Proc. Nati. Acad. Sci. USAVol. 91, pp. 11904-11908, December 1994Biochemistry

Protein footprinting by the combined use of reversible andirreversible lysine modifications

(protein contacts/vaccinia virus topoisomerase/lysine-speciflc endoproteinase/polypeptide end-labeling/citraconic anhydride)

Ryo HANAI AND JAMES C. WANGDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138

Contributed by James C. Wang, August 12, 1994

ABSTRACT A two-step lysine-modification procedure hasbeen devised to chemically footprint protein surfaces involvedin macromolecular interactions. A protein tagged at one par-ticular end, in the free state or in a complex, is first treatedlightly with a reversible lysine-modifying reagent. The proteinis then unfolded and treated extensively with an irreversiblelysine reagent to block those lysines that did not react previ-ously; next, the first lysine modification is reversed, and alysine-specific endoproteinase is used to cleave the taggedpolypeptide at the deblocked lysines. Separation of the proteo-lytic products by size and identification ofthe tagged fragmentsmap the positions of these lysines. In this procedure, thereversible lysine reagent serves as the chemical footprintingagent, as cleavage of the polypeptide ensues only at the sites ofreaction with this reagent. Lysines involved in macromolecularcontacts are identified from differences in proteolytic patternsof the tagged protein when the first lysine modification is donewith the protein in the free form and in a complex. Applicationof the method to vaccinia virus topoisomerase identifies anumber of lysines that are involved in its binding to DNA.

Multicomponent assemblies of macromolecules are ofteninvolved in biological transactions, and well-known exam-ples include the nucleoprotein complexes in replication,transcription, translation, site-specific recombination, andRNA splicing. Various "footprinting" approaches have beendevised and used extensively in mapping the topography ofnucleic acids in such complexes (1-8). In the direct foot-printing approach, which is rooted in the degradative meth-ods of nucleic acid sequencing, a polynucleotide is labeled atone particular end, and complexes of this end-labeled poly-nucleotide are subjected to chemical, enzymatic, or photo-chemical reactions that result in or lead to cleavage of thepolynucleotide at the sites ofreactions; these sites are readilydeduced from the sizes of the labeled cleavage products, andthe dependence of the reactivities at the various sites alongthe polynucleotide on complex formation provides a footprintof the bound macromolecule. Instead of direct end-labeling,a set of polynucleotides possessing a unique end can also beindirectly labeled by hybridization with a labeled probe, thesequence ofwhich is complementary to that of a region closeto the unique end of the polynucleotide (9). In addition tofootprinting based on the degradative nucleotide-sequencingapproach, the copying methods of nucleic acid sequencingare also extensively used: a primer of a specific sequencecomplementary to a short stretch of the polynucleotide isextended by a DNA polymerase to the various sites ofreactions in the footprinting treatments; sizing of these ex-tended chains, in turn, reveals the positions of the reactionsites.

Although nucleic acid footprinting based on template copy-ing cannot be applied to polypeptides, the degradative se-quencing approaches are readily extendible. Radiolabeling ofthe amino terminus of a polypeptide was used in the mappingof sites accessible to various endoproteinases (10), andpolypeptides immunologically tagged at either the amino orthe carboxyl end were used in the proteolytic probing of anATP-dependent conformational change in yeast DNA topo-isomerase II (11). These approaches represent, respectively,direct and indirect end-labeling of a polypeptide (see thereferences in ref. 11 for additional examples of taggingpolypeptide chains). The availability of sequence-specificendoproteinases, such as thrombin, factor Xa, and enteroki-nase, and protein kinases, such as heart muscle kinase,provide additional tools for generating and labeling polypep-tides with distinctive ends.

In contrast to the wide applications of various nucleic acidfootprinting methods, protein footprinting has rarely beenused in the study of protein-protein or protein-nucleic acidcontacts. There appear to be two major hurdles in proteinfootprinting. (i) Proteins are intricately folded, and thisfolding severely restricts accessibility of the proteins toendoproteinases. (ii) Unlike the case with nucleic acids, fewreagents are known to modify proteins in their native statesso that cleavages ofthe polypeptides at the modification sitescan be effected subsequently. As a consequence, proteolysisand laborious analysis of the resulting peptic fragments areusually necessary to identify the sites of modifications.On the basis of the above considerations, we have devised

a two-step procedure for mapping lysines that are likely to bepresent at protein-protein or protein-nucleic acid interfaces.An appropriately end-labeled native protein, in the free stateor in a complex, is first treated lightly with a reversible lysinereagent. The protein is then denatured and treated exten-sively with an irreversible lysine reagent to modify thepreviously unmodified lysines. Reversal of the first lysinereaction, followed by digestion with an endoproteinase thatcleaves specifically at unmodified lysines, provides a way ofidentifying the lysines that react differently to the reversiblereagent when the protein is free by itself and when it ispresent in a complex. In this two-step lysine-modificationapproach, the reversible lysine reagent serves as the foot-printing reagent, as cleavage of the polypeptide would even-tually ensue only at its reaction sites; the irreversible lysinereagent serves the purpose of blocking proteolytic cleavageat sites that don't react in the first step. To illustrate the useof this approach, we report here the identification of severallysines in vaccinia virus topoisomerase that are protected bybinding of the enzyme to DNA.

MATERIALS AND METHODSOverexpression and Purification of Immunotagged Vaccinia

Virus Topoisomerase. Escherichia coli plasmid clonespA9topo (12) and pETtopo-Phe274 (13) for the phage T7RNApolymerase-based overexpression ofwild-type vaccinia virus

11904

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Proc. Natl. Acad. Sci. USA 91 (1994) 11905

topoisomerase and its Tyr-274 -- Phe (Y274F) derivative, inwhich the active-site tyrosine Y274 is replaced by a phenyl-alanine, were provided by B. Moss (National Institutes ofHealth) and S. Shuman (Sloan-Kettering). Several inducibleexpression systems were used to overexpress the wild-typeand mutant vaccinia virus topoisomerase in E. coli; thehighest yields, -2.5 mg ofpurified wild-type enzyme or 10mgof purified Y274F mutant protein per liter of.culture, wereobtained with pRH3-VVTOP1 (14) clones in which the codingsequences of the viral protein, together with a region con-taining a phage T7 promoter, a lac operator, and a ribosome-binding site from pET11 (15), were placed downstream of atac promoter (16). The nucleotide sequence between theShine-Dalgarno motif AGGA of the tac promoter and thestart of the viral enzyme-coding sequences is 5'-AACAGC-GATGTCGGTACCCGGGGATCTCGATCCCGCGAAAT-TAATACGACTCACTATAGGGGAATTGTGAGCG-GATAACAATTCCCCTCTAGAAATAATTTTGTT-TAACTTTAAGAAGGAGATATACAT-3', where theunderlined 21-mer is a second lac operator separated by 64 bpfrom the lac operator sequence in the upstream tac promoter.The last or 314th codon of the vaccinia virus topoisomerase,GGA, was replaced by GGG. This codon was followed by thenucleotide sequence 5'-GCCGCTCTAGTAIACLC.AAL-GACGTCCCGGACTACGCCCCTCGATGA(stopcodon)CTAGAACTAGTGGATCCTCTAGAGTCGACCT-GCAGGCATGCAAGCTT-3', in which the underlined se-quence encodes the immunotag YPYDVPDYA, a nonapep-tide found in influenza virus hemagglutinin that is recognizedby a mouse monoclonal antibody 12CASAb (17). E. coli strainBL21 (15) harboring the expression plasmids, which alsocarry an ampicillin-resistance marker and a lacI gene with aniq promoter up-mutation, was grown in Luria broth/ampicillin, and expression of the vaccinia protein was in-duced by the addition of isopropyl -3-D-thiogalactoside to 1mM at a cell density of w2 x i0o per ml. Cells were harvested2.5 hr after induction, and vaccinia virus topoisomerase or itsY274F derivative was purified according to the procedure ofMorham and Shuman (18). Purified proteins were stored in 50mM Tris HCl, pH 7.5/100 mM NaCl/1 mM dithiothreitol/1mM EDTA/50%o (vol/vol) glycerol at -200C.

Identification of Lysines Involved in Binding of VacciniaVirus Topolsomerase to DNA. The Y274F mutant of the viralenzyme, immunotagged at its carboxyl terminus, was used inthese experiments. The protein and plasmid DNA weredialyzed separately against 20 mM sodium phosphate, pH8.3, before reaction with citraconic anhydride (19). Each100-pi reaction mixture contained the viral protein at -0.5mg/ml (14 ,uM or 0.5 mM in viral protein lysines) and 100 bpof pBluescript DNA per virus topoisomerase molecule in theDNA-containing samples. Citraconic anhydride (Pierce) wasrapidly diluted to 0.5 mM by adding 4.5 pl of the reagent to100 ml of 20 mM sodium phosphate, pH 8.6, while the bufferwas under vigorous stirring at room temperature. The dilutedreagent was immediately delivered to a set of three reactionmixtures to give a citraconic anhydride/lysine molar ratio of0, 0.05, or 0.1. The mixtures were kept at room temperaturefor 30 min, and an excess ofTris-HCl buffer, pH 8.5, was thenadded to each to quench the reaction. The reaction mixtureswere dialyzed against 20 mM sodium phosphate, pH 8.2. To1 vol of each dialyzed solution, 0.8 vol of 7 M guanidiniumchloride, buffered with 20mM sodium phosphate to pH 8.44,and 0.4 vol of 1% N-hydroxysuccinimide acetate (Sigma)/dioxane were added to unfold the polypeptide chain and toacetylate lysines (20) that were not citraconylated. Thereaction was allowed to proceed at room temperature for 30min and was then terminated by addition of excess Trisbuffer. Citraconylated lysines were then deblocked by dia-lyzing the protein solutions against 30%o acetic acid for 24 hrat room temperature (21, 31). The dialysis buffer was changed

to 100 mM Tris*HCl, pH 8.0, and samples were recoveredafter SDS addition to -0.2%, which dissolved proteinaceousprecipitates that had formed in some samples during dialysisagainst acetic acid. The recovered samples were concen-trated under reduced pressure in a spinning evaporator(Savant) and dialyzed against 100mM Tris HCl, pH 8.5. Twomicroliters of a 50 pg/ml stock solution of endoproteinaseLys-C (Sigma) was then added to each reaction mixturecontaining =5 pg of vaccinia virus topoisomerase in a 100-A4vol, and the mixture was kept at 370C for 20 hr before analysisby gel electrophoresis.

Separation and Detection of Imm ragments.Peptide samples thus prepared were separated by discontin-uous SDS/PAGE, using 15% polyacrylamide as the resolvinggel. Protein bands were transferred onto poly(vinylidenedifluoride) membrane (Immobilon-P; Millipore), and the im-munotagged fragments were detected by the use ofthe mousemonoclonal antibody 12CASAb specific to the flu virushemagglutinin tag (17). Horseradish peroxidase-conjugatedgoat anti-mouse immunoglobulin (Bio-Rad) and reagents formonitoring the peroxidase by chemiluminescence (Amer-sham) were used according to manufacturers' instructions.Chemiluminescence was detected on photographic film (Re-flection; DuPont). When vaccinia virus topoisomerase with-out the immunotag was used, no signal was detectable.Peptide length markers were generated by partial degradationof the immunotagged protein either with cyanogen bromide,which cleaves polypeptides at methionines (22), or with2-nitro-5-cyanobenzoic acid (Aldrich), which cleaves poly-peptides at cysteines (23). The fragmented polypeptides weresubsequently modified with N-hydroxysuccinimide acetateto acetylate the lysines (20), so as to directly compare theirelectrophoretic mobilities with those of the immunotaggedpolypeptides from the footprinting treatments.

RESULTS

Epitope-Tagging of Vaccinia Virus Topolsomerase. Vac-cinia virus topoisomerase is 314 amino acids in length (24) andis the smallest member of a subfamily of DNA topoi-somerases represented by eukaryotic DNA topoisomerase I.The viral enzyme and eukaryotic DNA topoisomerase I arehomologous in their primary sequences and are mechanisti-cally similar in their catalysis of DNA-strand breakage,rejoining, and passage (25). The viral enzyme exhibits astrong sequence specificity (26), and its catalysis of DNA-strand breakage and rejoining involves the formation of anenzyme-DNA covalent complex between Tyr-274 of theenzyme and a DNA 3'-phosphoryl group (13, 27). ReplacingTyr-274 by a phenylalanine abolishes the catalytic activity ofthe enzyme, but its DNA-binding ability is unaltered (26, 28).Immunotagging of the enzyme and its Y274F derivative

was accomplished by the fusion of a 15-amino acid peptideAALVXEXDVPDXAPR to the carboxyl terminus of theenzyme. This extraneous peptide contains a nonapeptideepitope of influenza virus hemagglutinin (underlined), whichis recognized by a mouse monoclonal antibody. Both epitope-tagged proteins were overexpressed in E. coli and purified tonear homogeneity. Assays of the relaxation of supercoiledDNA showed that addition of the epitope to the wild-typeenzyme has little effect on its enzymatic activity (data notshown). From this result, it can be inferred that addition ofthe same tag to the Y274F mutant protein does not alter itsDNA-binding characteristics.

Successive Use ofReversible and Irreversible Lysine-SpecificReagents in Probing Lysines that are Accessible to the Revers-ible Reagent. Fig. 1 depicts the results of a typical experimentusing the two-step lysine-modification method of proteinfootprinting. Samples ofimmunotagged vaccinia virus Y274Fprotein were treated as specified below. After discontinuous

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11906 Biochemistry: Hanai and Wang

- ~ ~ ~ ~ ~

4,

_;wAMOAOL,O

- ;'' w F S _.~~~__

40 a 46 i._

N

VW

.,p-u1I Ori1

FIG. 1. Immunochemical detection of epitopically tagged vac-

cinia virus topoisomerase after various treatments. Polypeptideswere resolved by size by discontinuous SDS/PAGE, and the taggedfiagments were immunochemically stained after transferring elec-trophoretically the peptides to a poly(vinylidene difluoride) mem-

brane. The position of the intact vaccinia virus protein is marked byan arrow in the left margin. Lane 1, treatment of the sample withcitraconic anhydride was omitted in the two-step lysine-modificationfootprinting procedure; lysines in the unfolded protein were acety-lated with N-hydroxysuccinimide acetate directly, and subsequentdigestion with endoproteinase Lys-C was done after dialysis against30%o (vol/vol) acetic acid. Lanes 2 and 3, fragments obtained bypartial cleavage at cysteine residues with 2-nitro-5-cyanobenzoicacid (lane 2) and at methionine residues by cyanogen bromide (lane

3); the dark band in lane 3 is composed of four bands discernible inlighter exposures, and these bands correspond to cleavages atMet-121, -123, -126, and -134. In a separate experiment, a smallfragment with an electrophoretic mobility corresponding to cleavageat Met-275 was also seen. Lanes 4 and 6, samples were first treatedwith a stoichiometric ratio of 0.05 (lane 4) or 0.1 (lane 6) citraconicanhydride/lysine. After acetylation of lysines in the unfolded proteinthat did not react with citraconic anhydride, the citraconylatedlysines were deblocked by dialysis against 30%o acetic acid; thesamples were then digested with Lys-C endoproteinase and ana-

lyzed. Lanes 5 and 7, samples treated in the same way as those oflanes 4 and 6, respectively, except that the citraconylation step wasdone in 5.6 M guanidinium chloride.

SDS/PAGE, the tagged polypeptides were detected immu-nochemically, using a monoclonal antibody specific to theC-terminal tag. In all lanes, the intact protein migrated to theposition marked by an arrow in the left margin of Fig. 1; minorbands with much lower mobilities and variable intensitieswere often seen in different lanes, and these bands probablyrepresent aggregated molecules. For the control sample inlane 1, treatment with citraconic anhydride was omitted: afterunfolding of the protein in guanidinium chloride and acety-lation of the lysines with N-hydroxysuccinimide acetate, thesample was subjected to the same decitraconylation andLys-C endoproteinase treatments as prescribed in the two-step lysine-modification footprinting procedure. Several dis-tinct proteolytic fragments were produced by these treat-ments. These fragments were probably the result of incom-plete acetylation at some of the lysines, but the possibilitiesof other contributory factors cannot be eliminated. Theproteolytic pattern was greatly altered by inclusion of thecitraconylation step (lanes 4 and 6): while the intact proteinremained as the major species, many more fragments with a

distinct pattern of intensities were seen in these and other

samples not shown here. The samples analyzed in lanes 4 and6 differed only in the amounts of citraconic anhydride used inthe first lysine-modification reaction: the reversible lysinereagent/lysine molar ratios were 0.05 and 0.1, respectively.The patterns of proteolytic fragmentation were very similar;moderate increments in intensities were observable for somebands in the latter sample.

Positions of the deblocked lysines corresponding to thevarious tagged fragments produced by treatment with en-doproteinase Lys-C were deduced from the known aminoacid sequence of vaccinia virus topoisomerase (24) and thesizes of the immunotagged fragments calculated from theirelectrophoretic mobilities. To provide calibration standardsin these calculations, the immunotagged viral protein wasfragmented by chemical cleavages at cysteines (lane 2) andmethionines (lane 3); in both treatments the lysines in thefragmented products were subsequently acetylated to makethe electrophoretic mobilities of these size markers directlycomparable to those of the immunotagged fragments result-ing from the footprinting treatments. Assignments of thedeblocked lysines corresponding to the immunotagged frag-ments resolved in lanes 4 and 6 are displayed in the rightmargin of Fig. 1. Most of these lysines can be assigned tospecific positions in the polypeptide chain; when severallysines are very close together, however, the resolution ofSDS/PAGE would limit assignment to one or more of thelysines within such a cluster, rather than to the individuallysines.To test whether there are lysines in the native viral protein

that are inaccessible to citraconic anhydride, treatment withthis reagent was also done while exposing the protein to 5.6M guanidinium chloride; the other steps in the procedurewere then carried out in the same way as described above(lanes 5 and 7). Only minor differences are seen between thetwo samples run in lanes 4 and 5 and between the two samplesrun in lanes 6 and 7. Surprisingly, unfolding the polypeptidebefore citraconylation reduced the intensity of the bandcorresponding to cleavages within the cluster of three ly-sines-Lys-65, Lys-74, and Lys-83. Careful inspection of theband from cleavage within the Lys-65/Lys-74/Lys-83 clusterof the native and guanidinium chloride-treated protein re-veals that when citraconylation was done with the unfoldedprotein (lanes 5 and 7), the band was discernibly a doublet;the lower band of the doublet lines up with the more intenseband seen in samples citraconylated in the native state (lanes4 and 6), but the upper band of the doublet was actuallyabsent in the lanes 4 and 6 samples. This difference suggeststhat Lys-65 is not accessible to citraconic anhydride when theprotein is in its native state, and the band seen in lanes 4 and6 is most likely due to cleavage at Lys-74, and perhaps atLys-83 as well; upon unfolding of the polypeptide, Lys-65becomes more accessible. Other differences in band inten-sities are also observable; intensities of bands correspondingto cleavage at Lys-149 and/or Lys-151, Lys-178, and Lys-202are higher, whereas that at Lys-249 and/or Lys-250 appearslower, when citraconylation was done with the protein in theunfolded rather than the native state (compare the patternsshown in lanes 4 and 5 and lanes 6 and 7 of Fig. 1).

Identification of Lysines that Are Likely to be Located at theInterface Between Vaccinia Virus Topolsomerase and DNA.When treatment of vaccinia virus topoisomerase with citra-conic anhydride occurred in the absence or presence ofDNA, differences in the proteolytic patterns are readily seenafter acetylation of the uncitraconylated lysines, reversal ofcitraconylation, and extensive digestion with endoproteinaseLys-C. In the immunoblot shown in Fig. 2, the lane 1 and lane2 samples were size markers derived from the virus topo-isomerase, as described above, and the next six lanes con-tained three pairs of samples treated with different amountsof citraconic anhydride in the absence or presence of a

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Proc. Natl. Acad. Sci. USA 91 (1994) 11907

1 4 5 67 8

_ + _ + +

-,-

- :39.~ ~ ~~ .

FIG. 2. Protection of lysyl e-amino groups in vaccinia virustopoisomerase from citraconylation by binding of protein to DNA.The Y274F mutant protein was treated with the reversible lysinereagent without or with DNA (as indicated, respectively, by a minusor plus sign above lanes 3-8); the three pairs of samples differed incitraconic anhydride/lysine molar ratios (O for lanes 3 and 4, 0.05 forlanes 5 and 6, and 0.1 for lanes 7 and 8). After acetylation of theunreacted lysines, decitraconylation was done before protein diges-tion with Lys-C endoproteinase and analysis of the proteolyzedsamples by SDS/PAGE. Bands with much reduced intensities in theplus DNA lanes in this and other experiments not shown are markedby arrowheads at left of lane 7. Lanes 1 and 2, size markers obtainedby partial cleavage at cysteines and methionines, respectively.

saturating amount ofDNA (indicated by a minus or plus signabove each lane). For the pair of samples analyzed in lanes3 and 4, treatment with citraconic anhydride was omitted; thefragmentation pattern was the same whether DNA wasabsent or present, as expected, and the pattern did not differsignificantly from that seen earlier in Fig. 1, lane 1. Distinctdifferences in the fragmentation patterns were seen whenreversible lysine modifications were carried out in the ab-sence (Fig. 2, lanes 5 and 7) or presence (lanes 6 and 8) ofDNA. These experiments were repeated several times, andbands at which intensity differences are readily visualized inall experiments are indicated by arrowheads at left of lane 7in Fig. 2. These bands correspond to cleavages at Lys-35,Lys-74 and/or Lys-83; two or perhaps all three lysines in theLys-133/Lys-135/Lys-138 cluster; Lys-213; and Lys-249and/or Lys-250. The intensities of several additional bandsalso appear reduced by DNA, although to a lesser extent thanthose specified above. Because of the difficulty of quantitat-ing bands by immunochemical detection, further experimentsare needed to ascertain whether lysines in addition to theones specified are also protected from citraconylation byDNA binding.

DISCUSSIONOur results illustrate the usefulness of chemical footprintingof proteins. A number of lysines in vaccinia virus topoisom-erase are protected from citraconylation by the binding ofDNA. Although protection of a given side chain from chem-ical attack by DNA binding could be attributed to an allo-steric conformational change of the protein, it seems morelikely that the protection of a group of lysines widely sepa-rated along the polypeptide chain is due to their localizationon the DNA-binding surface of the protein. For Lys-35 andLys-74, this interpretation is consistent with the knowncrystal structure of a 77-amino acid amino-terminal fragmentof the viral topoisomerase: Lys-35 and Lys-74 are exposedsurface residues on the same side of the small fragment (14).Furthermore, it is known that deletion of the first 74 aminoacids of the vaccinia enzyme abolishes its DNA-binding

ability (18). We believe that in addition to Lys-35 and Lys-74,two or three lysines in the cluster Lys-133/Lys-135/Lys-138,as well as Lys-213 and Lys-249 and/or Lys-250, are alsolocated on the DNA-binding surface, as their reactivity withcitraconic anhydride is significantly reduced in the presenceof DNA. We are uncertain whether Lys-7 and Lys-10 areinvolved in DNA binding, as partial cleavage at these posi-tions yields tagged fragments insufficiently resolved from theintact polypeptide, the very high intensity of which in theinmunoblots obscures the presence of any adjacent bands.Cleavage at any one of the six lysines beyond Lys-250 in thecarboxyl terminus of the protein is also not detectable in ourexperiments, as the immunotagged products of cleavage atthese lysines are too small to be retained on the membraneused in the immunoblotting experiments. Some of theselysines are most likely to be involved in DNA binding as well,as the active-site tyrosine, Tyr-274, is located in this region(13, 27).

In principle, probing ofthe surface lysines can also be donewith a single lysine-modification reagent. The native proteincan be treated extensively with the reagent, and, afterunfolding the protein and partial proteolysis with a lysine-specific endoproteinase, the appropriately tagged fiagmentscan be resolved; lysine modification at a particular site wouldcorrespond to the absence of a particular tagged fragment.The two-step procedure we have devised has the distinctadvantage, however, in that the native protein is only treatedvery lightly with the footprinting reagent; on average, lessthan one lysine per protein molecule is treated with thereversible lysine reagent. This light treatment not only min-imizes structural perturbation of a complex being probed butalso allows the application of functional selections to thereaction products. Lightly citraconylated vaccinia virus to-poisomerase, for example, can be selected for its ability toform the covalent DNA-protein complex (28, 29). Proteinsfrom the two populations, those capable and those incapableof forming the complex, can be separately processed todetermine which lysines are probably required for catalysis.Mapping of functionally selected populations of a lightlymodified protein is equivalent to the binding-interferencetype of experiments with DNA (1).We have selected lysine modifications in testing the chem-

ical footprinting of proteins primarily because of the specificreactivity of the lysine e-amino group and the availability ofwell-characterized reversible and irreversible lysine re-agents. The abundance of lysine in many proteins and thetendency oflysyl groups to be located on surfaces rather thanin the interior also make lysyl groups attractive targets inprotein footprinting. In vaccinia virus topoisomerase, 34 of atotal of 314 amino acids are lysines. Among these, 8 are tooclose to either end ofthe polypeptide to be readily detectable;most, if not all, of the remaining 26 lysines appear accessibleto citraconic anhydride, the footprinting reagent selected.

Further technical improvements of the method are clearlyfeasible. The rapid progress in mass spectrometry of proteins(30) makes the method an attractive alternative to the reso-lution of tagged polypeptides by SDS/PAGE, and furtherdevelopment of reagents for probing amino acids other thanlysine should be fruitful. We believe that the chemicalmapping of protein-protein and protein-nucleic acid inter-faces at the amino acid-sequence level should be valuable inthe study of macromolecular interactions.

We thank Drs. B. Moss and S. Shuman for providing clonesencoding vaccinia virus topoisomerase, Dr. Paul Caron for a plasmidclone carrying the flu virus hemagglutinin nonapeptide-coding se-quence, and Dr. J. Carey for communicating to us her procedure ofdeblocking citraconylated lysine residues. This work was supportedby a grant from the U.S. Public Health Service (GM24544).

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