When X-rays modify the proteinstructure: radiation damage at workOliviero Carugo1,2and Kristina Djinovic´ Carugo31Departme...
enables structural characterization of an intact, non-reduced protein structure. This technique, termed here‘multi-crystal...
beams have been experimentally measured. Theinteraction between X-rays and solid-state matter isnevertheless a more comple...
displacement parameters (B factors) or result in a locallyinaccurate stereochemistry with a concomitant risk ofmisinterpre...
species are found at the active site of SOD [42]; copper-and zinc-containing SODs (CuZnSODs) have copper as thecatalytical...
These observations indicate that caution is required inusing crystallographic results in biochemistry and mol-ecular biolo...
21 Penner-Hahn, J.E. et al. (1989) Low-temperature X-ray adsorptionspectroscopy of plastocyanin: evidence for copper-site ...
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2005 when x rays modify-protein_structure_radiationd_amage at work

  1. 1. When X-rays modify the proteinstructure: radiation damage at workOliviero Carugo1,2and Kristina Djinovic´ Carugo31Department of General Chemistry, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy2National Laboratory TASC, INFM, Area Science Park Basovizza, 34012 Basovizza, Trieste, Italy3Max F. Perutz Laboratories, University Departments at Vienna Biocenter, Institute for Biomolecular Structural Chemistry,University of Vienna, Campus Vienna Biocenter 6/1, Rennweg 95b, A-1030 Vienna, AustriaThe majority of 3D structures of macromolecules arecurrently determined by macromolecular crystallogra-phy, which employs the diffraction of X-rays on singlecrystals. However, during diffraction experiments, theX-rays can damage the protein crystals by ionizationprocesses, especially when powerful X-ray sources atsynchrotron facilities are used. This process of radiationdamage generates photo-electrons that can get trappedin protein moieties. The 3D structure derived from suchexperiments can differ remarkably from the structure ofthe native molecule. Recently, the crystal structures ofdifferent oxidation states of horseradish peroxidase andnickel-containing superoxide dismutase were deter-mined using crystallographic redox titration performedduring the exposure of the crystals to the incident X-raybeam. Previous crystallographic analyses have notshown the distinct structures of the active sitesassociated with the redox state of the structural featuresof these enzymes. These new studies show that, forprotein moieties that are susceptible to radiationdamage and prone to reduction by photo-electrons,care is required in both the design of the diffractionexperiment and the analysis and interpretation.IntroductionX-ray crystallography is rapidly evolving as a standardtechnique in biochemistry and molecular biology. Impress-ive progress has been made during the past two decades,including recombinant-DNA technology and other tech-niques for sample preparation, computational proceduresto solve the phase problem (see Glossary) and refine 3Dstructures, and tunable synchrotron beam lines to collectdiffraction data [1]. Crystallography is becoming a sourceof information not only to interpret but also to predictbiological features of proteins, such as function [2] andinteraction networks [3]. Sequence analyses and data-bases are increasingly being replaced by 3D-structureanalyses and databases owing to their high informationcontent at atomic level [4,5]. However, large macromol-ecular assemblies and membrane proteins [6] remainproblematic because they are difficult to handle andcrystallize and the necessary diffraction quality is oftendifficult to obtain. Several structural genomics initiativeshave been launched with the dual aim of determining the3D structures of entire proteomes [7,8] and of developingnew technologies to enable high-throughput analyses[9,10]. Use of synchrotron X-ray sources has made acollection of extremely good diffraction data available,which might result in detailed stereochemical character-izations of the proteins: in a few cases, when atomicresolutions (1.2 A˚ or better) are reached [11], very detailed3D structural information can be obtained.More than ten years ago, Henderson estimated that acrystal cryo-cooled at 77 K would survive approximatelyone day in an X-ray beam at a second generationsynchrotron facility and five years on a rotating anodesource, corresponding to an absorbed dose of 2!107Gy[12]. Because the X-ray beams generated at modernsynchrotron facilities deliver such a dose in standarddata-collection experiments [13], radiation damage is aprominent issue in modern structural biology, and X-raybeams at synchrotron radiation facilities often need to beattenuated or defocused [14].Several papers have been published on the observationof radiation damage during the diffraction experiments[15–18] and on the possible use of the radiation damageitself to determine the crystal structures of biologicalmacromolecules (radiation-damage-induced phasing) [19].However,experimentalartefactsarestillpossibleandcanbecaused by the interaction between the incident X-rayphotons and the molecules from which the crystals are built.A special case of radiation damage is photo-reduction ofmetals in metalloproteins. Photo-reduction of metals inproteins and of free metal ions in an aqueous solution wasstudied by extended X-ray absorption fine structure(EXAFS) and by X-ray absorption near-edge structure(XANES) [20–24], which showed that these events takeplace when samples are irradiated with intense X-raybeams. The stereochemistry of a metal centre, whichaccepts electrons emitted by the protein and the solventatoms in the crystal as a consequence of their excitation bythe intense incident X-ray beam, cannot be determined byroutine crystallographic experiments [25]. A proteinmoiety is progressively reduced during the data collectionand the resulting 3D model can be considerably differentfrom the native structure.A modification of the standard X-ray diffraction data-collection procedures has had recent success [25] – itCorresponding authors: Carugo, O. (carugo@tasc.infm.it), Carugo, K.D.(kristina.djinovic@univie.ac.at).Available online 8 March 2005Review TRENDS in Biochemical Sciences Vol.30 No.4 April 2005www.sciencedirect.com 0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.02.009
  2. 2. enables structural characterization of an intact, non-reduced protein structure. This technique, termed here‘multi-crystal data-collection strategy’ (Figure 1), has beenapplied to study the various redox states that areaccessible to the haem moiety in horseradish peroxidaseand to the nickel in a nickel-containing superoxidedismutase [25–26].Here, we review radiation damage and its impact on thebiological information extracted from the crystal struc-tures of horseradish peroxidase and nickel-containingsuperoxide dismutase.X-rays on crystals: radiation damageWhen a crystal is exposed to an X-ray beam, the latter canbe absorbed and diffracted (Rayleigh scattering) by theordered and periodical arrangement of the electrons of theatoms present in the crystal [27]. It is this phenomenonthat enables determination the 3D structure of proteinsand other molecules once the intensities of the diffractedGlossaryB factors: In addition to the spatial position of the atoms, crystallographyenables the determination of the atomic-displacement parameters, usuallyreferred to as B or thermal factors, which monitor the average positionalspreading of the atom around its equilibrium position.Compton scattering: Inelastic scattering of an X-ray photon by an electron.X-rays transfer part of their energy to the electrons and the scattered photonshave wavelengths longer than the photons of the incident X-rays.Incident X-rays: TheX-rayswithwhichproteincrystalsareirradiatedindiffractionexperiments. X-rays interact with electrons in atoms and an X-ray photon isdeflected away from its trajectory because of its collision with an electron.Phase problem: The interaction between the incident X-rays and the crystalgives rise to characteristic diffraction patterns that are unique to the crystal.Such patterns are characterized by the intensities of the diffracted beams, whichare measured experimentally, and their associated phases, which are notmeasurable. The phase information that is lost during the diffractionexperiment must be recovered by computational and/or experimentalmethods; this is termed the phase problem. It is the fundamental problem ofany crystallographic structure determination.Photo-reduction: A reduction reaction induced by light. Sometimes theexpression ‘photo-reduction’ is also used to indicate reductions in which thesubstrate is not electronically excited by light. In these cases the expressionphoto-induced reduction is more appropriate.Rayleigh scattering: Elastic (or coherent) scattering of an X-ray photon by anelectron. X-rays do not transfer part of their energy to the electrons and thescattered photons have the same wavelength of the incident X-raysSealed tubes: In a sealed tube, a cathode emits electrons that get acceleratedbecause of the high potential with respect to the metal anode. They travel invacuum and reach the anode (copper or molybdenum speed. Most of theelectron energy is converted into heat, which is removed by cooling with water.A small amount of the energy is emitted as X-rays, the sharp peaks in thespectrum are due to electron transitions between orbitals of the anode material.Rotating anode: The heating of the anode caused by the electron beam limitsthe maximum power of the tube. Power loading can be improved if the anode isa rotating cylinder instead of a fixed piece of metal.Tunable X-ray sources at synchrotron radiation facilities: In a synchrotron ring,the charged particles (electrons or positrons) circulate in high vacuum atvelocities close to the speed of light and emit a spectrum of electromagneticradiation when they pass through a magnetic field. X-rays are electromagneticradiation in the wavelength range 1000–0.1 A˚ . A tunable X-ray source at asynchrotron radiation facility enables selection of desired wavelengths.Radiation-damage-induced phasing: This method exploits specific structuralchanges caused by powerful X-ray beams at synchrotron sources in biologicalmacromolecules. These changes are employed to derive phase informationneeded to solve the phase problem.Photo-ionization: This is the process in which an atom or a molecule is ionizedby photons.Non-Bragg diffuse scattering: The non-Bragg reflections, or diffuse scattering,are produced by the non-periodical components of the protein crystal andcontains information about the inter- and intra-molecular motion in the crystal.Cryo-cooling data-collection methods: The single crystal is flash-cooled to atemperature of w100 K before being exposed to the incident radiation and keptunder cryogenic conditions during the whole experiment using a cold nitrogenstream. The chemical reactions, and diffusion of the products, which areresponsible for the radiation damage, are slowed down and, in some cases,entirely stopped at low temperatures, resulting in an increased lifetime of asingle specimen in the X-ray beam.Multi-crystal data-collection strategy: A series of diffraction experiments isperformed on a number of crystals. The resulting datasets are then combined toobtain a set of complete datasets, each corresponding to a nearly constant degreeof absorbed X-ray irradiation. Thus, it is possible to refine a 3D model for manysteps of the reaction induced by the irradiation, similar to a redox titration takingplace in a test tube. By monitoring the oxidation state of the active site byelectronic absorption microspectroscopy [33] it is then possible to assign theoxidation state to each of the 3D structures that have been refined.EXAFS: Extended X-ray absorption fine structure is a technique that uses X-rayabsorption measurements in the region up to 1000 eV over the absorption edgeof an atom to yield information about the local coordination environment of theatom under study.XANES: X-ray absorption near-edge structure is a technique that uses X-rayabsorption measurements in the region near the absorption edge (withinseveral tens of eV) of an atom to determine the local structure around the atomunder study.TiBSIncident X-raysProteincrystalDiffracted X-raysSubset 1time t1dose d1Subset 2time t2dose d2Susbset 3time t3dose d3321CrystalTime t3Dose d3subset 1Dose d1Dose d2Time t2Time t1Time t2Dose d2subset 2Dose d3Dose d1Time t1Time t3Time t1Dose d1subset 3Dose d2Dose d3Time t3Time t2(a)(b)Figure 1. Multi-crystal data-collection strategy. (a) In a crystallographic data-collection procedure, the X-rays diffracted by the crystal are measured in asequential manner. The crystal is rotated in such a way that its orientation, relativeto the detector (which is fixed), changes. Therefore, some of the diffraction data(subset 1) are measured at time t1, others (subset 2) at time t2 and others (subset 3)at time t3. We limit this example to three subsets of data for simplicity, althoughmuch higher values can be used. This implies that the diffraction data of subset 1experience radiation dose d1, those of subset 2 take up dose d2, and those of subset3 dose d3. (b) By using three different crystals it is possible to make three completedata collections in such a way that all subsets of diffracted X-rays (subsets 1, 2and 3) can be measured at the same time (t1, t2 or t3). In other words, it is possible toconstruct three complete datasets, the first of which experiences radiation dose d1,the second radiation dose d2 and the third radiation dose d3. Therefore, it is possibleto have an entire dataset collected at time t1 that experienced radiation dose d1(pink), an entire dataset collected at time t2 which experienced radiation dose d2(blue), and another entire dataset collected at time t3 that took up radiation dosed3 (orange). Each of these reconstructed datasets enables, through crystallographicanalyses, the structural effects of the radiation damage occurring during theexposure of the crystals to the incident X-rays to be monitored.Review TRENDS in Biochemical Sciences Vol.30 No.4 April 2005214www.sciencedirect.com
  3. 3. beams have been experimentally measured. Theinteraction between X-rays and solid-state matter isnevertheless a more complex phenomenon.Besides the elastic Rayleigh scattering that causes thediffraction, the incident X-ray photons can lose some oftheir energy upon interaction with matter (inelasticCompton scattering) and they can also transfer theirenergy and ionize atoms (photo-ionization). In this case,the resulting photo-electrons, which become solvated,initiate photochemical processes that chemically modifythe molecules within the crystal, which typically containsw50% of water. Solvated electrons might cause breakageof X–H (XZS, O, N or C) bonds of the molecules in thecrystal, with consequent formation of highly reactivespecies such as hydroxyl and hydrogen radicals, whichcan initiate a myriad of reactions. An insidious effect ofthese solvated electrons travelling within the crystalspecimen occurs when they are trapped within molecularmoieties that can be reduced to stable products. Forexample, if a metal ion in a high oxidation state is presentin the crystal, it might easily accept these solvatedelectrons and, thus, reduce its oxidation state. Theresulting 3D structure could reflect an altered sample,different from the original material used at the beginningof the experiment.Incident photons have sufficient energy to photo-ionizeatoms within the crystal and cause degradation. Degra-dation of the crystal can be monitored by, for example,periodically measuring reference diffraction intensities,which are often observed to decrease with time. Suchdiffraction decay is caused by radiation damage anddepends on the incident flux and energy of the radiation,and is faster when highly intense X-ray sources atsynchrotron radiation facilities are used. It is, to a lesserextent, also observed in experiments with low intensityX-ray sources that are used in smaller laboratories (sealedtubes or rotating anodes).Apart from the Compton and Rayleigh scattering,some of the ‘non-diffraction’ phenomena that occur in acrystal exposed to X-rays can be exploited to extractbiological information: for example, the non-Braggdiffuse scattering can provide information about intra-molecular dynamics [28,29].Several stratagems can be used to reduce radiationdamage during data collection, the most common beingcryo-cooling data-collection methods [30], which areroutinely used in macromolecular crystallography. Nota-bly, cryo-cooling methods are key features, together withtunable X-ray sources at synchrotron radiation facilities,that enable the collection of diffraction data at severalwavelengths for use in phasing procedures. These exper-iments require long exposures to X-rays and have hadconsiderable impact on state-of-the-art macromolecularcrystallography during the past decade [31,32].Systematic analyses of the radiation damageAlthough cryo-cooling significantly reduces the radiationdamage during X-ray diffraction experiments, proteincrystals still decay when exposed to ionizing radiation.Several studies have been devoted to the analysis ofradiation damage in protein crystals, and have revealedthat this is not a stochastic phenomenon; rather, itconcentrates at particular sites of the protein molecule.Thus far, several proteins have been systematicallyanalysed: Torpedo californica acetylcholinesterase, henegg white lysozyme, winged bean chymotripsin inhibitorand myrosinase [15–17]. A series of X-ray diffractiondatasets were taken consecutively from the same crystaland the structures given by each dataset were determined.The comparison of the refined structures with increasingexposures showed that there are two main types ofresidues that are affected by the ionizing radiation.Disulfide bonds progressively break, and glutamate andaspartate side-chains have a tendency to decarboxylate.The results agreed with radio- and photochemical studiesof both proteins and model compounds in solution [33],showing that the ionizing radiation produces the samekind of structural modifications in samples, both insolution and crystalline aggregate state. The photo-electrons directly generated by X-ray absorption eitherin the bulk solvent or in the protein [16] seem to beresponsible for such degradation and have been shown tobe mobile at cryo-temperatures within proteins [34]. Uponirradiation, the disulfide bond (RSSR) forms a radicalanion (RSSR%K) that might subsequently be protonated(RSSRH%). This dissociates into a thiol (RSH) and thiylradical (RS%) [35]. Other modifications, such as the loss ofhydroxyl groups from tyrosine and of the methylthio groupfrom methionine, were also observed [15,16]. The solventaccessibility does not seem to be directly correlated tospecific radiation damage on protein residues per se,although there are semi-quantitative indications thatflexible regions are more affected than those that adoptordered a-helical or b-stranded secondary structure [18].It also seems that less-stable disulfide bonds dissociatemore easily [16]. It has been suggested that disulfidebonds or metal bio-sites in structures deposited in theProtein Data Bank [36] require re-investigation becausethey might be severely affected by the exposure of proteincrystals to X-rays [16].Thorough analyses have shown structural changesand/or chemical modifications in bacteriorhodopsin crys-tals exposed to the X-ray radiation during diffractionexperiments [37]. A diffraction experiment conceptuallysimilar to the multi-crystal data-collection strategy, butusing several zones on a single crystal, was employed tominimize the extent of radiolysis during the exposure toX-rays; this enabled the study of only selected light-induced conformational changes of bacteriorhodopsin.Careful experimentalists, aware of possible subtlestructural changes of photo-active yellow protein (PYP)caused by radiation damage, have used experimentalphasing techniques [38], together with the computationalcorrections for radiation damage, based on the use of acontrol dataset. This elegant method has identified a newphoto-intermediate of PYP.X-ray titrations of protein structuresThe specific radiation damage described might hinder thecorrect description of the molecular details. For example,the decarboxylation of glutamate and aspartate side-chains during data collection might influence their atomicReview TRENDS in Biochemical Sciences Vol.30 No.4 April 2005 215www.sciencedirect.com
  4. 4. displacement parameters (B factors) or result in a locallyinaccurate stereochemistry with a concomitant risk ofmisinterpretation of the results. Careful X-ray diffractionand spectroscopic experiments on green fluorescent protein(GFP) have shown that illumination with visible or UV lightcauses a photo-conversion of GFP in a one-photon process,which is complemented by decarboxylation of the glutamicresidue in the active site [39]. To confirm that thedecarboxylation was not due to X-ray-induced structuralchange, a diffraction dataset on a low intensity X-ray sourcewas collected to compare the structure with that obtainedform the X-ray high-intensity experiment.Another relevant problem arises in redox enzymes, inwhich the oxidation state of the metal ions at the active sitecould be affected by photo-electrons or free radicals,modifying the active site stereochemistry. Changes tostereochemistry caused by altered oxidation state can befollowed by X-ray diffraction experiments that employ themulti-crystal data-collection strategy [25] (Figure 1). In thisway, it is possible to obtain complete diffraction datasets bycollecting subsets ofdata acquired fromseveralcrystals thathave taken up the same radiation dose. The completedataset obtained at low dose enables the analysis of thematerial that did not suffer radiation damage. Completedatasets, which bear increasing radiation doses, can beassembled in a similar manner and used to analyse theprogressive effects of the radiation damage on the 3Dfeatures. This sort of X-ray titration can also be elegantlycomplemented by microspectrophotometric experiments, inwhich optical spectra are recorded in the course of the X-raydata collection or ‘off line’: the polarized absorption studiesprovide a rigorous experimental tool to correlate theobserved structures and fine structural changes with theirphysico-chemical features [40].Unstable horseradish peroxidase redox intermediatesHorseradish peroxidase is an archetypal haem-containingenzyme that uses hydrogen peroxide (H2O2) or alkylperoxide to oxidize a variety of organic and inorganiccompounds. The stable oxidation states of this enzyme areillustrated in Figure 2; there are five stable intermediatesin two possible catalytic cycles [25]. Although there are nosevere experimental problems in determining the crystalstructures of the chemically reduced ferrous form or theground-state ferric form, the structures of intermediatestates – compound I, II and III – have not been determineduntil recently: by applying the multi-crystal data-collec-tion strategy, the structure of the catalytic pathway ofhorseradish peroxidase has been analysed for the firsttime. In classical data-collection experiments, compoundIII is rapidly reduced to the ferrous form once exposed toX-rays; analogously, compound I is reduced to compoundII, and compound II to the ferric form.The Fe–O distances determined crystallographically(1.7, 1.8 and 1.8 A˚ for compound I, II and III, respectively)agree well with those derived from EXAFS (1.64, 1.93 and1.72 A˚ for compound I, II and III, respectively) [40].Although the region above the haem plane does not hostsolvent molecules in the ferric and ferrous forms, incompound III, a superoxide radical anion binds to themetal with one of its oxygen atoms in a bent conformationand is stabilized by three hydrogen bonds that its otheroxygen atom forms with Arg38, His42 and a watermolecule. In compounds I and II, a ferryl oxygen atom iscoordinated by the iron in the haem group, and forms twohydrogen bonds with Arg38 and a water molecule, which,in turn, interacts with His42 via another hydrogen bond.The structures of active sites of the five stable oxidationstates of are illustrated in Figure 3.The analysis of compound III also revealed themechanism of the four-electron reduction of oxygen towater, which causes the transformation of compound III tothe ferrous form of the horseradish peroxidase (Figure 4).The O–O bond of the superoxide ligand breaks and theresulting water molecule forms a hydrogen bond with theferryl-like oxygen that keeps its coordination position closetotheiron.AtahigherX-raydose,theFe–Obondbreaksanda penta-coordinated iron centre forms, with the iron slightlymoving below the haem plane. Interestingly, these are theonlymodificationsintheproteinthatareobserved.Disulfidebonds, for example, are unaffected by any measurable radi-ation damage, suggesting that the metal bio-site is muchmore reactive than the rest of the molecule.Nickel superoxide dismutase: war and peace betweenspectroscopy and crystallographyOxygen-metabolizing organisms have to face the toxicityof superoxide radicals (O2K) [41] that are generated by asingle-electron transfer to dioxygen. Superoxide dismutases(SOD) enzymes, which are active in the defence againstoxidative stress, keep the concentration of O2Kat controlledlow levels, thus, protecting biological molecules from oxi-dative damage. SODs are classified according to the metalspecies that acts as the redox-active centre to catalyse thedismutation reaction 2O2KC2HC/O2CH2O2. Three metalTiBSFe (III)Ferric form(ground state)Fe(II)Ferrous formFe(III)Compound IIIOO•–HOO•Fe(IV)Compound Iπ radical cationO•+Fe(IV)Compound IIOH+H+H+e–, H+e–, H+e–, H+H2OH2OH2O2O2Figure 2. Stable oxidation states of the active site of horseradish peroxidase. Theperoxidase catalytic cycle, via compounds I and III, is depicted on the right side(purple arrows). The oxidase catalysis cycle, via the ferrous form and compound III,is shown on the left side (green arrows).Review TRENDS in Biochemical Sciences Vol.30 No.4 April 2005216www.sciencedirect.com
  5. 5. species are found at the active site of SOD [42]; copper-and zinc-containing SODs (CuZnSODs) have copper as thecatalytically active metal, whereas manganese- and iron-containing SODs (MnSODs and FeSODs, respectively)host manganese and iron, respectively. Recently, a newSOD with Ni3Cat its active site (NiSOD) was purifiedfrom several Streptomyces species [43–45]. X-ray absorp-tion spectroscopy [46] and electron paramagnetic reson-ance (EPR) spectroscopy on the resting enzyme revealedthe involvement of sulfur ligands and at least one axialnitrogen-ligand in the Ni coordination. The structure ofNiSOD (from S. seoulensis) in two independent crystalforms [47], determined by classical X-ray diffractionexperiments, showed a square-planar Ni coordination forthe amino group of His1, the amide group of Cys2 and twothiolate groups Cys2 and Cys6 (Figure 5). The imidazole ofHis1 was not found to coordinate the Ni but was, instead,involved in hydrogen-bonding interactions to neighbour-ing residues. By contrast, EPR spectra confirmed thepresence of an axial N-ligand at pHz5. A subsequentmulti-crystal data-collection strategy revealed the activesite of NiSOD in three stages of its oxidation state:oxidized (Ni3C), intermediate and reduced (Ni2C)(Figure 6). Similarly, the crystal structure of a homologousNiSOD from S. coelicolor, determined to high resolution(1.3 A˚ ) in a standard X-ray diffraction experiment [48],showed a partially reduced metal site, with the His1 side-chain in a double conformation corresponding to ligandingand non-liganding orientations, implying oxidized andreduced states of the nickel ion in the active site. Thisfinding suggests that the application of multi-crystal data-collection strategy would enable the ‘isolation’ of the pureoxidized and reduced form of the enzyme.Concluding remarks: final perplexities and caveatsDetailed studies have shown that the initial effectsradiation damage do not affect the overall diffractionquality of the crystals, which therefore do not showsymptoms of lethal illness. The photo-electrons and theradicals formed by the absorption of photons by the crystalcan initiate chemical reactions that are not spreadstochastically within the crystal but tend to cluster inprotein moieties, causing specific damage observed asablation of carboxylate groups from aspartate and gluta-mate residues, breakage of disulfide bonds and reductionof metal centres. A thorough understanding of theseprocesses becomes particularly relevant when some of theafflicted residues are involved in crucial functional roles ofthe protein, such as, for example, in GFP [39].The case of redox enzymes is also pertinent. There isevidence that some of the oxidation states that are stablein horseradish peroxidase and NiSOD cannot bestructurally characterized with routine crystallographictechniques because the oxidized forms can be quicklyreduced, probably by photo-electrons. The resulting 3Dmodels could, in such cases, correspond to one ore morestates that have evolved during exposure to the X-raybeam. An ingenious multi-crystal data-collection strategyhas been proposed, and successfully applied, to determinedistinct redox forms of horseradish peroxidase and,subsequently of NiSOD, the metal centres of which aretoo sensitive to radiation damage to be treated routinely.Arg38Arg38Arg38 Arg38 Arg38His42 His42 His42His42His42His170 His170 His170 His170 His170AcetateWater WaterWaterCompound III (1h57) Compound II (1h55)Compound I (1hch)Ferric form (1h5a) Ferrous form (1h58)Figure 3. Crystal structures of the active site of horseradish peroxidase in the various stable oxidation states. The PDB codes are given in parentheses. The iron cation isindicated by a magenta sphere and the water molecules are indicated by red spheres. Hydrogen bonds are indicated by broken lines.Fe(III)His170H+H+Fe(III)His170•+Fe(III)His170OH•His170Fe(III)+ 2H2OFe(II)His170e– + H+e– + H+e– + H+e– + H+H2OH2OH2OH2OO•TiBSO2–Figure 4. Mechanism of reduction of horseradish peroxidase from compound III tothe ferrous form.Review TRENDS in Biochemical Sciences Vol.30 No.4 April 2005 217www.sciencedirect.com
  6. 6. These observations indicate that caution is required inusing crystallographic results in biochemistry and mol-ecular biology, in particular when molecules under studyare highly sensitive to radiation. Many inaccurate resultshave been deposited in the Protein Data Bank; is it, thus,justified to take the PDB files as a standard of truth? Someresults might host experimental artefacts and couldpropagate errors in the Protein Data Bank.What we study and even what we teach could be, atleast in part, wrong. For example, interpretation of thestructural features of the blue copper protein active sitesmight need to be revisited. Their oxidized, copper(II) formsare considered strong oxidizing agents because their metalbio-sites are constrained to adopt a stereochemistry thatis more appropriate for the reduced, copper(I) state [49].It can be hypothesized that the oxidized, copper(II)forms are, at least partially, reduced during diffractionexperiments [23,24].Obviously, this does not imply that the entatic statetheory that relates reactivity with stereochemical strain[50] must be reconsidered; we would like to point out thatcaution should be applied when using crystallographicinformation in biological interpretations. Despite excitingprogress in theory and methods, experimental artefactsare still possible, and synergy between crystallographicand spectroscopic methods is certainly necessary [51].Limiting and controlling radiation damage remains, ofcourse, essential to crystallography. Scavengers canintercept photo-induced radical species and reduce radi-ation damage when introduced appropriately in thecrystal sample [52], and nanotechnology-based templatescan give higher resistance to intense beams [53]. Radi-ation damage can, however, be an intrinsically interestingphenomenon that might give crucial functional infor-mation when exploited suitably, but could be misleadingwhen ignored or overlooked.References1 Hendrickson, W.A. (2000) Synchrotron crystallography. TrendsBiochem. Sci. 25, 637–6432 Kinoshita, K. and Nakamura, H. (2003) Protein informatics towardsfunction indentification. Curr. Opin. Struct. Biol. 13, 396–4003 Aloy, P. and Russell, R.B. (2002) Interrogating protein interactionnetworks though structural biology. Proc. Natl. Acad. Sci. U. S. A. 99,5896–59014 Lo Conte, L. et al. (2002) SCOP database in 2002: refinementseccomodate structural genomics. Nucleic Acids Res. 30, 264–2675 Shepherd, A.J. et al. 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This milestonein electronic scientific, technical and medical publishing means that researchers around the globe will be able to access an unsurpassedvolume of information from the convenience of their desktop.The rapid growth of the ScienceDirect collection is due to the integration of several prestigious publications as well as ongoing additionto the Backfiles - heritage collections in a number of disciplines. The latest step in this ambitious project to digitize all of Elsevier’sjournals back to volume one, issue one, is the addition of the highly cited Cell Press journal collection on ScienceDirect. Also availableonline for the first time are six Cell titles’ long-awaited Backfiles, containing more than 12,000 articles highlighting important historicdevelopments in the field of life sciences.www.sciencedirect.comReview TRENDS in Biochemical Sciences Vol.30 No.4 April 2005 219www.sciencedirect.com