小角X射线散射实验中蛋白质溶液的辐照损伤及防护方法

随着同步辐射光源（尤其是目前快速发展的第四代同步辐射光源）技术的进步,可用于实验的辐射通量越来越高,实验样品（特别是蛋白质等生物大分子样品）受到的辐照损伤也越来越严重。在全球现有的同步辐射装置上,蛋白质等生物大分子溶液专用小角X射线散射（SAXS）实验站的光子通量基本上都在1013cps量级。在如此高的通量下,蛋白质等生物大分子溶液样品在实验测量中受到的辐照损伤极其严重。如果没有有效的辐照防护措施,蛋白质溶液样品在毫秒级辐照时间内便会辐照损伤,导致不能获取有效的实验数据。辐照损伤严重制约了SAXS实验技术在蛋白质溶液样品方面的应用。因而,认识蛋白质溶液样品辐照损伤的产生机理、影响因素、判断标准,以及有效降低辐照损伤程度、延缓辐照损伤产生时间的方法,对于蛋白质等生物大分子溶液的散射实验具有重要的指导意义。本文在简要概述生物大分子溶液样品辐照损伤产生机理、影响因素、辐照剂量等基本概念的基础上,重点综述了同步辐射SAXS实验中辐照损伤的判断标准和防护措施。此外,本文还对比了各种防护措施的优缺点,讨论了在建HEPS新光源中SAXS束线可用的散射数据采集时间,指出辐照损伤防护剂是有价值的研究方向...     

Shedding UV light on the phase problem

In this issue of Structure, Nanao and Ravelli (2006) describe the use of UV-induced radiation damage (UV-RIP) to solve the phase problem for proteins, employing single-wavelength X-ray radiation, without the need for derivatization. This should also permit data collection for many proteins on home sources, without travel to a synchrotron.     

'Cool' crystals: macromolecular cryocrystallography and radiation damage

Macromolecular crystals commonly suffer rapid radiation damage during room temperature X-ray data collection. Therefore, data are now routinely collected with the sample held at around 100K, significantly reducing secondary radiation damage, and usually resulting in higher resolution and better quality data. At synchrotron sources, the frequent observation of radiation damage even at cryotemperatures has prompted the development of exciting new experiments aimed at characterising and reducing this damage, and using it for structure determination and enzymatic studies. Current research into cryotechniques seeks to understand the basic physical and chemical processes involved in flash-cooling and radiation damage, which should eventually enable the rational optimisation of cryoprotocols.     

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.     

Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: purification, crystallization and structure determination.

We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 A resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 A resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.     

Specific radiation damage can be used to solve macromolecular crystal structures

The use of third generation synchrotron sources has led to renewed concern about the effect of ionizing radiation on crystalline biological samples. In general, the problem is seen as one to be avoided. However, in this paper, it is shown that, far from being a hindrance to successful structure determination, radiation damage provides an opportunity for phasing macromolecular structures. This is successfully demonstrated for both a protein and an oligonucleotide, by way of which complete models were built automatically. The possibility that, through the exploitation of radiation damage, the phase problem could become less of a barrier to macromolecular crystal structure determination is discussed.     

Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules

Background

Synchrotron radiation facilities are pillars of modern structural biology. Small-Angle X-ray scattering performed at synchrotron sources is often used to characterize the shape of biological macromolecules. A major challenge with high-energy X-ray beam on such macromolecules is the perturbation of sample due to radiation damage.

Results

By employing atomic force microscopy, another common technique to determine the shape of biological macromolecules when deposited on flat substrates, we present a protocol to evaluate and characterize consequences of radiation damage. It requires the acquisition of images of irradiated samples at the single molecule level in a timely manner while using minimal amounts of protein. The protocol has been tested on two different molecular systems: a large globular tetremeric enzyme (β-Amylase) and a rod-shape plant virus (tobacco mosaic virus). Radiation damage on the globular enzyme leads to an apparent increase in molecular sizes whereas the effect on the long virus is a breakage into smaller pieces resulting in a decrease of the average long-axis radius.

Conclusions

These results show that radiation damage can appear in different forms and strongly support the need to check the effect of radiation damage at synchrotron sources using the presented protocol.

     

Monitoring and validating active site redox states in protein crystals

High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV-visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

Genomic instability, bystander effects and radiation risks: implications for development of protection strategies for man and the environment

The last few years has seen what people are now referring to as a "shifting Paradigm" in our way of thinking about radiation effects on biological systems. The concept of the central role of DNA damage due to double strand breaks induced by a radiation "hit" has been itself hit by many studies showing persistent effects in the distant progeny of radiation exposed cells. This phenomenon is known as radiation induced genomic instability. More recently evidence has been accumulating that not even the parent cell need be exposed to radiation (the bystander effect). The new paradigm suggests that cellular stress responses or damage signalling through a range of signal transduction pathways are involved and that cell-cell contact or secretion of damage signalling molecules can induce responses in undamaged and unirradiated cells. Are these new effects relevant to risk assessment, or does it matter HOW radiation affects cells if we have good epidemiological evidence of which to base our risk estimates? The aim of this paper is to introduce the new concepts and to consider reasons why they might alter our methods of risk estimation. The paper also considers the impact of the new concepts on environmental protection and discusses the need for research in the field of comparative radiobiology if we are to develop policies which can adequately protect biodiversity.     

Stopped-flow solution scattering using synchrotron radiation: Apparatus,data collection and data analysis

We have constructed an experimental system, under remote control, for stopped-flow X-ray scattering using synchrotron radiation. It has been used, in conjunction with an annular detector and its associated electronics, to obtain good scattering curves, with time-slices as short as 200 ms, in a new study of the dissociation of the enzyme complex aspartate transcarbamylase. The data have been analysed by new statistical methods, and they agree well with the results from parallel chemical quench experiments. For studying dissociation reactions, stopped-flow X-ray scattering is a quite practical method, which need not use very much more material than conventional stopped-flow experiments.     

Clustered DNA damage induced by heavy ion particles.

Clustered DNA damage (locally multiply damaged site) is thought to be a critical lesion caused by ionizing radiation, and high LET radiation such as heavy ion particles is believed to produce high yields of such damage. Since heavy ion particles are major components of ionizing radiation in a space environment, it is important to clarify the chemical nature and biological consequences of clustered DNA damage and its relationship to the health effects of exposure to high LET particles in humans. The concept of clustered DNA damage emerged around 1980, but only recently has become the subject of experimental studies. In this article, we review methods used to detect clustered DNA damage, and the current status of our understanding of the chemical nature and repair of clustered DNA damage.     

When X-rays modify the protein structure: radiation damage at work

The majority of 3D structures of macromolecules are currently determined by macromolecular crystallography, which employs the diffraction of X-rays on single crystals. However, during diffraction experiments, the X-rays can damage the protein crystals by ionization processes, especially when powerful X-ray sources at synchrotron facilities are used. This process of radiation damage generates photo-electrons that can get trapped in protein moieties. The 3D structure derived from such experiments can differ remarkably from the structure of the native molecule. Recently, the crystal structures of different oxidation states of horseradish peroxidase and nickel-containing superoxide dismutase were determined using crystallographic redox titration performed during the exposure of the crystals to the incident X-ray beam. Previous crystallographic analyses have not shown the distinct structures of the active sites associated with the redox state of the structural features of these enzymes. These new studies show that, for protein moieties that are susceptible to radiation damage and prone to reduction by photo-electrons, care is required in both the design of the diffraction experiment and the analysis and interpretation.     

Developments in fiber diffraction.

Improved specimen preparation methods, third generation synchrotron sources, new data processing algorithms and molecular dynamics refinement techniques are, together, allowing the high-resolution structure determination of larger and larger macromolecular complexes by fiber diffraction. New synchrotron sources are also making possible both time-resolved studies and studies of ordered fibers only a few microns in diameter.     

Biological applications of synchrotron radiation infrared spectromicroscopy

Extremely brilliant infrared (IR) beams provided by synchrotron radiation sources are now routinely used in many facilities with available commercial spectrometers coupled to IR microscopes. Using these intense non-thermal sources, a brilliance two or three order of magnitude higher than a conventional source is achievable through small pinholes (< 10 μm) with a high signal to-noise ratio. IR spectroscopy is a powerful technique to investigate biological systems and offers many new imaging opportunities. The field of infrared biological imaging covers a wide range of fundamental issues and applied researches such as cell imaging or tissue imaging. Molecular maps with a spatial resolution down to the diffraction limit may be now obtained with a synchrotron radiation IR source also on thick samples. Moreover, changes of the protein structure are detectable in an IR spectrum and cellular molecular markers can be identified and used to recognize a pathological status of a tissue. Molecular structure and functions are strongly correlated and this aspect is particularly relevant for imaging. We will show that the brilliance of synchrotron radiation IR sources may enhance the sensitivity of a molecular signal obtained from small biosamples, e.g., a single cell, containing extremely small amounts of organic matter. We will also show that SR IR sources allow to study chemical composition and to identify the distribution of organic molecules in cells at submicron resolution is possible with a high signal-to-noise ratio. Moreover, the recent availability of two-dimensional IR detectors promises to push forward imaging capabilities in the time domain. Indeed, with a high current synchrotron radiation facility and a Focal Plane Array the chemical imaging of individual cells can be obtained in a few minutes. Within this framework important results are expected in the next years using synchrotron radiation and Free Electron Laser (FEL) sources for spectro-microscopy and spectral-imaging, alone or in combination with Scanning Near-field Optical Microscopy methods to study the molecular composition and dynamic changes in samples of biomedical interest at micrometric and submicrometric scales, respectively.     

Time-resolved protein crystallography.

Advances in synchrotron radiation technology have allowed exposure times from protein crystals of the order of milliseconds to be used routinely, and in exceptional circumstances exposure times of 100 ps have been obtained. However, many data sets take seconds to record because of the slow time scale of film change or crystal reorientation or translation when more than one exposure is required. This problem has been addressed by Amemiya et al. (1989). There has been considerable progress in methods to initiate reactions in protein crystals, especially the development of photolabile caged compounds but also temperature jump, pH jump, and diffusion. Although flash lamps deliver pulses of 100 mJ/ms, often several pulses are required to release sufficient product, and reaction initiation can take several seconds. Laser illumination can provide more powerful input, but the laser must be accommodated within the restricted space at the synchrotron station. The requirement to maintain synchrony among the molecules in the crystal lattice as the reaction proceeds and to ensure that the lifetime of intermediates is longer than data collection rates emphasizes the need for chemical characterization of the reaction under study. As Ringe advocated in the studies with chymotrypsin, it may be more profitable to devise conditions under which certain intermediates along the reaction pathway accumulate in the crystal and to record these in a series of discrete steps rather than continuous monitoring of the reaction. The Laue method is limited to those proteins that give well-ordered crystals and problems of transient disorder on initiation of reaction and problems of radiation damage need to be overcome or avoided by suitable experimental protocols.(ABSTRACT TRUNCATED AT 250 WORDS)     

Astronomical Sources of Circularly Polarized Light and the Origin of Homochirality

Possible astronomical sources of ultraviolet circularly polarized light(UVCPL) which might be responsible for enantiomeric selection in interstellarorganic molecules are considered, Synchrotron radiation from magnetic neutronstars has been suggested as a possible source of UVCPL. However, synchrotronradiation in these situations is not predicted to be strongly circularlypolarized. Very few such sources show optical synchrotron radiation and in thefew that do circular polarization has not been observed. Magnetic white dwarfsand white dwarf binaries (Polars) can be highly circularly polarized but anyeffect on molecular clouds and star formation regions must rely on rare chance encounters. Recent observations show that substantial levels of circularpolarization are present in reflection nebulae in star formation regions. Thismechanism produces polarized light exactly when and where it is needed inregions where star formation is occurring and organic molecules are known to be present.     

Synchrotron radiation circular dichroism spectroscopy of proteins: secondary structure, fold recognition and structural genomics

Recent developments in instrumentation and bioinformatics show that the technique of synchrotron radiation circular dichroism spectroscopy can provide novel information on protein secondary structures and folding motifs, and has the potential to play an important role in structural genomics studies, both as a means of target selection and as a high-throughput, low-sample-requiring screening method. This is possible because of the additional information content in the low-vacuum ultraviolet wavelength data obtainable with intense synchrotron radiation light sources, compared with that present in spectra from conventional lab-based circular dichroism instruments.     

Radiation damage in macromolecular cryocrystallography

X-ray radiation damage to cryocooled ( approximately 100 K) macromolecular crystals has emerged as a general problem, especially since the advent of third generation synchrotron undulator sources. Interest in understanding the physical and chemical phenomena behind the observed effects is growing rapidly. The specific structural damage seen in electron density maps has to be accounted for when studying intermediates, and can sometimes be related to biological function. Radiation damage induces non-isomorphism, thus hampering traditional phasing methods. However, specific damage can also be used to obtain phases. With an increased knowledge of expected crystal lifetime, beamline characteristics and types of damage, macromolecular crystallographers might soon be able to account for radiation damage in data collection, processing and phasing.     

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.     

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.