FormaldehydeCrosslinking Journal of Biological Chemistry

Formaldehydecrosslinking Journal Of Biological Chemistry-Free PDF

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MINIREVIEW Formaldehyde Crosslinking,Formaldehyde Reactivity with Proteins. As studies of formaldehyde reactivity became more sophisti. cated it was found that conditions that more closely resemble. those used for crosslinking components in cells yield a subset of. the products identified in the earlier studies 17 Using model. peptides formaldehyde was found to react with N terminal. amino groups and side chains of cysteine histidine lysine tryp. tophan and arginine 10 Reaction products were in some. cases influenced by the peptide sequence yielding intramolec. ular crosslinks as well as linkages of the N terminus and histi. dine asparagine glutamine tryptophan tyrosine and arginine. residues to glycine molecules added to the reaction 10. Despite the long incubation time 48 h adducts were not. detected between glycine and peptide cysteine or lysine resi. dues Subsequent work employing model substrates along with. formaldehyde concentrations and reaction times more in line. with those used with cells identified a smaller subset of formal. dehyde reaction products involving lysine tryptophan and cys. teine side chains as well as the peptide N terminus 17 Such. Downloaded from http www jbc org by guest on November 19 2020. studies have often been motivated by interest in developing. techniques for analysis of native protein complex subunit com. position As discussed in more detail below the rapid reactivity. FIGURE 1 Graphic depicting the main aspects of formaldehyde reactivity. in cells The dashed arc represents cell or nuclear membranes which are of formaldehyde with cellular constituents suggests that cells. thought to be highly permeable to formaldehyde red circles The thick black are highly permeable to formaldehyde and the requirement for. curved line represents DNA shown assembled as nucleosomes light gray cir. cles A chromatin interacting factor is schematized in cyan with other part crosslinked groups to be closely apposed makes formaldehyde a. ner proteins shown in dark blue and purple Small molecules such as glycine good candidate for capturing macromolecular complexes in. and Tris that react with formaldehyde and can therefore quench reactivity vivo containing specific but unstably bound subunits which. with cellular constituents are shown as green circles Formaldehyde can cross. link macromolecules together as well as modify exposed groups on macro can then be analyzed by mass spectrometry 18. molecules forming a product species potentially stabilized by reactivity with In discussing the complexity of crosslinked complexes. a quencher Quenchers are ordinarily added to the extracellular milieu and. may exert their main effects outside the cell formed by incubation of cells with formaldehyde it is impor. tant to distinguish between two types of complexity The first is. the chemical complexity arising from the multiplicity of mac. romolecular functional groups that can potentially react with. formaldehyde and the second is the complexity associated with. the types and numbers of macromolecules crosslinked to each. other Although formaldehyde can potentially generate a great. variety of chemically distinct products in vitro the biologically. relevant chemical complexity is in all likelihood simpler under. incubation conditions more typically used for analyses of mac. romolecular complexes in vivo This is due to several factors. including a lowered effective formaldehyde concentration in. cells when compared with most model experiments in vitro. limiting the ability of formaldehyde to locate and interact with. a functional group Although there is much greater macromo. lecular diversity in cells than in typical in vitro experiments. native macromolecules likely provide a smaller range of chem. FIGURE 2 Chemical reactions occurring during formaldehyde cross ically reactive groups than model substrates used in vitro As. linking of biomolecules Formaldehyde crosslinking of biomolecules discussed below N terminal amino groups may be less avail. occurs in two steps First formaldehyde reacts with a relatively strong. nucleophile most commonly a lysine amino group from a protein This able and side chains are less accessible to formaldehyde cross. reaction forms a methylol intermediate that can lose water to yield a Schiff linking due to protein tertiary structure in native proteins. base an imine Second the Schiff base reacts with another nucleophile. possibly an amino group of a DNA base to generate a crosslinked product These factors would decrease the proportion of potentially. This second nucleophile might also be from another protein the same chemically reactive groups and allow for a smaller less diverse. protein as the first nucleophile a quencher molecule or another endog set of chemical products in vivo For instance reactivity with. enous small molecule and therefore a protein DNA crosslink is only one of. many possible products All of the reactions in this two step process are native proteins is limited to those nucleophilic groups that are. reversible which is a key feature of formaldehyde crosslinking for chro accessible to formaldehyde and indeed studies exploring dif. matin capture A specific example of a protein DNA crosslink is shown The. atoms are color coded to match those of Fig 1 cyan protein red formal ferential formaldehyde reactivity have been used to provide. dehyde and black DNA insight into enzyme structure and catalytic function 19 Sol. OCTOBER 30 2015 VOLUME 290 NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 26405. MINIREVIEW Formaldehyde Crosslinking, vent accessible lysine residues have been found to provide the tional changes are required to allow reaction of DNA bases with. most reactive functional groups in native proteins and more formaldehyde i e full extrahelical extrusion of a DNA base may. over modification of native proteins by formaldehyde does not not be required The rates of formaldehyde reactivity with. appear to perturb tertiary structure very much 20 This is naked DNA in vitro were found to be orders of magnitude. consistent with early work in the chromatin field that estab below diffusion limited rates although these studies make it. lished that lysine residues are the predominant sites of forma clear that reaction conditions can have large effects on reactiv. tion of methylene bridges in histone complexes such studies ity Indeed it was recognized early on that it would be difficult. led to the suggestion as well that formaldehyde crosslinking to extrapolate rates of reaction obtained in relatively simple in. does not in general perturb protein structure 21 The appar vitro systems to other more complex systems let alone in vivo. ent preference of formaldehyde for accessible lysine residues 11. may explain in part why formaldehyde has emerged as the. crosslinker of choice for trapping protein DNA complexes as Capture of Protein DNA Complexes. lysine residues are common mediators of interactions with The early use of formaldehyde as a probe of macromolecular. DNA 22 The differential reactivity of accessible groups on structure led to the discovery that formaldehyde can crosslink. protein surfaces has also been explored to understand how histones to DNA 38 Retrieval of the crosslinked complexes. formaldehyde fixation impacts epitope recognition by antibod and analysis of the associated DNA then gave birth to the ChIP. ies 23 Of note the potential for formaldehyde to affect anti assay 14 39 40 which has become ubiquitous in the chroma. body recognition could possibly impact a wide range of exper tin field in a multitude of variations 41 49 Although ChIP. iments that require quantification of recovered fixed material assays performed without crosslinking have proven valuable for. Downloaded from http www jbc org by guest on November 19 2020. by immunoprecipitation Conditions can often be worked out analyses of stable chromatin complexes 50 crosslinking has. such that formaldehyde treatment does not adversely impact made it possible to identify interactions that would not other. antibody recognition 24 25 but to our knowledge this has wise withstand the isolation procedure Given the central utility. not been examined in great detail in the chromatin field Impor of crosslinking and its critical role in establishing many of the. tantly the apparent predominance of a subset of reactive sites principles underlying the current understanding of chromatin. on macromolecules under typical experimental conditions structure and function a clear picture of formaldehyde chem. does not suggest that overall crosslinking complexity in cells is istry is critical to ensure that any biases resulting from formal. necessarily simple Although in vivo crosslinking is probably dehyde crosslinking are taken into account. predominated by a subset of the chemical products observed in The ability of formaldehyde to crosslink amino acids to DNA. vitro there is potential for macromolecules to become cross bases has been examined systematically in vitro In comparing. linked together in multiple ways and in multiple combinations the products of reactions containing lysine cysteine histidine. forming larger daisy chained structures that complicate in vivo or tryptophan with each of the four DNA bases the highest. crosslinking results Indeed there is some evidence that form yield of crosslinked product was obtained with lysine and deox. aldehyde treatment of cells can result in higher order chroma yguanosine 51 consistent with lysine being the most reactive. tin or nuclear structures whose formation may yield misleading among residues in native proteins as described above Similar. interpretations of chromatin association data by trapping fac results were obtained using short peptides and trinucleotides. tors within dense crosslinked networks 4 26 51 In the context of protein DNA interactions the first. chemical step could involve reaction with an amino acid side. Formaldehyde Reactivity with DNA chain in a protein the protein N terminus or an amino or imino. Formaldehyde reacts with amino and imino groups of DNA group on a DNA base importantly however the amino. bases and extensive studies have been performed to document group on the lysine side chain is a better nucleophile than are. the kinetic and thermodynamic aspects of such reactions 11 the amino imino groups on DNA bases whose lone pair elec. 12 27 29 Although formaldehyde reactivity with proteins trons are delocalized in the aromatic ring For this reason it. does not appear to perturb protein tertiary structure formalde seems reasonable to speculate that in most crosslinked protein. hyde reactivity with DNA is notably different as covalent mod DNA complexes a Schiff base is formed on a lysine residue first. ification of DNA bases requires disruption of base pairing in followed by nucleophilic attack by the DNA base held in prox. duplex DNA and in fact formaldehyde was used in pioneering imity to the side chain resulting in a methylene bridge. studies to probe DNA melting 30 34 Modified bases are thus Interestingly and in line with this idea formaldehyde reac. precluded from base pairing and promote further DNA dena tivity with DNA was stimulated substantially by adding amino. turation 30 This likely occurs to some extent in stretches of acids or histones to an in vitro reaction resulting in stable prod. naked DNA in cells treated with formaldehyde although under ucts that in some cases contained both DNA and the protein or. typical conditions employed for in vivo studies the recovered amino acid 52 The 20 30 fold stimulation in the reaction. DNA is by and large suitable for enzymatic manipulation 35 rates observed in these early experiments by the addition of. Formaldehyde modification of naked DNA in vitro may be glycine or lysine for example was striking furthermore form. more extensive 36 Conformational changes in DNA that pro aldehyde crosslinking of proximal functional groups on spe. mote formaldehyde reactivity have been referred to as DNA cific stable macromolecular complexes presumably can occur. breathing or base flipping Measurement of the rates of such even faster because of the constrained physical proximity of the. spontaneous conformational changes is an active area of inves reacting species 53 In addition to the ubiquity of lysine side. tigation 37 and it is unclear what specific DNA conforma chains in DNA binding proteins for interaction with the phos. 26406 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 NUMBER 44 OCTOBER 30 2015. MINIREVIEW Formaldehyde Crosslinking, phate backbone the DNA bases provide a high density of proteins by mass spectrometry but this strategy has nonethe. amino and imino groups along the length of the nucleic acid less been successfully employed for repeated DNA sequences. These two features may contribute to the relatively higher yield such as telomeres 75 77 and in model in vitro systems 36 as. of protein DNA crosslinks when compared with protein pro well as for single copy and multicopy regions in yeast 75 76. tein crosslinks as measured by conjugation of chromatin regu 78 80 The relatively mild formaldehyde reaction conditions. latory complexes that interact indirectly with DNA 54 It has used to maximize crosslinking of physiologically relevant asso. been observed that for some transcriptional co regulators pro ciations and avoid spurious ones may yield a relatively low pro. tein protein crosslinks are not efficiently detected between fac portion of formaldehyde crosslinked material and thereby con. tors that interact with chromatin indirectly when using chro tribute to the technical challenges of this kind of approach 36. FormaldehydeCrosslinking deeper and more comprehensive understanding of the effects mediated by formaldehyde in cells The following discussion provides a framework for under standing aspects of formaldehyde function when used to trap macromolecular complexes in cells with the main features shown in Fig 1 Beginning with basic chemical reactivity this

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