Metalloproteomics: forward and reverse approaches in metalloprotein structural and functional characterization

About one-third of all proteins are associated with a metal. Metalloproteomics is defined as the structural and functional characterization of metalloproteins on a genome-wide scale. The methodologies utilized in metalloproteomics, including both forward (bottom-up) and reverse (top-down) technologies, to provide information on the identity, quantity, and function of metalloproteins are discussed. Important techniques frequently employed in metalloproteomics include classical proteomic tools such as mass spectrometry and 2D gels, immobilized-metal affinity chromatography, bioinformatic sequence analysis and homology modeling, X-ray absorption spectroscopy and other synchrotron radiation based tools. Combinative applications of these techniques provide a powerful approach to understand the function of metalloproteins.     

Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis

An estimated half of all proteins contain a metal, with these being essential for a tremendous variety of biological functions. X-ray crystallography is the major method for obtaining structures at high resolution of these metalloproteins, but there are considerable challenges to obtain intact structures due to the effects of radiation damage. Serial crystallography offers the prospect of determining low-dose synchrotron or effectively damage free XFEL structures at room temperature and enables time-resolved or dose-resolved approaches. Complementary spectroscopic data can validate redox and or ligand states within metalloprotein crystals. In this opinion, we discuss developments in the application of serial crystallographic approaches to metalloproteins and comment on future directions.     

Imaging and speciation of trace elements in biological environment

Mineral elements, often at the trace level, play a considerable role in physiology and pathology of biological systems. Metallogenomics, metalloproteomics, and metallomics are among the emerging disciplines which are critically dependent on spatially resolved concentration maps of trace elements in a cell or tissue, on information on chemical speciation, and on that on metal-binding coordination sites. The mini-review discusses recent progress in analytical techniques for element profiling on the genome scale, biological trace element imaging, and probing, identification and quantification of chemical species in the biological environment. Imaging of the element distribution in cells and tissue sections is becoming possible with sub-micrometer spatial resolution and picogram-level sensitivity owing to advances in laser ablation MS, ion beam and synchrotron radiation X-ray fluorescence microprobes. Progress in nanoflow chromatography and capillary electrophoresis coupled with element specific ICP MS and molecule-specific electrospray MS/MS and MALDI enables speciation of elements in microsamples in a complex biological environment. Laser ablation ICP MS, micro-SXRF, and micro-PIXE allow mapping of trace element distribution in 1D and 2D proteomics gels. The increasing sensitivity of EXAFS and XANES owing to the use of more intense synchrotron beams and efficient focusing optics provide information about oxidation state, fingerprint speciation of metal sites and metal-site structures.     

Structure and composition of myelinated axons: a multimodal synchrotron spectro-microscopy study

We report elemental mappings on the sub-cellular level of myelinated sciatic neurons isolated from wild type mice, with high spatial resolution. The distribution of P, S, Cl, Na, K, Fe, Mn, Cu was imaged in freeze-dried as well as cryo-preserved specimen, using the recently developed cryogenic sample environment at beamline ID21 at the European Synchrotron Radiation Facility (ESRF). In addition, synchrotron radiation based Fourier transform infrared (FTIR) spectromicroscopy was used as a chemically sensitive imaging method. Finally single fiber diffraction in highly focused hard X-ray beams, and soft X-ray microscopy and tomography in absorption contrast are demonstrated as novel techniques for the study of single nerve fibers.     

Medical physics aspects of the synchrotron radiation therapies: Microbeam radiation therapy (MRT) and synchrotron stereotactic radiotherapy (SSRT)

Stereotactic Synchrotron Radiotherapy (SSRT) and Microbeam Radiation Therapy (MRT) are both novel approaches to treat brain tumor and potentially other tumors using synchrotron radiation. Although the techniques differ by their principles, SSRT and MRT share certain common aspects with the possibility of combining their advantages in the future. For MRT, the technique uses highly collimated, quasi-parallel arrays of X-ray microbeams between 50 and 600 keV. Important features of highly brilliant Synchrotron sources are a very small beam divergence and an extremely high dose rate. The minimal beam divergence allows the insertion of so called Multi Slit Collimators (MSC) to produce spatially fractionated beams of typically ∼25–75 micron-wide microplanar beams separated by wider (100–400 microns center-to-center(ctc)) spaces with a very sharp penumbra. Peak entrance doses of several hundreds of Gy are extremely well tolerated by normal tissues and at the same time provide a higher therapeutic index for various tumor models in rodents. The hypothesis of a selective radio-vulnerability of the tumor vasculature versus normal blood vessels by MRT was recently more solidified.SSRT (Synchrotron Stereotactic Radiotherapy) is based on a local drug uptake of high-Z elements in tumors followed by stereotactic irradiation with 80 keV photons to enhance the dose deposition only within the tumor. With SSRT already in its clinical trial stage at the ESRF, most medical physics problems are already solved and the implemented solutions are briefly described, while the medical physics aspects in MRT will be discussed in more detail in this paper.     

Research at the European Synchrotron Radiation Facility medical beamline.

The application of synchrotron radiation in medical research has become a mature field of research at synchrotron facilities worldwide. In the relatively short time that synchrotrons have been available to the scientific community, their characteristic beams of UV and X-ray radiation have been applied to virtually all areas of medical science which use ionizing radiation. The ability to tune intense monochromatic beams over wide energy ranges differentiates these sources from standard clinical and research tools. At the European Synchrotron Radiation Facility (Grenoble, France), a major research facility is operational on an advanced wiggler radiation beamport, ID17. The beamport is designed to carry out a broad range of research ranging from cell radiation biology to in vivo human studies. Medical imaging programs at ID17 include transvenous coronary angiography, computed tomography, mammography and bronchography. In addition, a major research program on microbeam radiation therapy is progressing. This paper will present a very brief overview of the beamline and the imaging and therapy programs.     

In vivo K-edge imaging with synchrotron radiation.

We present in this paper two imaging techniques using contrast agents assessed with in vivo experiments. Both methods are based on the same physical principle, and were implemented at the European Synchrotron Radiation Facility medical beamline. The first one is intravenous coronary angiography using synchrotron radiation X-rays. This imaging technique has been planned for human studies in the near future. We describe the first experiments that were carried out with pigs at the ESRF. The second imaging mode is computed tomography using synchrotron radiation on rats bearing brain tumors. Owing to synchrotron radiation physical properties, these new imaging methods provide additional information compared to conventional techniques. After infusion of the contrast agent, it is possible to derive from the images the concentration of the contrast agent in the tumor area for the computed tomography and in any visible vessel for the angiography method.     

Functional lung imaging with synchrotron radiation: Methods and preclinical applications

Many lung disease processes are characterized by structural and functional heterogeneity that is not directly appreciable with traditional physiological measurements. Experimental methods and lung function modeling to study regional lung function are crucial for better understanding of disease mechanisms and for targeting treatment. Synchrotron radiation offers useful properties to this end: coherence, utilized in phase-contrast imaging, and high flux and a wide energy spectrum which allow the selection of very narrow energy bands of radiation, thus allowing imaging at very specific energies. K-edge subtraction imaging (KES) has thus been developed at synchrotrons for both human and small animal imaging. The unique properties of synchrotron radiation extend X-ray computed tomography (CT) capabilities to quantitatively assess lung morphology, and also to map regional lung ventilation, perfusion, inflammation and biomechanical properties, with microscopic spatial resolution. Four-dimensional imaging, allows the investigation of the dynamics of regional lung functional parameters simultaneously with structural deformation of the lung as a function of time. This review summarizes synchrotron radiation imaging methods and overviews examples of its application in the study of disease mechanisms in preclinical animal models, as well as the potential for clinical translation both through the knowledge gained using these techniques and transfer of imaging technology to laboratory X-ray sources.     

Ab initio self-consistent x-ray absorption fine structure analysis for metalloproteins

X-ray absorption fine structure is a powerful tool for probing the structures of metals in proteins in both crystalline and noncrystalline environments. Until recently, a fundamental problem in biological XAFS has been that ad hoc assumptions must be made concerning the vibrational properties of the amino acid residues that are coordinated to the metal to fit the data. Here, an automatic procedure for accurate structural determination of active sites of metalloproteins is presented. It is based on direct multiple-scattering simulation of experimental X-ray absorption fine structure spectra combining electron multiple scattering calculations with density functional theory calculations of vibrational modes of amino acid residues and the genetic algorithm differential evolution to determine a global minimum in the space of fitting parameters. Structure determination of the metalloprotein active site is obtained through a self-consistent iterative procedure with only minimal initial information.     

Purification of recombinant growth hormone by clear native gels for conformational analyses: preservation of conformation and receptor binding

Most protein preparations require purification steps prior to biophysical analysis assessing protein stability, secondary structure and degree of folding. It was, therefore, the aim of this study to develop a system to separate and purify a protein from a commercially available medicinal product, recombinant human growth hormone (rhGH) and show preservation of conformation and function following the gel-based procedure. The rhGH was run on clear native (CN) gels and recovered from the gels by electroelution using D-Tube Dialyzer Midi under rigorous cooling. Melting point studies indicated preservation of the structural integrity. This finding was confirmed by synchrotron radiation circular dichroism spectroscopy (SRCD) revealing an identical folding pattern for the sample before and after electrophoretic separation and purification. Synchrotron small-angle X-ray scattering (SAXS) indicated that the sample was folded and monomeric, both before and after separation and purification, and that its shape corresponded well to the known crystal structure of GH. Binding properties of rhGH to a receptor-model system before and after clear native electrophoresis were comparable. This analytical and preparative approach to purify and concentrate a protein preserving conformation and function may be helpful for many applications in analytical, protein and stereochemistry.     

The use of metalloproteins as standards for X-ray microanalysis of biological material

Thin films of metalloprotein, deposited directly onto carbon-formvar-coated electron microscope grids, provide a potentially useful, but little developed, type of standard for biological transmission X-ray microanalysis. The possibility of using various metalloproteins was investigated in terms of their suitability as standards, and compared with an evaporated film of gelatin-salt mixture. The metalloproteins that were selected comprised conalbumin (containing detectable S, Fe), hemocyanin (S, K, Ca, Fe and Cu), insulin (S, Zn), phosvitin (P, Ca, Fe, and Zn) and alpha-lactalbumin (S, Ca). In each case, the metalloprotein preparation was homogeneous at the microlevel (unlike evaporated gelatin-salt mixtures), and was stable in the electron beam up to a current of 120 nA, over a lifetime of 200 s.     

Establishing isostructural metal substitution in metalloproteins using 1H NMR, circular dichroism, and Fourier transform infrared spectroscopy.

Far-UV CD, 1H-NMR, and Fourier transform infrared (FTIR) spectroscopy are three of the most commonly used methods for the determination of protein secondary structure composition. These methods are compared and evaluated as a means of establishing isostructural metal substitution in metalloproteins, using the crystallographically defined rubredoxin from Desulfovibrio gigas and its well-characterized cadmium derivative as a model system. It is concluded that analysis of the FTIR spectrum of the protein amide I resonance represents the most facile and generally applicable method of determining whether the overall structure of a metalloprotein has been altered upon metal reconstitution. This technique requires relatively little biological material (ca. 300 micrograms total protein) and, unlike either CD or 1H-NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.     

Metal imaging in non-denaturating 2D electrophoresis gels by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for the detection of metalloproteins

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was developed as a powerful analytical technique for metal imaging of 2D gels for the detection of metalloproteins in rat kidney after electrophoretic separation. Protein complexes, extracted with water, were separated in their native state in the first and second dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, manganese and lead, were monitored by LA-ICP-MS after gel ablation by a focused laser beam in a way that the total surface of a selected fragment of the gel was totally ablated. The metal distribution of this part of the gel was then constructed by plotting the metal (isotope) signal intensity as a function of the x,y (isoelectric point, molecular mass) coordinates of the gel. The proteins at locations rich in metals were cut out, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Metalloproteomics: high-throughput structural and functional annotation of proteins in structural genomics

A high-throughput method for measuring transition metal content based on quantitation of X-ray fluorescence signals was used to analyze 654 proteins selected as targets by the New York Structural GenomiX Research Consortium. Over 10% showed the presence of transition metal atoms in stoichiometric amounts; these totals as well as the abundance distribution are similar to those of the Protein Data Bank. Bioinformatics analysis of the identified metalloproteins in most cases supported the metalloprotein annotation; identification of the conserved metal binding motif was also shown to be useful in verifying structural models of the proteins. Metalloproteomics provides a rapid structural and functional annotation for these sequences and is shown to be approximately 95% accurate in predicting the presence or absence of stoichiometric metal content. The project's goal is to assay at least 1 member from each Pfam family; approximately 500 Pfam families have been characterized with respect to transition metal content so far.     

Improved normal tissue protection by proton and X-ray microchannels compared to homogeneous field irradiation

The risk of developing normal tissue injuries often limits the radiation dose that can be applied to the tumour in radiation therapy. Microbeam Radiation Therapy (MRT), a spatially fractionated photon radiotherapy is currently tested at the European Synchrotron Radiation Facility (ESRF) to improve normal tissue protection. MRT utilizes an array of microscopically thin and nearly parallel X-ray beams that are generated by a synchrotron. At the ion microprobe SNAKE in Munich focused proton microbeams (“proton microchannels”) are studied to improve normal tissue protection. Here, we comparatively investigate microbeam/microchannel irradiations with sub-millimetre X-ray versus proton beams to minimize the risk of normal tissue damage in a human skin model, in vitro. Skin tissues were irradiated with a mean dose of 2 Gy over the irradiated area either with parallel synchrotron-generated X-ray beams at the ESRF or with 20 MeV protons at SNAKE using four different irradiation modes: homogeneous field, parallel lines and microchannel applications using two different channel sizes. Normal tissue viability as determined in an MTT test was significantly higher after proton or X-ray microchannel irradiation compared to a homogeneous field irradiation. In line with these findings genetic damage, as determined by the measurement of micronuclei in keratinocytes, was significantly reduced after proton or X-ray microchannel compared to a homogeneous field irradiation. Our data show that skin irradiation using either X-ray or proton microchannels maintain a higher cell viability and DNA integrity compared to a homogeneous irradiation, and thus might improve normal tissue protection after radiation therapy.     

Multimodal analysis of metals in copper–zinc superoxide dismutase isoforms separated on electrophoresis gels

It has been suggested that copper–zinc superoxide dismutase (CuZnSOD) isoforms of distinct isoelectric point (pI) could result from differences in their metallation state. Our aim was then to develop and validate analytical methods for the determination and understanding of metallation states in human CuZnSOD isoforms. To avoid metal losses during sample preparation steps, CuZnSOD isoforms were separated according to their pI using non-denaturing isoelectric focusing (IEF) gel electrophoresis. Metal quantification was directly performed in-gel. Cu/Zn ratios of CuZnSOD isoforms were quantified by Particle-Induced X-ray Emission (PIXE) and Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS). Cu/Zn ratios were measured close to the value of 1 as expected from the known stoichiometry of CuZnSOD with slight, but statistically significant, differences between acidic and basic isoforms. Overall, this study demonstrates that metal quantification can be performed directly on metalloproteins separated on electrophoresis gels.     

同步辐射方法在生命科学中的应用

同步辐射方法在生命科学研究中有着十分重要的应用。上海光源是我国建造的第一个第三代同步辐射装置,本文结合上海光源首批建造的光束线站,介绍了几类同步辐射实验方法在生命科学中的应用。     

同步辐射在显微CT中的应用

计算机X射线断层成像技术（CT）是利用X射线的穿透能力对物体进行扫描,所得信号经过反投影的算法而得到物体二维分布的一种成像方法,已经在医学诊断、工业探伤等领域广泛应用。但是由于实验室光源的低通量,光源点大小及其单色性等限制了其向高分辨发展,通常其分辨率在0．5mm左右。利用微焦点X射线源作为光源的显微CT分辨率可以达到微米量级,但是由于其光通量低且为非单色光,对不同样品有不同程度的束线硬化,影响了其真实分辨率。同步辐射作为一种新兴的光源有高亮度、高光子通量、高准直性、高极化性、高相干性及宽的频谱范围的特点,配合高分辨的X射线探测器,可以发展同步辐射显微CT,其分辨率可达10μm以下。利用同步辐射的高空间相干性开展位相衬度显微CT的研究,对低吸收物质也可以清晰三维成像。新建的上海光源的X射线成像及生物医学应用线站开展了三维显微CT方面的研究,经过初步试验,得到了较好的结果。