in STINT-NMR the spectral complexity is minimized because only the target protein is labeled with NMR-active nuclei, which leaves the interactor protein(s) cryptic. This method can be combined with genetic and molecular screens to provide a structural foundation for proteomic studies. The protocol takes 4 d from the initial transformation of the bacterial cells to the acquisition of the NMR spectra.