Radiolytic Protein Footprinting with Mass Spectrometry to Probe the Structure of Macromolecular...

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Radiolytic ProteinFootprinting with MassSpectrometry to Probe theStructure ofMacromolecular ComplexesKeiji Takamoto and Mark R. ChanceCase Center for Proteomics, Case Western Reserve University, Cleveland,Ohio 44106; email: [email protected]

Annu. Rev. Biophys. Biomol. Struct.2006. 35:251–75

The Annual Review ofBiochemistry is online atbiochem.annualreviews.org

doi: 10.1146/annurev.biophys.35.040405.102050

Copyright c© 2006 byAnnual Reviews. All rightsreserved

1056-8700/06/0609-0251$20.00

Key Words

hydroxyl radical footprinting, computational modeling,protein-protein interactions, mass spectrometry, radiolysis, Fentonchemistry

AbstractStructural proteomics approaches using mass spectrometry are in-creasingly used in biology to examine the composition and struc-ture of macromolecules. Hydroxyl radical–mediated protein foot-printing using mass spectrometry has recently been developed todefine structure, assembly, and conformational changes of macro-molecules in solution based on measurements of reactivity of aminoacid side chain groups with covalent modification reagents. Accuratemeasurements of side chain reactivity are achieved using quanti-tative liquid-chromatography-coupled mass spectrometry, whereasthe side chain modification sites are identified using tandem massspectrometry. In addition, the use of footprinting data in conjunc-tion with computational modeling approaches is a powerful newmethod for testing and refining structural models of macromoleculesand their complexes. In this review, we discuss the basic chemistryof hydroxyl radical reactions with peptides and proteins, highlightvarious approaches to map protein structure using radical oxidationmethods, and describe state-of-the-art approaches to combine com-putational and footprinting data.

251

First published online as a Review in Advance on February 28, 2006

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Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 252CHEMISTRY OF HYDROXYL

RADICAL FOOTPRINTING . . . 253Generation of Hydroxyl Radicals . . 254Reactions of Hydroxyl Radicals

with Peptides and Proteins . . . . . 254MASS SPECTROMETRY

METHODS FORQUANTITATIVEFOOTPRINTING . . . . . . . . . . . . . . 257Quantification of Peptide

Oxidation with LC-MS . . . . . . . . 257Confirmation of Peptide Identify

and Determination ofModification Site by MS/MS. . . 259

Examples of Footprinting toExamine Protein Structure . . . . . 261

Radiolytic Footprinting ofCytochrome c . . . . . . . . . . . . . . . . . 261

Radiolysis by Electric Dischargewithin ESI Ion Source . . . . . . . . . 261

Fenton/Photochemical HydroxylRadical Footprinting . . . . . . . . . . 262

Probing Actin Structure andInteractions by SynchrotronX-Ray Footprinting . . . . . . . . . . . 262

The Future: Hybrid Approachesthat Combine Experimental andComputational Data . . . . . . . . . . . 264

Structure Modeling byFootprinting and Ab InitioModeling . . . . . . . . . . . . . . . . . . . . . 264

Structure Modeling of NucleicAcid–Protein Interactions . . . . . . 265

Structure Modeling ofMg2+-G-Actin . . . . . . . . . . . . . . . . 266

CONCLUSION . . . . . . . . . . . . . . . . . . . . 267Time-Resolved Methods . . . . . . . . . . 267Hydroxyl Radical Site Sources . . . . 267Computational Approaches . . . . . . . 268

INTRODUCTION

Advances in protein structure determina-tion and computational modeling mediatedby structural genomics initiatives throughoutthe world promise to correlate sequence andstructure for most protein domains withinthe next five years (10, 16, 15). Coincidentwith progress toward this milestone is the re-alization of the importance of macromolec-ular interactions and even the fundamentalsignificance of large macromolecular com-plexes in all major normal and aberrant bi-ological functions (29). Solving the structureand connecting structure to function for theselarge complexes are two of the most importantchallenges in structural biology today. Unlikesolving the structure of protein domains orshort nucleic acids that contain tertiary struc-ture, this effort is far from high throughputand likely involves a combination of com-putational and experimental approaches tai-lored specifically to the problem at hand. Thishybrid approach involves traditional crystal-lography or NMR approaches, where thishigher-resolution data on complexes or theirsubcomponents are computationally coupledto lower-resolution data from methods suchas cryo-electron microscopy, electron tomog-raphy, cross-linking, fluorescence resonanceenergy transfer, and spin label EPR methods(34, 36, 91).

Another well-established method forprobing macromolecular structure in solu-tion is footprinting, a term coined by Galasand Schmitz in their landmark papers on thesubject (26, 92). Initially the technique wasinvented to map the sites of DNA-proteininteraction (9, 26, 52); the protein-dependentattenuation of DNA reactivity towarddimethyl sulfate modification and DNase Idigestion marked the binding site throughhigh-resolution gel readouts of the data. Thetool quickly became a standard method for

252 Takamoto · Chance

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probing DNA-protein interaction (26, 30,92, 115) and determining RNA structure anddynamics (75, 119). Hydroxyl radical has beenaccepted as an ideal reagent for nucleic acidfootprinting experiments after Tullius andDombroski (113) introduced Fe(II)-ethyl-endiaminetetraacetate (EDTA) Fenton-Haber-Weiss chemistry as the radicalgenerator. The use of gamma rays as a radiol-ysis source to generate hydroxyl radicals wassubsequently established. The introductionof high-flux synchrotron X rays for hydroxylradical generation enabled millisecond time-scale footprinting, which was used to examineMg2+-dependent RNA folding (18, 94).

The development of protein footprint-ing techniques lagged behind that of nucleicacids, as the first report of protein footprint-ing did not appear until 1988 (100). Paral-leling the development of nuclease cleavagemethods for DNA, this early approach uti-lized limited proteolysis of proteins followedby SDS polyacrylamide gel electrophoresis(SDS-PAGE) to separate the cleaved frag-ments. Compared with nucleic acids gel meth-ods, which have single-nucleotide resolution,protein gel methods are cruder, providing lessspatial resolution in the probe of structure.The development of tagged Fe(II)-EDTA(84) and free Fe(II)-EDTA Fenton chemistry(46) was an improvement; however, backbonecleavage with Fenton, although nonspecific, isrelatively inefficient. The development of newanalytical technologies for examining pro-teins, particularly the use of mass spectrom-etry (MS) for the detection of cleaved frag-ments or irreversibly modified peptides afterproteolysis, improved the spatial resolution ofthe technique considerably (45, 104).

Although the hydroxyl radical has attrac-tive features in that solvent accessibility is con-veniently probed, the intrinsic inefficiency ofbackbone scission is a serious drawback for theconduct of footprinting experiments. How-ever, examination of the well-established lit-erature concerning metal-catalyzed oxidationand radiolysis of peptides and proteins sug-gests that side chain modification by hydroxyl

Hydroxyl radical:highly reactivespecies used forfootprintingtechniques

EDTA: ethylendi-aminetetraacetate

Radiolysis: thechemical reactioncaused by theabsorption ofhigh-energy photons

SDS-PAGE: SDSpolyacrylamide gelelectrophoresis

Fenton chemistry:well-known chemicalreaction used togenerate hydroxylradical based ontransition metal(Fe2+)-mediatedhomolysis ofperoxides

Tandem massspectrometry(MS/MS):technique used toidentify peptidesequences in whichthe peptide ion isseparated byfirst-stage MS andthen introduced intocollision cell orreaction chamber tobreak down ions

radical should be efficient and rapid (27). Thechemistry of amino acid and peptide oxida-tion using MS detection was investigated sub-sequent to radiolysis (32, 70). These experi-ments showed that in dilute aqueous solutionoxidative modification of side chains is ob-served in considerable excess compared withbackbone scission or cross-linking. Tandemmass spectrometry (MS/MS) methods werefound to be ideal for examining the specificsites of oxidation (13, 64, 70). Thus the reac-tivity of side chains to hydroxyl radical attackand the attenuation of this reactivity as a func-tion of ligand binding, unfolding, or macro-molecular interactions signal surface accessi-bility changes at the defined sites in question.

Over the past five years, extensive stud-ies of the radiolysis chemistry of amino acids,peptides, and proteins using MS-based de-tection and comparisons of these recent datato the extensive literature available (33, 78,123–127) have resulted in the development ofhydroxyl-radical-mediated protein footprint-ing as an effective method for probing pro-tein structure (13, 31, 34, 37–39, 62–64, 69,87). In addition to radiolytic approaches, hy-droxyl radical protein footprinting methodsbased on chemical generation of hydroxylradicals by both Fenton chemistry (96) andUV photolysis of hydrogen peroxide havebeen described (97, 98). This review describesthe current state of hydroxyl radical proteinfootprinting and outlines likely directions ofits future development. “Hydroxyl radical–mediated protein footprinting” as discussedin this review emphasizes modification-basedfootprinting approaches; we do not includeextensive discussions of “cleavage-based foot-printing” methods in this review.

CHEMISTRY OF HYDROXYLRADICAL FOOTPRINTING

Hydroxyl radicals have significant advantagesover other footprinting reagents. Althoughselected chemical modification and cleavagereagents have been used successfully for pro-tein footprinting (45, 60, 104, 111, 118), they

www.annualreviews.org • Hydroxyl Radical Footprinting 253

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Fe2+ + H2O2 Fe3+ + - OH +

.OH

Reductant (ascorbate, thiol)

Fenton chemistry

Radiolysis of water

+ +

slowveryfast

H2O

H2O. +

H2O*

H2Oe- H3O

+ .OH e-

aq+

H.

H.

+ -OH.OH+

H2O

Ionizingradiation

Figure 1Schematicrepresentation ofhydroxyl radicalgeneration byFenton chemistryand radiolysis ofwater by ionizingradiations. EDTAchelates Fe2+ toprovide anelectrically neutralspecies, preventingspecific interactionwith proteins. It isnot directly involvedin chemistry.

are often limited by the number of reac-tive sites available or by the large size ofthe reagent compared with hydroxyl radicals.The major advantages of hydroxyl radicalsare as follows. First, these radicals have vander Walls area and solvent properties simi-lar to those of water molecules. This makeshydroxyl radicals ideal as solvent accessibil-ity probes. Second, they are highly reac-tive species that have well-understood chem-ical selectivity. This is of great importancefor a protein footprinting strategy that at-tempts to increase the number of target sitesto maximize the available structural informa-tion. Third, they can be generated safely andconveniently under a wide range of solutionconditions.

Generation of Hydroxyl Radicals

Hydroxyl radical is generated mainly usingtwo methods (other methods can be used as

well and are described below) (7). The firstmethod is transition metal-dependent chem-ical generation from peroxide and the secondmethod is radiolysis of water (Figure 1). Eachmethod has advantages over the other. Amongthe chemical methods, the most frequentlyused is Fe(II)-EDTA Fenton chemistry (113),which requires commonly available chem-icals that are cheap and easy to handle.The EDTA chelate tends to neutralize thetransition metal’s positive charge; however,specific interactions of the chelate withmacromolecules can bias the local reactivityobserved. On the other hand, radiolysis of wa-ter using gamma or X rays generates radicalsisotropically in the solution and does not re-quire the addition of any chemicals, althoughbuffering of any solution for biochemical con-ditions of interest is desirable. Radiolysis canalso be used to carry out footprinting in vivo(81). If high-flux X rays from synchrotronsources are employed, millisecond exposuresare sufficient for footprinting such that mil-lisecond time resolution footprinting can becarried out (94).

Reactions of Hydroxyl Radicals withPeptides and Proteins

Initially, hydroxyl radicals for protein foot-printing were used to nonspecifically cleavethe backbone (2, 3, 24, 46, 48, 83) in conjunc-tion with SDS-PAGE to separate the cleav-age products. The chemistry involves the hy-drogen abstraction from the Cα carbon andsubsequent reaction of the radical specieswith oxygen leading to backbone cleavage,ultimately generating a new N-terminal end(Scheme 1) (27, 28). However, the reaction

NH

CH

C

O

R1

- -

=

-

-- NH

CH

C

O

R2

- -

=

-

- NH

C . C

O

R1

- -

=

-

-- NH

CH

C

O

R2

- -

=

-

-

.OH HOH O2

NH

C

C

O

R1

- -

=

-

-- NH

CH

C

O

R2

- -

=

-

-

.O

O

--

NH

C

C

O

R1

- -

=

-

-- NH

CH

C

O

R2

- -

=

-

-.

O

-

1/2 O2

O2 HO2

NH

C

O = C =

R1

-

-

- N CH

C

O

R2

- -

=

-

-O

= +

H2O

H2N CH

C

O

R2

- -

=

-

-+CO2

Scheme 1The reactionmechanism ofpeptide backbonescission byhydroxyl radicalattack underaerobic conditions.


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of side chains with hydroxyl radicals occursat rates 10 to 1000 times faster than the ab-straction of hydrogen from the Cα carbon(27, 70, 124). Thus, side chains are prefer-able as probes for the study of protein struc-ture. Quantitative mass spectrometric analy-ses of the modified protein fragments providea footprinting approach that has been usedto probe protein structure and protein-ligandand protein-protein interactions (34, 37–39,62–64, 69, 87, 96–98).

To define the side chain probe set for pro-tein footprinting, the reactions of proteinsand amino acids have been investigated ex-tensively using MS under aerobic (27, 70,123–127) or anaerobic conditions (27, 33, 70,78). The reactions of hydroxyl radicals withaliphatic side chains are initiated by hydro-gen abstraction from side chain carbon atoms.Under aerobic conditions this carbon radi-cal eventually reacts with oxygen and formshydroxyl or ketone groups (in Ala the sidechain is converted to an aldehyde) througha peroxide radical intermediate with mass in-creases of 16 or 14 Da, respectively (Figure 2).Reactions of hydroxyl radical with aromaticrings involve the addition of hydroxyl radicalto the ring; subsequent reaction of the radi-cal species with oxygen results in +16-Da orhigher integer multiple (e.g., +32, +48) in-creases in mass. Acidic residues react with hy-droxyl radical and lose CO2, resulting in analdehyde with an overall reduction in massof 30 Da. The basic amino acids also reactwith hydroxyl radical and give a rise to uniqueproducts. The reaction of lysine is identicalto that of aliphatic residues. Histidine under-goes a complicated set of reactions, includ-ing ring opening, which generates multipleproducts. Arginine can suffer +16 or +14-Damass changes when the β- or γ-carbon expe-riences hydrogen abstraction. However, whenthe δ-carbon is attacked, a guanidino elimina-tion reaction follows, resulting in a productwith a 43-Da reduction in mass. This reac-tion has consequences for the mass spectro-metric analysis, as a strong positive chargeis lost in the reaction; this may influence

digestion with trypsin and make detectionof reaction product difficult in positive ionmode MS.

The sulfur-containing residues are highlyreactive toward hydroxyl radicals. The studyof these residues revealed that Met is con-verted to sulfoxide (+16-Da) fairly easy (butnot to +32 with equal efficiency) and thatCys is converted primarily to the negativelycharged cysteinic acid (+48-Da), which isnot easily detected in positive ion modes.Figure 2 summarizes the reactions of hy-droxyl radical with many of the amino acidside chains. The uniqueness of the chemicalproducts can in some cases identify the oxi-dation sites on the basis of characteristic masssignatures.

The usefulness of a side chain in footprint-ing experiments depends on both its ability toreact with hydroxyl radicals and the ease of de-tection of the reaction products in MS experi-ments (124). The relative reactivity of the sidechains under aerobic conditions using MS de-tection is as follows: Cys > Met > Trp > Tyr >

Phe > Cystine > His > Leu ∼ Ile > Arg ∼Lys ∼ Val > Ser ∼ Thr ∼ Pro > Gln ∼ Glu >

Asp ∼ Asn > Ala > Gly. The residues Gly,Ala, Asp, and Asn are not useful as probes be-cause of their low reactivity (Gly is similar inreactivity to backbone carbon atoms). Ser andThr, though as reactive as Pro, a probe knownto be useful, have reaction products that aredifficult to detect. Thus, 14 of 20 residuescan be routinely used for protein footprint-ing. These 14 residues cover ∼65% of the se-quence of the typical protein; thus structuralresolution of the technique is reasonably good(127).

The sulfur-containing residues (Met andCys) are susceptible to secondary oxidationafter hydroxyl radical exposure; radiolysis cangenerate less-reactive and long-lived radicalspecies such as hydrogen peroxide and re-lated peroxide radicals. If these species arenot quenched, the observed oxidation ex-tent of peptides can increase during sam-ple processing, including the protease diges-tion and chromatographic steps. In particular,


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CH3

CH2

CH2

CH3

CH2

HCOH

CH3

CH2

C=O+

+16-Da +14-Da

Aliphatic

Aromatic

OH

+16-Da

Arginine

CH2

CH2

CH2

C

NH

HN NH2

C

CH2

CH2

HO

-43-Da

Histidine

N

NH

CH2

HN

NH

CH2

ONH

CH2

OO

H

CH2

O

OH

NH2

CH2

O

OH

CH2

O

+16-Da +5-Da

-10-Da -23-Da -22-Da

HN

CH2

HN

CH2O

HN

CH2O

C=OH

+16-Da +32-Da

Tryptophan

CH2

S

CH2

CH3

CH2

O=S=O

CH2

CH3

CH2

S=O

CH2

CH3

Methionine

+16-Da +32-Da

SH

CH2

O=S

CH2

OH

O=S=O

CH2

OH

Cysteine

+32-Da +48-Da

(CH2)

C

CH2

O OH

(CH2)

C

O H

Glutamic (aspartic) acid

-30-Da

Figure 2The reactionproducts by aminoacid side chainsoxidationsubsequent toradiolysis. Onlymajor products andsome minorproducts areshown. Lysineundergoes areaction identicalto that of aliphaticside chains.

methionine is known for its susceptibilityto such oxidation. Also, in Fenton chem-istry, hydrogen peroxide must be removedor quenched right after reaction completion.Such secondary oxidation can be avoided by

the addition of reducing species such as me-thionine in its amino acid or amide forms (inexcess) to compete with unwanted secondaryoxidation of methionine and cysteine residuesin digested peptides or protein species (126).


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Alternatively catalase can be added to scav-enge excess peroxide.

MASS SPECTROMETRYMETHODS FOR QUANTITATIVEFOOTPRINTING

Mass spectrometric readouts of oxidized pro-teins provide the analytical basis for determin-ing solvent accessibility according to peptidereactivity and provide structural resolutionby MS/MS methods. Accurate determinationof the extent of oxidation provides the basisfor sensitive measures of structural changesin ligand binding and in protein interactions.We outline the specific protocols required inorder to achieve quantitative results fromhydroxyl radical footprinting experiments.

Quantification of Peptide Oxidationwith LC-MS

The target protein subjected to hydroxyl rad-ical oxidation is analyzed by MS subsequentto protease digestion. These procedures arebased on those developed for deuterium ex-change experiments (50, 59). As the radiolyticmodifications of proteins are covalent and sta-ble (unlike in deuterium exchange), subse-quent analyses are straightforward and rel-atively easy to carry out. One result of thisis that samples can be frozen after footprint-ing for later analysis. Both specific and non-specific proteases can be selected or used intandem at a range of temperatures or diges-tion times to generate peptide fragments ofinterest. In addition, strong reducing agents[e.g., TCEP; Tris(2-carboxyethyl)phosphine]can be used to digest heavily disulfide-bondedspecies with long incubation times.

Proteolysis is generally performed individ-ually with a set of specific proteases such astrypsin, Asp-N, or Glu-C in order to maxi-mize sequence coverage. Complete coveragefor large proteins using MS is not easy owingto inadequate digestion and poor ionizationof certain peptides. However, 80% coverageor more is generally achievable. The resultant

Hydroxyl radicalfootprinting:methods used toprobesolvent-accessiblesurface area ofmacromolecules

LC-MS: liquid-chromatography/mass spectrometry

HPLC:high-performanceliquidchromatography

Selected ionchromatogram(SIC): the timecourse record of ionintensity withparticular m/z value

mixture of protein fragments (digested pep-tides) is analyzed by liquid-chromatography(LC)-MS (Figure 3). Reverse-phase high-performance liquid chromatography (HPLC)separates the digested peptides and also sep-arates radiolytically modified peptides fromtheir unmodified parent peptides. The modi-fied peptides are generally eluted earlier thanthe unmodified peptides for simple alcoholor keto additions; however, the identity ofall modified and unmodified peptides shouldbe confirmed by MS/MS. The abundance ofeach peptide is calculated from the selectedion chromatogram (SIC) peak area. As SICmonitors the intensity of specific m/z (mass-to-charge ratio) ions as a function of reten-tion time, integration of the peak area in SICgives a total current of selected ion. How-ever, a set range of the retention time (deter-mined empirically) within the chromatogramis used for each experiment to ensure con-sistency of quantitation for the modified andunmodified species. Also, multiple modifiedspecies with separate or multiple modifica-tions may be present; these should all be quan-tified and added into the sum total modified.Then, the fraction of unmodified peptide iscalculated from the integrated intensity val-ues of the modified and unmodified peptides.To provide oxidation rate data that emphasizethe intact population, we calculate the frac-tion unmodified peptide and monitor the lossof the unmodified fraction. The dose responsecurves are generated by the fraction unmodi-fied peptide calculated at multiple time pointsof oxidation. Multiple independent experi-ments are globally fit to provide the oxida-tion rate constants using nonlinear regression.The data obey a pseudo first-order reaction;deviation from these kinetics is observed at in-creased exposure times and indicates overoxi-dation of the sample. The longest time pointsthat evidence such overoxidation should be re-moved from the fits to provide the best data.Although the full dose response experiment islabor intensive, the rate constants calculatedby this protocol are reliable measures of pep-tide reactivity.


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Digestedpeptides

Exposure to hydroxyl radicals and subsequent proteolysis

Modification

.OH Proteolysis

Control

.OH Proteolysis

Complex

Protected

Protected

MS analysis

[M+H]+

Inte

nsit

y

m/z

[M+16+H]+

Inte

nsit

y

m/z

Inte

nsit

y / io

n c

urr

en

t

Retension

time

m/z

Total ion current Rt1 Rt2Mass spectrum

at retension time Rt2

Mass spectrum

at retension time Rt1

SIC

HPLC chromatogram, Ion chromatogram and mass spectrum

HPLCchromatgram

Oxidizedpeptide A

Peptide A

Unmodifiedpeptide

Modifiedpeptide

. OH

do

sag

e

Unmodifiedpeptide

Unmodifiedpeptide

Modifiedpeptide

Modifiedpeptide

Fractional = unmodified

peptide


peptide


peptide

Complex

Control

Dosage (time of exposure)

Lo

g (

fracti

on

al u

nm

od

ifie

d)

y=e-kD k; rate constant

D; dosage of .OH

Calculation of rate constants from dose response curve

Selected ion chromatograms


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Confirmation of Peptide Identify andDetermination of Modification Siteby MS/MS

An important aspect of analysis is, of course,confirmation that a peak with a particularm/z value belongs to a peptide expected fromthe sequence based on the expected diges-tion pattern. In addition to confirmation ofpeptide identity, structural resolution requiresone to determine the modification site(s) onthe peptide. The fragmentation of peptides bycollision-induced dissociation (CID) is stud-ied extensively and reliable for this purpose(54, 56, 105, 108, 129). The translational ki-netic energy of selected ions is converted tointernal energy of the ions (for low-energycollisions this energy is mostly vibrational),inducing cleavage of the peptide backbone.The fragments are termed a-, b-, and c-series(for N-terminal fragments) or x-, y-, and z-series (for C-terminal fragments) dependingon the location of the bond cleavage (89). Themost frequently observed ion types in low-energy collisions are b- and y-series ions, de-pending on the position of charged residues inthe sequence. By examining the mass differen-tials of ion peaks observed in the MS/MS spec-trum, one can deduce the sequence of pep-tide (with exceptions for isobaric amino acidsor posttranslational modifications). Thus, byperforming an MS/MS scan for a particularm/z ion, the identity of peptide can be deter-mined or confirmed. If the peptide has a mod-ification site generated by oxidation, MS/MSscan also identifies the footprinting proberesidue. For example as shown in Figure 4,

Collision-induceddissociation (CID):the technique usedfor MS/MS togenerate fragmentions in whichtranslational andkinetic energy ofions are converted tointernal energy bycollision with gaswithin the collisionchamber

the peptide with sequence ASDFGHK wouldhave a y-series of fragment ions with m/z of y1

147.1, y2 284.2, y3 341.2, y4 488.3, y5 603.3,and y6 690.3 from a singly charged precur-sor ion of m/z 761.4. These m/z values givemass differences of 137, 57, 147, 115, 87, and71 that correspond to residues H, G, F, D, S,and A, respectively. If the peptide has one ox-idation adduct due to alcohol formation, theselected precursor ion will have m/z 777.4 forthe singly charged ion (761.4 + 16). If oneobserves an MS/MS spectrum with y1 147.1,y2 284.2, y3 341.2, and y4 + 16 504.3, thisindicates that the +16 modification is on thefourth residue of the peptide, as the first threey-type ions are unshifted compared with tan-dem spectra of the unmodified ion whereasthe y4 ion retains the modification and isshifted. Observation of additional fragmentions of y5 + 16 603.3 and y6 690.3 + 16 sup-ports this assignment. That the fourth residuefrom the C terminus is F, a residue highlylikely to be oxidized by +16, also supports theassignment of F as the probe residue. The ac-tual spectrum can be more complex, as modi-fications may occur at multiple residues. Also,the doubly charged, modified ion at 388.7 canbe selected and fragmented to monitor the b-type ions in this case, for which b1-b3 shouldbe unshifted relative to the unmodified ionand b4-b7 would retain the modification.

A summary of the experiment is shown inFigure 4. We can determine the modifica-tion site and confirm the identity of peptide;however, the CID process and spectrum aresemiquantitative at best, so that it is risky to

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3Hydroxyl radical footprinting: data collection and data analysis. Top panel: Protein is exposed tohydroxyl radical and modified covalently. The resulting protein sample is then digested by protease orchemical cleavage to fragments that are suitable in size for mass spectrometry. The experiment is carriedout for each individual protein and for the protein complex. In a tight binding interface, some regions areprotected from hydroxyl radical attack. Middle panel: Peptides are separated by liquid chromatographyand introduced into a mass analyzer. The selected ion chromatograms (SIC) are constructed for each ion(with particular mass) as a function of retention time. By monitoring the mass and time, we know whatspecies appears at what retention time. By integrating peak areas in SIC, we can calculate the totalindicated ion abundance. Bottom panel: The determinations of modification rates are performed bycalculating the “loss of intact peptide” in order to maximize the interrogation of intact material.


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CN C

R2

O

-

=

-

HH

-

- -

- CN C

R4

O

-

=

-

HH

-

- -

- OHC C

R1

O

-

=

-

H

-

-

-H2N CN C

R3

O

-

=

-

HH

-

- -

-

a1 c1b1 a2 c2b2 a3 c3b3

x3 z2y3 x2 z2y2 x1 z1y1

Nomenclature of fragment ions

ASDFGHK

147.1

284.2

341.2

488.3 603.3 690.3

761.4

Precursor Ion [M+H]+

m/z

Inte

nsity

y1

y2

y3

y4 y5 y6

0 100 200 300 400 500 600 700 800

K H G F D S A

MS/MS spectrum of CID fragment ions(Identification of peptide and determination of modification sites)

ASDF(+O)GHK

147.1

284.2

341.2

504.3 619.3 706.3

777.4Precursor Ion [M+16+H]+

m/z

Inte

nsity

y1

y2

y3

y4+16 y5+16 y6+16

0 100 200 300 400 500 600 700 800

K H G F+16 D S A

Estimation of contribution of each modification sitein the case of multiple sites in peptides

* * * *F E L K Y E S P N0.3s-1 0.4s-1

0.2s-1 0.4s-1 0.1s-1

Trypsin

V8

Tryptic peptides have two sites in sequence

V8 protease peptides reveals rate constants of F and P

∴ F = 0.2s-1, L = 0.1s-1, Y = 0.3s-1, P = 0.1s-1

Figure 4Analyses by MS/MS. Top panel: The nomenclature for fragment ions. The a-, b-, c-series and x-, y-,z-series of ions are N- and C-terminal fragments, respectively. The numbers 1, 2, 3 represent how manyresidues are in the fragment from N or C terminus of the peptide. Middle panel: Interpretation ofMS/MS data. The schemes illustrate the “ideal” CID spectra for hypothetical peptide ASDFGHK. Theupper spectrum shows the cartoon of CID data of unmodified peptide, and the lower panel illustrates theCID spectrum of modified peptide. By comparing two spectra with sequence, one can determine thelocation of the modification site. Bottom panel: The possible determination of contributions ofmodification sites in the case of multiple modifications is observed.


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estimate the relative contribution of multipleresidues in the case of multiple modificationsites. Multiple enzymatic digestion can givesome clues as to the relative contribution ofindividual residues. If, as a result of peptidemapping, using different enzymes digestionsites can isolate subfragments, it is sometimespossible to estimate the contribution fromeach site. The bottom panel in Figure 4 ex-plains one possible approach along these lines.However, it is highly sequence dependent andnot always applicable.

Examples of Footprinting to ExamineProtein Structure

In this section we show how hydroxyl radi-cal footprinting methods are applied to probeprotein structure and protein interactions.The method is still relatively new comparedwith hydroxyl radical nucleic acids footprint-ing; however, it is growing in acceptance anduse. Different groups have developed varia-tions of the technique to adapt to specific bi-ological problems of interest. Many of theseare discussed below.

Radiolytic Footprinting ofCytochrome c

Anderson’s group has been developing ananaerobic radiolysis method that utilizesnonsolvent-exchangeable H/D exchange me-diated by hydroxyl radical oxidation (32, 33,78). Radiolysis leads to hydrogen abstractionfrom Cα (or side chain carbon atoms) andforms radicals at these positions. This radi-cal is then repaired by dithiothreitol that hasbeen equilibrated with D2O under anaero-bic conditions. The –SH group has been re-placed as –SD; thus when the deuteron repairsthe radical a nonexchangeable deuterium ad-dition event has occurred providing a masstag for the solvent-accessible site (Scheme 2).This data was compared to the analysis of ra-diolytic modification of cytochrome c underaerobic conditions (79). The study also in-cludes analyses of oxidation mechanisms of

ESI: electrosprayionization

NH

CH

C

O

R1

- -

=

-

-- NH

C . C

O

R1

- -

=

-

--

.OD HOD

R-SH

R-SDD2O

eq.

NH

CD

C

O

R1

- -

=

-

--

R-S.

NH

CH

C

O

HR1

- -

=

-

-- NH

CH

C

O

.R1

- -

=

-

--

.OD HOD

NH

CH

C

O

DR1

- -

=

-

--

Scheme 2The mechanism ofhydroxylradical–mediateddeuterium exchangeof nonsolventexchangeablebackbone/side chainhydrogen atoms.

certain residues. Anderson and coworkers uti-lized 50%18O-labled H2O as a source of rad-icals generated by gamma-ray irradiation todifferentiate the reaction directly by hydroxylradical (contains 18O) and through oxygen(mainly 16O). As expected from previous lit-erature (70), no +18-Da oxidation of Tyr wasobserved, indicating that the oxidation sourceis molecular oxygen. They also found a +14-Da modification product of Tyr that was pre-sumably the carbonyl form. They concludedthat the oxidation of Tyr itself is mediated bythe addition of radical but that carbonyl oxy-gen is exchanged with solvent during subse-quent analyses.

Radiolysis by Electric Dischargewithin ESI Ion Source

Maleknia & Downard (71) have utilized anelectrospray ionization (ESI) ion source as ahydroxyl radical generator. This electrochem-ical process is a third method that generateshydroxyl radicals. The high voltage given tothe electrospray tip (8 kV) while using oxy-gen as a nebulizer gas in the ESI ion sourceproduces hydroxyl radicals through genera-tion of active oxygen species by electrochem-ical reactions. The chemistry of the reactionswith amino acids is apparently the same asthe chemistry of aerobic hydroxyl radical re-actions. The advantage of this technique isthat no special instruments are needed to per-form the modification reaction. Especially inthe case of intact protein, it is just a routineionization reaction without any additional


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ES-FT-ICR-MS:electrospray Fouriertransform ioncyclotron resonancemass spectrometry

experiments. The group successfully studiedsome examples that illustrate interactions ofpeptide and protein (121, 122). In order toretrieve the information of modification sites,the sprayed sample from the ESI ion sourceneedle needs to be collected and condensed.This is a major drawback of this technique.There is also the question of whether thestructures of proteins are native under sucha severe electrochemical condition within theaerosol. In spite of these limitations, it is anuseful technique that can be performed withinthe regular ESI ion source.

Fenton/Photochemical HydroxylRadical Footprinting

Hettich’s group is also developing hydroxyl-radical-based footprinting techniques. Fen-ton chemistry (96) and photochemical-basedapproaches (97, 98) have been reported bythe group. Fenton chemistry is a conve-nient method that generates hydroxyl radi-cals without special equipment and is suitedfor the static analyses of structures of macro-molecules. This technique has been used forstructural analyses of nucleic acids for years (4,12, 43, 44, 106, 112, 113, 119); however, theyare the first group to apply Fenton chemistry–based hydroxyl radical footprinting to pro-teins. They probed apomyoglobin solutionstructure using Fenton chemistry and ana-lyzed the products by ES-FT-ICR-MS (elec-trospray Fourier transform ion cyclotron res-onance mass spectrometry) or LC-MS/MS.They reported that apomyoglobin’s structureby NMR is consistent with their footprintingexperimental results. NMR studies showedan overall structure for apomyoglobin that isglobular and rigid except in selected regions.Interestingly, although W7 and L11 are notsolvent accessible for X-ray crystal structuresof native myoglobin, Hettich and cowork-ers observed oxidative modifications for theseresidues. As NMR analyses reported that theregion that covers these two residues are dis-ordered in solution, it is clear from footprint-ing that the residues are much more accessible

than in the myoglobin crystal structure, con-sistent with the NMR data.

In spite of the success of this study, theyallow that the Fenton chemistry requirementof Fe2+ with EDTA and relatively high con-centration of ascorbate may affect the struc-ture of proteins for some experiments. Withthose concerns as background, they have useda photochemical method for generating hy-droxyl radicals (97, 98). UV-induced homol-ysis of hydrogen peroxide is well known andwas adopted as a mean of hydroxyl radical gen-eration. Hettich and coworkers took full ad-vantage of state-of-the-art FT-MS (Fouriertransform mass spectrometry) instrumenta-tion and investigated the relationship betweenaccessible surface area and the extent of oxida-tion. The relation between the two was seen tobe linear for both the entire protein (until co-operative oxidation is observed at high doses)and peptides digested by protease. They (97)reported that peptides missing in LC-MS/MSexperiments can be found in direct infusionES-FT-MS owing to the higher resolutionand wider dynamic range. As mass spectrom-eters are continuing to evolve, it is feasibleto expect that detection limits and sequencecoverage will improve. The latest work fromthis group is also intriguing (98). They com-pared the footprinting data of guanidine-HCl denatured protein with that of nativeprotein. The footprinting results were usedas structural restraints to examine structuresthat are computationally predicted for Sml1Pprotein. This subject is discussed furtherbelow.

Probing Actin Structure andInteractions by Synchrotron X-RayFootprinting

Our group has been examining actin structureby utilizing synchrotron X-ray footprinting.Actin is fundamental for eukaryotic cell motil-ity and its interconversion to actin filamentsis tightly regulated by binding to a numberof actin binding proteins (49). In spite of itsimportance, the structure of free monomeric


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Figure 5Thethree-dimensionalstructure of actin.The ATP, metal ion,and residues thatexperience theprotection uponexchange of metalion from Ca2+ toMg2+ are indicatedby sticks and thesurrounding spheres.

actin bound to the biologically relevant Mg2+

ion has not been determined by X-ray crys-tallography or NMR. There is a good reasonfor the failure of these structural determina-tions. The nature of actin prevents the at-tempt because it polymerizes at relatively lowconcentrations (in the few micromolar rangefor Mg+2 actin, depending on solution con-ditions). Also, actin expressed in Escherichiacoli is toxic, making the production of labeledmaterial for NMR experiments difficult andexpensive. Thus, methods such as footprint-ing become quite valuable in providing struc-tural information for actin and its complexeswith binding proteins (35, 38). Figure 5 showsthe overall structure of the actin monomer.The actin monomer is composed of two do-mains, and each domain is subdivided into twosubdomains. The nucleotide binding pocketand metal binding site are located at the bot-tom of the large cleft. Although numerous X-ray crystal structures of monomeric actin arebound to Mg2+ nucleotide, all of them includeother bound ligands or proteins that preventpolymerization. These structures are similarto the structure of monomeric actin bound toCa2+ nucleotide, which requires much higheractin concentrations for polymerization.

The structural differences observed be-tween Ca+2-ATP and Mg+2-ATP actin us-ing hydroxyl radical footprinting are sup-ported by numerous other biophysical andbiochemical experimental results such as lim-ited proteolysis (19, 103) and fluorescencestudies (25, 74, 116, 130). The characteris-tic difference in structure of Mg2+ comparedwith the Ca2+ form of monomeric actin re-vealed by footprinting is closure of its largecleft. The sites of protection for Mg2+ ver-sus Ca2+ actin are located primarily at theedge or within the large cleft (Figure 5). Pro-tection sites located on residues Met-44 andMet-47 of subdomain 2 and residues 200–202 and Pro-243 of subdomain 4 are sugges-tive of contact formation at the cleft edges.The high degree of protection (>80% re-duction in reactivity) within the large cleftresidues Leu-67 and Tyr-69 strongly indicatesthe burial of those residues by complete clo-sure of large cleft. There are also observedchanges in hydroxyl radical reactivity in N-terminal (Phe-21) and C-terminal (His-371,Cys-374, and Phe-375) regions that are partof subdomain 1 but are linked to other subdo-mains. Because the other regions of the pro-tein do not show changes in reactivity with


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GS1: gelsolinsegment 1

Comparativemodeling:computationalmodeling techniquesthat take advantageof sequence andstructural similarities

Ab initio modeling:computationalmodeling techniquesfor determiningstructure in whichstructural models arecomputed byphysical parameterswith certain degreeof simplification(force field)

hydroxyl radicals, it is unlikely that the pro-tein structure undergoes large-scale reorga-nization. Although the N- and C-terminalregions’ changes in reactivity are not fully un-derstood, it is clear that the large cleft ex-periences Mg+2-dependent closure, thus ex-plaining hydroxyl radical footprinting results.The ability of footprinting to reveal metal-ion-dependent structural changes was also re-vealed in a study of Ca2+-dependent gelsolinactivation (62, 63).

Our study of actin also involves the exam-ination of protein-protein interactions. Actininteracts with a number of proteins thatcontrol biological processes such as elonga-tion or severing of F-actin filaments. In thecourse of our actin monomer structure stud-ies, the structures of Ca2+- and Mg2+-boundactin monomer interacting with gelsolin seg-ment 1 (GS1) were investigated. Protectionswere observed at positions where contacts areformed in crystal structures, confirming thatthe structure of actin/GS1 complex in solu-tion is similar to the observed crystal struc-tures. Another actin protein of interest isthe actin monomer’s interaction with cofilin.Cofilin binds tightly to actin monomers andfilaments and facilitates filament severing. Ithas been predicted to bind to actin at the siteof GS1 binding. However, our recent dataindicate an entirely different binding mode(K. Takamoto & M. R. Chance, unpublisheddata). The complementary binding surface ofactin on cofilin has been previously mapped(38). With this new protection data on actin,a model of the actin/cofilin complex may beconstructed.

The Future: Hybrid Approaches thatCombine Experimental andComputational Data

We have described examples of hydroxyl rad-ical protein footprinting section and their usein mapping solvent-accessible surface area.The underlying chemistry has been thor-oughly investigated, and the method workswell for probing structural changes and

protein-protein interactions. Computationalmodeling, both comparative modeling andthreading, can predict a wide range of proteinstructures and can operate to predict structuredata on a large scale (15, 16, 22). Althoughits accuracy of prediction is being improved,experimental data can supplement the predic-tions, especially when appropriate templatesare not available or are distantly related to thetarget protein (128). To speed structure so-lution, there is a growing interest in hybridmethods that represent a marriage betweenexperimental and computational approaches.More precisely, we wish to include explicitexperimental constraints within the computa-tional structure-prediction and modeling pro-grams.

Hydroxyl radical footprinting data haveserved as excellent constraints for RNA struc-ture predictions (85, 86, 110). Similar ap-proaches can be applied in the case of pro-tein footprinting data. These approaches arewell precedented for proteins; for example,numerous studies combine the experimentaland computational approaches using NMRdipolar-coupling data (23, 39, 51, 55, 77, 82,88, 98, 117, 120). However, there are few pub-lished works that take this approach using hy-droxyl radical footprinting data.

Structure Modeling by Footprintingand Ab Initio Modeling

As mentioned above, Hettich’s group hascarried out footprinting experiments on theSml1p protein. They combined hydroxyl rad-ical footprinting data and an ab initio model-ing strategy. Sml1p is a small protein com-posed of 104 amino acids but does notshare enough sequence similarity with anyother protein families for accurate compara-tive modeling (76, 90, 109). Threading meth-ods (57, 61) have also failed to provide a rea-sonably good template structure (42). Thus,their only choice for computational model-ing was an ab initio strategy (6, 61, 102). Us-ing the HMMSTR (11)/Rosetta (101) server,they retrieved five structures and compared


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the structures with hydroxyl radical footprint-ing data in order to assess the models. Becausethey compared footprinting data of native anddenatured structures, they could easily assignthe residues within the hydrophobic cores,e.g., these residues were reactive only in thedenatured state. This is a strong constraintto evaluate predicted structures. They foundnone of the models to be consistent with ex-perimental data, which is not surprising be-cause it is often difficult to predict hydropho-bic contacts by ab initio methods (1). Becausepartial NMR data that reported the secondarystructure was available, they reconstructed themodel manually. The final result was satisfac-tory. This work is novel in terms of compu-tational modeling using an ab initio modelingapproach and filtering the predicted structureby experimental data. The difficulty lies incomputational modeling, not in filtering pre-dicted structures. As the authors pointed out,it is important to integrate the experimentalconstraints into the modeling process itself in-stead of using them as a filter.

Structure Modeling of NucleicAcid–Protein Interactions

The human adenovirus protease (AVP) playsan important role in the control of infec-tious virion synthesis. The protease requirestwo cofactors for complete activation and ac-tivity. One cofactor is an 11-residue, highlypositively charged peptide called pVIc andthe other is viral DNA. Although each cofac-tor can activate the protease by itself, whencombined, the increase of kcat/Km approaches30,000-fold. The crystal structure of proteaseand the protease/peptide complex has beensolved (5, 72); however, the structure of thecomplex with viral DNA and the ternary com-plex have not. In order to understand themechanism of activation, it is important tolocate the binding site of DNA. Thus, hy-droxyl radical footprinting was used to locatethe DNA binding site on the protein surface.The AVP was exposed to the synchrotron Xray in the presence or absence of DNA or pVIc

AVP: humanadenovirus protease

and also as a ternary complex with both co-factors. Strong DNA-dependent protectionsites were identified at residues Phe-86, Pro-101, Tyr-175, and Pro-183. Those residuesexperience almost complete burial upon DNAbinding. Another fascinating finding was thatsome residues are more exposed to solvent byDNA binding. Those residues (Cys-40, Trp-55, Met-56, and Trp-60) are located near theactive site within the cleft between two do-mains of AVP. Thus, DNA binding causesthe opening of the cleft and possibly increasessubstrate access to the active site. Also, con-served positively charged residues on the pro-tein surface are likely to interact with DNAphosphates through electrostatics and con-served aromatics on the surface. From theseobservations, it was concluded that DNA in-teracts with AVP and pVIc by base stack-ing with aromatic residues that are protectedupon DNA binding and also through electro-static interactions between basic residues inAVP and pVIc. A model of DNA was man-ually built on the AVP/pVIc cocrystal struc-ture to satisfy the hydroxyl radical footprint-ing data and locate the backbone phosphateand conserved basic residues using programO (58) by rotating the DNA backbone an-gles. The model (Figure 6) satisfies the ob-served protections mediated by DNA bindingand also gives an insight into how DNA bind-ing induces changes in the active-site cleft.The DNA wraps around AVP/pVIc and workslike a strap, pulling the two domains open.Binding of pVIc alone causes some degreeof changes in the structure and viral DNAalone can bind to AVP. Together, pVIc pro-vides more interaction sites for DNA such asa highly positively charged C-terminal sideand C-terminal Phe for stacking interaction.Thus, the interaction is stronger and the effectis dramatic in terms of the allosteric changesobserved by footprinting that are correlatedwith significant changes in enzymatic activ-ity. Modeling based on the footprinting dataprovided a proposed DNA contact surfaceand explained the allosteric mechanisms ofactivation.


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Figure 6Thethree-dimensionalmodel of AVPbound to pVIc andDNA cofactors.The residues thatare more exposedto solvent uponactivation arecolored pink. DNAis illustrated as astick-and-ballmodel within glassspheres.

Structure Modeling of Mg2+-G-Actin

The structure of Mg2+-G-actin has not beensolved. We have probed the solution structureof Mg2+-G-actin utilizing hydroxyl radicalfootprinting and concluded that the large cleftbetween two domains must be closed. Thisconclusion has many biological consequences,as it directly affects processes such as F-actinassembly and ATPase activity. In order to vi-sualize the closed structure of actin, we haveconducted a thorough inspection of solvedcrystal structures, analyzed the deviationswithin these solved structures using molecularviewer VMD (53) and in-house Tcl scripts forVMD (K. Takamoto & M. R. Chance, unpub-lished results), and built a model consistentwith crystallographic parameters and foot-printing data (K. Takamoto & M. R. Chance,unpublished results). The structure of actin iscomposed of two domains, the large and smalldomains. The small domain is subdivided intoSD1 and SD2. The significant motions ofSD2 relative to SD1 and some interesting dif-ferences in the geometry of side chains withinthe nucleotide binding pocket are observed byanalyses of these crystal structures. Also, therelative positions of the large and small do-

mains can vary. Those motions are rigid-bodyhinge movements rather than shear move-ments or reorganization of the subdomains.This observation is consistent with hydroxylradical footprinting results that do not indi-cate any large-scale reorganization within thesubdomains.

The protections within the large cleft be-tween two domains (Leu-67 and Tyr-69) canbe explained only by rigid-body movementof SD2. Also, the fact that blockage of thesmall cleft between SD1 and SD3 prohibitspolymerization suggests that relative move-ment of large and small domains also playsan important role for polymerization. Thisis true for binding of large proteins such asGS1 (73) and vitamin D binding protein (80)and for smaller ligands (66, 107) that shouldnot block the assembly of monomers. Thus,the strategy for the modeling included (a)move domain/subdomains as rigid bodies, (b)move SD2 relative to SD1, and (c) move thelarge domain relative to SD1 (within smalldomain). Those movements must satisfy hy-droxyl radical footprinting results. The largedomain moves toward SD1, and SD2 movestoward the large domain. We have generated


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Figure 7The comparison ofthe crystal structure(1YAG; gold ) andmodel ofMg2+-G-actin(silver). Protectionswithin SD4 andD-loop are shown asglowing spheres withsticks inside. Thepink spheres indicatethe positions incrystal structure1YAG and the cyanspheres indicate theresidues within themodel.

a series of movements by combining two pos-sible movements and have filtered them withrespect to how the initial models satisfied thefootprinting data. The best structure was thenoptimized by scripts within VMD and us-ing MODELLER 7v7 (90) to correct stereo-chemical parameters. The resulting structuralmodel is shown in Figure 7.

There are some insights revealed by themodel. The large domain has moved to-ward SD1 in order to satisfy the hydroxylradical data. This domain motion changesthe geometry within the nucleotide bindingcleft and brings the side chains closer to themetal ion, which is more consistent with theshorter bond lengths likely for Mg2+ bind-ing. The coordination states of metal and oxy-gen atoms within those side chains changefrom outer-sphere to inner-sphere coordina-tions, and we suggest this is one driving forcefor domain movement. The change in ge-ometry also changes the relative positions ofAsp-154 and γ-phosphate, making Asp-154a prime candidate for the catalytic residueas suggested (93). This study shows the po-tential power of hydroxyl radical footprint-ing to provide unique structural informa-tion relevant to the biological function ofactin.

CONCLUSION

Time-Resolved Methods

Similar to the developments that led fromstatic nucleic acid footprinting to millisec-ond time-resolved approaches (8, 95), pro-tein footprinting needs to be extended suchthat seconds to milliseconds timescale reac-tions could be routinely monitored. Proteinfootprinting, using either cleavage methods(46) or modification (63), can provide reliablebiophysical isotherms in equilibrium experi-ments. The next step would involve deliver-ing the radical dose in a short time by usingan appropriate mixing device (8, 95). Fentonapproaches are suitable for monitoring reac-tions that are on the minutes timescale, per-oxynitrite as a radical source could reduce thetimescales to seconds, and synchrotron exper-iments can push the timescale to milliseconds(8, 95). Alternatively, photolysis of peroxide, ifdeveloped with modern UV laser technology,could attain seconds to millisecond timescalesas well.

Hydroxyl Radical Site Sources

One disadvantage of bulk footprinting ex-periments is that the footprinting data does


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not have a three-dimensional context. Teth-ered footprinting approaches developed byDatwyler & Meares (20, 21) were designedto generate radicals from a specific site; themigration of these radicals to adjacent sitesprovided proximity information and helpedmap protein-protein interfaces. The mappingwas relatively imprecise because these meth-ods used gels. A more recent set of experi-ments by the Vachet group (67, 68) has usedthe intrinsic metal atom of proteins along withoxidation and MS to map residues in the metalbinding site on the basis of observed side chainreactivity. A union of these two methods, inwhich sites are engineered in proteins (typi-cally by introducing single Cys residues) andcoupled to Fenton-type reagents, may pro-vide sources of radicals that map intra-atomicdistances within a protein and interatomicdistances within a complex. A challengeof such approaches would be to developcalibration experiments such that the re-activity could be converted into soft dis-tance constraints. If successful, such technolo-gies could be competitive with cross-linkingapproaches.

Computational Approaches

This review highlights some of the first pub-lished attempts to explicitly use solution foot-printing data combined with computationalmethods to refine structure or evaluate com-peting structural models (39, 41, 98). To makethese approaches more valuable, progress intwo separate areas is needed. First, althoughthe relationship of solvent accessibility andside chain reactivity is demonstrable, its quan-titative basis is far from being well under-stood. Progress in relating measured changesin reactivity to explicit changes in solvent ac-cessibility would provide specific constraintsfor protein modeling. In addition, model-ing programs could be improved to allow fora more flexible inclusion of surface acces-sibility and/or distance constraints, particu-larly with respect to providing seamless usercontrol over the weighting factors appliedto specific constraint terms in the modelingprocess (98). As these improvements accrue,oxidative footprinting approaches using MSwill play an increasingly important role inunderstanding the structure and dynamics ofmacromolecules and their assemblies.

SUMMARY POINTS

1. The hydroxyl radicals are generated mainly by two methods; transition metal-mediated chemical methods (e.g., Fenton chemistry) or radiolysis of water by X orgamma rays.

2. The hydroxyl radicals react with peptide/protein side chains 10 to 1000 times fasterthan the backbone and form stable covalent modification products.

3. The reaction products of hydroxyl radicals are digested by proteases, analyzed byLC-MS, and sequenced by MS/MS.

4. The surface accessibility information is retrieved in quantitative fashion by calculationof rate constants from dose-response curves.

5. The footprinting data are a source of constraints for computational modeling ofmacromolecules.

6. Experimental and computational approaches in combination are an emerging newtechnique for protein structure prediction.


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ACKNOWLEDGMENTS

This research is supported in part by The Biomedical Technology Centers Program of theNational Institutes for Biomedical Imaging and Bioengineering (P41-EB-01979).

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Radiolytic Protein Footprinting with Mass Spectrometry to Probe the Structure of Macromolecular...

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