Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

Transcription

1 Control of electrosttic interctions etween F-ctin nd geneticlly modified lysozyme in queous medi Lori K. Snders*, Wujing Xin*, Cmilo Guáquet*, Michel J. Strohmn*, Chuck R. Vrsich*, Erik Luijten*, nd Gerrd C. L. Wong* Deprtments of *Mterils Science nd Engineering, Physics, nd Bioengineering, The Beckmn Institute for Advnced Science nd Technology, University of Illinois t Urn Chmpign, Urn, IL Edited y Michel J. Welsh, University of Iow College of Medicine, Iow City, IA, nd pproved August 21, 27 (received for review June 26, 27) The im for deterministic control of the interctions etween mcroions in queous medi hs motivted widespred experimentl nd theoreticl work. Although it hs een well estlished tht like-chrged mcromolecules cn ggregte under the influence of oppositely chrged condensing gents, the specific conditions for the stility of such ggregtes cn only e determined empiriclly. We exmine these conditions, which involve n interply of electrosttic nd osmotic effects, y using well defined model system composed of F-ctin, n nionic rod-like polyelectrolyte, nd lysozyme, ctionic gloulr protein with chrge tht cn e geneticlly modified. The structure nd stility of ctin lysozyme complexes for different lysozyme chrge mutnts nd slt concentrtions re exmined y using synchrotron x-ry scttering nd moleculr dynmics simultions. We provide evidence tht supports structurl trnsition from columnr rrngements of F-ctin held together y rrys of lysozyme t the threefold interstitil sites of the ctin sulttice to mrginlly stle complexes in which lysozyme resides t twofold ridging sites etween ctin. The reduced stility rises from strongly reduced prtitioning of slt etween the complex nd the surrounding solution. Chnges in the stility of ctin lysozyme complexes re of iomedicl interest ecuse their formtion hs een reported to contriute to the persistence of irwy infections in cystic firosis y sequestering ntimicroils such s lysozyme. We present x-ry microscopy results tht rgue for the existence of ctin lysozyme complexes in cystic firosis sputum nd demonstrte tht, for wide rnge of slt conditions, chrge-reduced lysozyme is not sequestered in ordered complexes while retining its cteril killing ctivity. ntimicroil cystic firosis self-ssemly smll-ngle x-ry scttering x-ry microscopy In the presence of multivlent ctions, strongly chrged nionic polyelectrolytes such s DNA nd F-ctin cn ssemle into densely pcked ggregtes (1). The existence of this counterintuitive like-chrge ttrction, which cnnot e explined within the frmework of men-field pproches such s the Poisson Boltzmnn theory (2, 3), hs motivted widespred experimentl nd theoreticl work (4 6). In the cse of F-ctin undles induced y multivlent ions, it hs een shown tht the ions within the undles re correlted nd hierrchiclly orgnized into density wves t polymer-length scles (7) ut remin liquid-like t moleculr-length scles (8). The prolem ecomes more complex when the mediting multivlent ctions re replced y sptilly extended mcroions, such s gloulr proteins. A mcroion is chrcterized y surfce chrge distriution nd hs its own counterions, s well s significnt excluded volume. In ddition, in the presence of slt there is strong interply etween electrosttic nd osmotic effects. For exmple, in previous work, we hve found tht ctin lysozyme complexes re stle up to considerly higher slt concentrtions thn would e expected, ecuse of osmotic pressure from ion prtitioning tht is constitutive of electrosttic inding etween oppositely chrged ojects (9, 1). The stility of these oppositely chrged polyelectrolyte protein complexes hs importnt iomedicl consequences. Accumultion of viscous mucus in pulmonry irwys is the primry cuse of long-term cteril infections nd eventully deth in cystic firosis (CF) (11). Anionic polyelectrolytes such s F-ctin (12) nd DNA (13, 14) re relesed into the irwy surfce liquid when neutrophils nd other cells lyse during the inflmmtory response. The concentrtion of F-ctin in the irwy is reported to e.1 5 mg/ml nd comprises 1% of totl leukocyte protein (12). Together with nionic mucins, these polyelectrolytes cuse the electrosttic ssemly of lrge ggregtes stilized y ctionic lignds, which commonly is oserved in CF sputum (15 17). It hs een suggested tht endogenous nticteril proteins, which re mostly ctionic (e.g., lysozyme, lctoferrin, -defensin, LL-37), constitute t lest portion of the lignds holding these nionic polyelectrolytes together (18). The concentrtion of the nticteril lysozyme is estimted t 1 mg/ml in the CF irwy (19). Becuse of their sequestrtion in polyelectrolyte complexes, the vilility of ntimicroil proteins such s lysozyme is diminished significntly, nd ntimicroil function in the irwy is impired correspondingly (2, 21). A iophysicl understnding of ntimicroil polyelectrolyte inding thus cn contriute to the rtionl design of therpeutic strtegies. In this rticle, we use geneticlly engineered lysozyme (22) with different monodisperse net chrges ( 9e, 5e, 3e) to understnd nd mnipulte the stility of self-ssemled ctin lysozyme complexes. Results from high-resolution synchrotron smll-ngle x-ry scttering (SAXS) nd moleculr dynmics (MD) simultions indicte tht, s the lysozyme chrge is reduced, ctin lysozyme complexes evolve from high-stility phse ( 9e lysozyme) to low-stility phse ( 5e nd 3e lysozyme): The evidence suggests tht the system evolves from complexes composed of hexgonlly coordinted columnr rrngements of F-ctin rods held together y 1D rrys of wild-type (WT) lysozyme t the threefold interstitil sites of the ctin sulttice to complexes in which the 1D rrys of chrgereduced lysozyme re rrnged t ridging sites etween pirs of ctin rods. Synchrotron x-ry microscopy using Fresnel zoneplte optics suggests tht the high-stility phse of ctin lysozyme complexes my occur in sputum collected from CF ptients, consistent with the expected ehvior of strongly sequestered endogenous lysozyme with 9e chrge. Interest- Author contriutions: L.K.S., W.X., E.L., nd G.C.L.W. designed reserch; L.K.S., W.X., C.G., M.J.S., nd C.R.V. performed reserch; L.K.S., C.G., E.L., nd G.C.L.W. nlyzed dt; nd L.K.S., E.L., nd G.C.L.W. wrote the pper. The uthors declre no conflict of interest. This rticle is PNAS Direct Sumission. Arevitions: CF, cystic firosis; SAXS, smll-ngle x-ry scttering; MD, moleculr dynmics. To whom correspondence my e ddressed t: Deprtment of Mterils Science nd Engineering, University of Illinois t Urn Chmpign, 134 W. Green Street, Urn, IL E-mil: gclwong@uiuc.edu or luijten@uiuc.edu. 27 y The Ntionl Acdemy of Sciences of the USA PNAS Octoer 9, 27 vol. 14 no cgi doi pns

2 S cttering I nten s it y ( ritrry units ) S cttering Inten s it y ( r itrry units ) Scttering Int ensit y ( r itrry units ) ingly, the low-stility phse contining chrge-reduced lysozyme dissocites t rod rnge of slt conditions, including the rnge reported for the irwy, so tht they re not sequestered y ctin in close-pcked complexes. This finding is independently confirmed y spectroscopic mesurements of the free lysozyme concentrtion in ctin lysozyme mixtures with lysozyme of different chrge. Moreover, cteril killing ssys show tht the chrge-reduced lysozyme mutnts cn retin most of the ntimicroil ctivity compred with the WT lysozyme ginst the PAO1 strin of Pseudomons eruginos, common opportunistic pthogen in CF, which suggests tht it is possile to simultneously minimize dventitious inding nd retin ctivity in chrge-optimized ntimicroils. c q r q z d q z q r Results nd Discussion WT Lysozyme nd F-Actin Self-Assemle into Stle Complexes t Physiologicl Conditions. First, we exmine the complextion ehvior of F-ctin with WT cteriophge T4 lysozyme. F-ctin is n nionic rod-like cytoskeletl polymer (liner chrge density e/.25 nm, persistence length 1 m). WT T4 lysozyme (dimensions Å 3 ) is ctionic gloulr protein with net chrge of 9e t physiologicl ph. In previous work (9, 1), we hve demonstrted tht F-ctin forms ordered ggregtes in the presence of hen egg-white lysozyme, which hs 3D structure different from WT T4 lysozyme (23). We confirm tht WT T4 lysozyme lso orgnizes F-ctin into undles. Fig. 1 shows representtive 2D SAXS diffrction pttern for prtilly ligned isoelectric lysozyme ctin undles proed with 3 3 m 2 synchrotron x-ry em, with ssocited 1D integrted intensity slices long the q z nd q r directions shown in Fig. 1.An inspection of the equtoril slice (q r ) shows correltion pek t q.71 Å 1 tht corresponds to inter-ctin pcking in the composite ctin lysozyme undles. Assuming the generic cse of hexgonl coordintion, this pek corresponds to n inter-ctin spcing of 12 Å (Fig. 1c). The inter-ctin spce fforded y this structure corresponds well to the size required y interstitil lysozyme ligned with its long xis prllel to the ctin rods. Other rrngements of lysozyme with different orienttions nd t different high-symmetry sites cnnot generte peks t this q position. In ddition to this diffrction feture nd wek mosicsmered intensity from the ctin form fctor (.113 Å 1 ), new correltion pek t.128 Å 1 is oserved long the meridionl (q z ) direction, which corresponds to correltion distnce of 49.1 Å, roughly the length of T4 lysozyme long its long xis nd consistent with the orienttion nd position of lysozyme inferred from the equtoril pek, indicting tht lysozyme is close-pcked long the ctin filments. These findings re in good ccord with those for complexes of ctin nd hen egg-white lysozyme (9, 1), supporting the generic nture of ctin lysozyme complextion. These x-ry oservtions re confirmed y comintion of grnd-cnonicl Monte Crlo simultions nd MD simultions of F-ctin undles to which neutrlizing mount of lysozyme hs een dded (1). Such complexes re found to hve vnishing net osmotic pressure (indicting stility) t n interctin spcing of 1 Å, in good greement with the SAXS results. Moreover, the simultions revel the sptil distriution of lysozyme within this complex (Fig. 1d). Under conditions of thermodynmic equilirium, the proteins re concentrted in the threefold interstitil sites etween the filments, in full greement with the structure inferred from the SAXS dt (Fig. 1c). The numericl clcultions lso explicitly confirm tht n even more expnded lttice structure, in which WT lysozyme is situted etween pirs of ctin filments, is thermodynmiclly unfvorle nd will contrct. In ddition, the simultions reproduce the close-pcked rrngement of lysozyme with its long xis prllel to the ctin xis. Experimentlly, we find tht this ctin lysozyme structure lso g 12 Å Si(111) Monochromtor Horiz./Vert. Slits e 3.1 g/l 2.6 g/l 2.1 g/l g/l Fresnel Zone Plte Order Sorting Aperture Smple CCD Detector Fig. 1. Synchrotron x-ry mesurements nd MD simultions demonstrte tht F-ctin nd WT lysozyme form stle, ordered complexes. () Synchrotron 2D x-ry diffrction pttern of prtilly ligned ctin WT lysozyme undles selfssemled in solution contining 1 mm NCl. () 1D integrted slices long q z (red line) nd q r (lck line) directions with rrows mrking the ctin ctin close-pcked undling pek (1) nd the lysozyme lysozyme correltion pek (2). (c) Proposed model of ctin WT lysozyme undle (end view) with WT lysozyme (ornge) pcked etween ctin filments (lue). The x-ry dt () indicte n inter-ctin spcing of 12 Å, providing sufficient spce for the presence of interstitil lysozyme. (d) Density plots from MD simultions showing the lysozyme distriution in the ctin WT lysozyme complexes without dded slt. The lysozyme is locted predominntly in the threefold interstitil regions. (e) Integrted diffrction dt show tht ctin lysozyme complexes re not significntly ffected y the presence of humn MUC5B frgments [t concentrtions of mg/ml (control) up to 3.1 mg/ml]. (f) 1D integrtion from x-ry microdiffrction mesurements of CF sputum (red line), which shows diffrction pek t q.7 Å, consistent with ctin lysozyme complexes, long with peks corresponding to self-ssemly from other mucus components. The locl chrcter of the ggregtes is demonstrted y the lck curve, which is smpled t 2 m from the smple shown in red ut displys no signs of ordered structure. (g) Schemtic digrm of the microdiffrction experiment t the Advnced Photon Source, Argonne, IL. is stle in the presence of mucins, such s purified humn MUC5B frgments, t physiologicl concentrtions nd tht MUC5B does not form complexes with lysozyme (Fig. 1e). The f APPLIED PHYSICAL SCIENCES Snders et l. PNAS Octoer 9, 27 vol. 14 no

3 Scttering Intensity (ritrry units) Scttering Int ensit y ( ritr ry units ) q r c q z d fctor of modertely twist-distorted ctin (25, 26) or my indicte possile dditionl self-ssemly from other mucus components. The diffrction fetures re highly sptilly loclized: Exmintion of representtive grid of microdiffrction dt shows tht ordered ggregtes hve typicl sizes of 1 2 m nd re emedded in disordered mtrix tht only exhiits low-q scttering from density fluctutions ut otherwise does not show discernle diffrction peks (Fig. 1f, lck line). This evidence rgues for the existence of ordered ctin lysozyme complexes in disordered nd heterogeneous CF mucus, the occurrence of which is unexpected. Becuse good surfce chrge-mtching etween ntimicroils nd F-ctin enhnces the stility of ound complexes in slt solutions through the mximiztion of counterion relese, potentil wy to dissolve such complexes is to destroy the chrge-mtching y modifying the chrge on lysozyme. 111 Å e 75 mm 5 mm 25 mm mm Fig. 2. Chrge-reduced lysozyme forms complexes with F-ctin tht re structurlly distinct from nd less stle thn those oserved for WT lysozyme. () Synchrotron 2D x-ry diffrction pttern of prtilly ligned ctin mutnt lysozyme undles. ( nd c) 1D integrted slices of ctin WT q z (red line), ctin mutnt q z (lck line), nd ctin mutnt q r (gry line) directions. A shift in the position of the ctin ctin undling pek to lower q, s indicted y the rrows (), suggests chnge in lysozyme coordintion in the complex from threefold sites (Fig. 1c) to twofold sites (c), thus shifting the inter-ctin spcing from 12Åto 111 Å. (d) Density plots showing the MD-simulted lysozyme distriution in the ctin lysozyme complexes without dded slt. In contrst to the WT lysozyme distriution (Fig. 1d), in the mutnt lysozyme ctin complexes (d) the lysozyme is found strongly concentrted round the ctin filments with enhnced concentrtion in the twofold ridging sites with the threefold sites eing nerly devoid of lysozyme. (e) 1D integrted slices of ctin triple-mutnt lysozyme ( 3e) in the q r direction showing the ctin ctin close-pcked undling pek t q.63 Å 1, which corresponds to n inter-ctin spcing of 114 Å. This phse dissocites upon the ddition of 25 mm NCl. ddition of up to 3 mg/ml of MUC5B mucin frgments hs no significnt effect on the diffrction signture of ctin lysozyme undles. A more stringent test of whether such complexes cn exist in CF irwy conditions, however, is direct exmintion of sputum smples from CF ptients. To investigte ordered structures within sputum, we employ x-ry microscopy techniques (Fig. 1 f nd g) in which.5.5 m 2 em is rstered cross sputum smples, nd the resultnt sptilly resolved diffrction dt re collected. In ddition to F-ctin nd lysozyme, CF sputum lso consists of wter, slts, DNA, vrious species of mucins, nd other polymeric components nd cell deris (24). Surprisingly, ordered structures indeed cn e oserved in CF sputum, despite its complex nd heterogeneous nture. We clerly oserve the presence of correltion pek t q.7 Å 1, which is close to the chrcteristic diffrction pek from ctin lysozyme complexes (Fig. 1f, red line). Diffrction fetures ner q.11 Å 1 my e ttriutle to the helix form MD Simultions Predict Tht Chrge-Reduced Lysozyme Forms Low-Stility Phse with Lysozyme t Twofold Bridging Sites. To exmine this hypothesis, we first employ MD simultions of corse-grined mixture of F-ctin nd lysozyme (1), where the net lysozyme chrge is reduced from 9e (the vlue of WT lysozyme t physiologicl ph) to 5e. To mintin chrgeneutrl mixture, the numer of lysozyme molecules in the simultion cell is incresed y fctor 9/5. We find tht, in the sence of externl slt, the chrge-reduced lysozyme still is le to undle the F-ctin. However, insted of the threefold coordintion oserved for the WT lysozyme (Fig. 1d), we now find strikingly different rrngement in which ech lysozyme hs twofold coordintion nd forms ridge etween pirs of ctin rods (Fig. 2d). This shift in geometry is ccompnied y shift in the inter-ctin seprtion, resulting in swelling of the undle. The swelling significntly reduces the prtitioning of ny dded monovlent slt nd thus gretly diminishes the stilizing effect oserved for WT lysozyme (9), s is confirmed in Fig. 3 (gry line), where the complex is found to e unstle upon ddition of even smll mounts of monovlent slt. Indeed, lredy in slt-free environment (Fig. 3, lck line), the net osmotic pressure only rely reches zero upon vrition of the ctin spcing nd displys very shllow negtive region, indicting low stility of the ctin lysozyme complex. It is instructive to compre the ionic distriutions within complexes tht re held together y WT nd chrge-reduced lysozyme, respectively (Fig. 4). Although the cross-sectionl distriution of nions is rther homogeneous nd similr in oth cses (Fig. 4 nd ), the ction distriutions re different. In the compct undles contining WT lysozyme, the ctions re concentrted etween pirs of ctin rods (Fig. 4c), llowing them to contriute to the effective inding, ut in the swollen undles with mutnt lysozyme of chrge 5e, the ctions re loclized completely round the ctin rods (Fig. 4d), preventing them from exerting ny inding effect. Wheres the present model cn provide qulittive picture, future improvements to these simultions could e mde with computtionl models tht include dditionl degrees of freedom to otin more ccurte description of the swelling ehvior in the presence of significnt concentrtions of slt. These findings suggest promising route towrd destiliztion of the ctin lysozyme complexes. Thus, we reduce the overll chrge of lysozyme through site-directed mutgenesis nd exmine how this ffects the electrosttic complexes. The monodispersity of these tilored mcroions in chrge s well s in size mkes them prticulrly suited for comprison to model systems nd gretly enhnces the predictive cpilities of the simultions cgi doi pns Snders et l.

4 O smotic Pressure (MP ) Actin-Actin Seprtion (Å) Fig. 3. Osmotic pressure of hexgonlly coordinted undle of mutnt ( 5e) lysozyme ctin filments in mm slt (lck line) nd 5 mm slt (gry line) solutions, s determined from MD simultions. Negtive pressure implies contrcting undle, wheres MP corresponds to stle undle. The zero crossing t seprtion ner 135 Å cn e contrsted with the 1-Å seprtion for the WT lysozyme ctin complex t mm slt s reported previously (9, 1). For the mutnt lysozyme ctin, the ddition of only 5 mm slt destilizes the undle (gry line). SAXS Revels Tht the Low-Stility Phse of Lysozyme Actin Complexes Is Destilized t Wide Rnge of Slt Conditions. Fig. 2 shows 2D SAXS dt from prtilly ligned isoelectric ctin lysozyme complexes mde with the K16E/R119E doule mutnt ( 5e), long with the diffrcted intensity integrted long wedges in the q z (Fig. 2, lck line) nd q r (Fig. 2, gry line) directions, proed y using 3 3 m 2 synchrotron x-ry em. Along the q r direction, strong diffrction feture is oserved t q.65 Å 1, corresponding to n expnded inter-ctin spcing of 111 Å. If we ssume tht the lysozyme remins t the threefold sites, this chnge would imply 27% Fig. 4. Ionic density distriutions (projected long the long xis of F-ctin) within ctin lysozyme ggregtes. ( nd ) The nionic distriution for stle ctin undle with WT lysozyme in the presence of 1 mm monovlent slt () nd for n ctin undle contining mutnt lysozyme in the presence of 5 mm monovlent slt (). (c nd d) The corresponding ction distriutions. Although the nion distriutions re homogeneous in oth ggregtes, the ction distriutions show strong modultion nd re different for oth cses. In the WT lysozyme ctin complex, the ctions re locted predominntly etween pirs of ctin filments (c), ut for the mutnt lysozyme ctin undle they re loclized round individul filments (d). Scttering Intensit y (ritrry units) wild type (+9) 2 mm 15 mm } mm } 1 mm 5 mm mutnt (+5) 2 mm 15 mm 1 mm 5 mm mm Fig. 5. Series of diffrction dt showing evolution of undle structure s function of NCl concentrtion for WT ( 9e) lysozyme ctin complexes () nd doule-mutnt ( 5e) lysozyme ctin complexes (). For the WT lysozyme ctin complexes (), the mximum undling occurs ner 125 mm NCl, wheres the mutnt lysozyme ctin undling pek () shows mximum t 5 mm NCl. increse in the lterl lysozyme size, which is unlikely given the two mino cid muttions. However, the expnded lttice spcing is consistent with the swelling oserved y the MD simultions (Fig. 2d), with lysozyme t the twofold ridging sites (depicted schemticlly in Fig. 2c). Along the q z direction, pek t q.126 Å 1 is oserved, reflecting periodic rrngement of lysozyme long the F-ctin xis tht is comprle to tht oserved for WT lysozyme (which shows pek t q z.128 Å 1 ). Similr results re oserved for two other 5e doule mutnts (K16E/K135E nd K16E/R154E), which suggest tht this structurl chnge is ttriutle to nonspecific electrosttic effects. Interestingly, n even more swollen version of the twofold ridging phse with further expnded lttice is oserved for the K16E/K135E/K147E triple mutnt with 3e chrge t zero dded slt (Fig. 2e). The intensity long the q r direction shows pek t q.63 Å 1 (inter-ctin spcing of 114 Å). The incresed ctin spcing compred with the 5e doule mutnts my indicte tht lysozyme is ridging F-ctin with the long xis of the lysozyme oriented t n ngle to tht of F-ctin in the composite undles. This phse, however, is unstle even for low concentrtions of dded slt: Fig. 2e shows tht the complex dissocites t 25 mm monovlent slt. As predicted y the simultions, the chnge from threefold to twofold coordintion of lysozyme within the undle leds to drstic chnge in the stility of the ctin lysozyme complex. Fig. 5 shows the SAXS spectr for electrosttic complexes formed etween F-ctin nd WT (Fig. 5) nd doule-mutnt (Fig. 5) lysozyme t incresing concentrtions of monovlent slt. For WT lysozyme, the undle formtion (nd lysozyme sequestrtion) is mximized etween 1 mm nd 15 mm monovlent slt. [Results for NCl re shown, ut the sme results lso re oserved for KCl (9, 1).] On the other hnd, for the doule mutnt ( 5e; Fig. 5), undle formtion is mximized etween mm nd 5 mm monovlent slt. The rnge of slt concentrtions for which undles form is lrger thn predicted y the simultions, which is consistent with the tendency of the corse-grined model, lredy oserved in ref. 1, to underestimte the precise rnge of stility. More importntly, however, the predicted trend is qulittively confirmed y the experiments, nd the mutnt lysozyme uninds from the ctin, nd thus is no longer sequestered, t the rnge of slt concentrtions expected in the CF irwy (5 15 mm). This finding suggests potentil method to mke nonstick ntimicroils tht will remin ctive in the electrosttic environment of the CF irwy. Direct Mesurement of Lysozyme Sequestrtion nd Antimicroil Activity. For ctin lysozyme complexes t slt concentrtions tht mimic the rnge of physiologicl conditions, we exmine whether APPLIED PHYSICAL SCIENCES Snders et l. PNAS Octoer 9, 27 vol. 14 no

5 Mterils nd Methods The methodology of the MD nd grnd-cnonicl simultions hs een provided in detil in ref. 1. The mutnt lysozyme ws modeled identiclly to the WT lysozyme (9, 1) ut with 2.5e chrge on ech of the two suunits. Bcteriophge T4 pseudo WT lysozyme crried chrge of 9e t neutrl ph nd hd dimensions of Å 3 nd moleculr weight of 18,7 (22). Expression nd purifiction procedures of the WT lysozyme nd its chrge-reduced mutnts were performed s reported (22). The chrge mutnts included three doule mutnts (K16E/R119E, K16E/R135E, nd K16E/ R154E) nd one triple mutnt (K16E/K135E/K147E); K, E, nd R denote the mino cids lysine, glutmic cid, nd rginine, respectively, nd, for exmple, R154E denotes muttion where the rginine t position 154 ws replced with glutmic cid. Monomeric ctin (G-ctin) (M r 43,) ws prepred from lyophilized powder of rit skeletl muscle (Cytoskeleton, Denver, CO) s previously reported (9, 1). The F-ctin lysozyme isoelectric point ws t molr rtio of 1.85:1 for F-ctin:WT nd 1:1 for F-ctin:doule mutnt. Typicl finl concentrtions were: F-ctin, 5.6 mg/ml; WT lysozyme, 3. mg/ml; nd doule mutnt, 5.4 mg/ml. These vlues re close to those found in the irwy. A series of smples ws prepred with the finl monovlent slt concentrtion rnging from mm to 2 mm. Lysozyme ctin complexes were seled in 1.5-mm qurtz cpillries (Hilgenerg, Mlsfeld, Germny) nd mixed thoroughly y centrifugtion. The pproximte smple volume in the cpillry ws 3 l. SAXS mesurements were performed with emline 4 2 t the Stnford Synchrotron Rdition Lortory (Plo Alto, CA) nd emline 12-ID-C t the Advnced Photon Source (Argonne Ntionl Lortory, Argonne, IL). For the Stnford Synchrotron Rdition Lortory experiments (incident x-ry wvelength Å), the scttered rdition ws collected y using MAR Reserch (Evnston, IL) chrge-coupled device (CCD) cmer (pixel size m 2 ). For the Advnced Photon Source experiments (incident x-ry wvelength 1.33 Å), the scttered x-rys were collected y using MAR Reserch 2D mosic CCD detector (pixel size m 2 ). The 2D SAXS dt from oth setups were checked for mutul consistency. Additionl detils hve een reported elsewhere (9, 1). In collortion with Donld Dvidson (Crle Clinic Assocition, Urn, IL) nd in ccordnce with institutionl review ord-pproved protocols t oth Crle Clinic nd t the University of Illinois t Urn Chmpign, we collected mucus from CF ptients who hd not een treted with DNse (Pulmozyme; Genentech, Inc., South Sn Frncisco, CA) for study y synchrotron x-ry microdiffrction. Ptients voluntrily expectorted 5 1 ml of sputum during respirtory therpy. After collection, the sputum smples were rpidly frozen nd stored t 8 C until use. Frozen smples were ultrmicrotomed nd seled etween kpton polymide film (Dupont, Wilmington, DE). Microdiffrction experiments of CF sputum smples were performed t emline 2-ID-D t the Advnced Photon Source y using em size of.5.5 m 2 focused with Fresnel zone plte in conjunction with n order-sorting perture. The smple-to- P erc e n t Surviv l l o ( cfu, % of co n tro l) C ys z y me Normlized C wild-type mutnt lysozyme only ctin-lysozyme wild-type mutnt wild-type mutnt Concentrtion (µg/ml) normlized c Fig. 6. Microequilirium dilysis experiments indicte tht chrge-reduced lysozyme is drsticlly less susceptile to ctin-induced sequestrtion thn WT lysozyme is, which is mesured y C lysozyme (C experimentl C theoreticl )/ C theoreticl, where C theoreticl is the expected concentrtion of fully dilyzed solution (.36 mg/ml) or 1 2 of the initil lysozyme concentrtion (.72 mg/ml). C lysozyme is shown for dilysis of.72 mg/ml lysozyme solutions in the sence of ctin () nd in the presence of 1.4 mg/ml F-ctin (). (c) Results for the ctin lysozyme system normlized y those for the lysozyme-only system. This normliztion corrects for potentil differentil dhesion of the different lysozyme to the dilysis memrne nd shows 17-fold decrese in sequestrtion for the mutnt (light gry) reltive to the WT (drk gry). (d) Results from nticteril killing ssys showing the percentge survivl of PAO1 cteri in the presence of incresing WT lysozyme (lck line) nd doulemutnt lysozyme (gry line) concentrtions. These results indicte tht, despite structurl chnges, the doule mutnt retins most of the WT nticteril ctivity. d the computtionl nd SAXS findings re reflected y the degree of lysozyme sequestrtion. Microequilirium dilysis experiments re used to mesure the extent to which WT nd mutnt lysozyme remin sequestered y F-ctin (Fig. 6 c). As seline mesurement, WT nd mutnt lysozyme re dilyzed for 12 h ginst uffer in the sence of F-ctin (Fig. 6). In this cse, the reltive concentrtion difference C lysozyme (C experimentl C theoreticl )/ C theoreticl is very smll, indicting tht oth WT nd mutnt lysozyme re nerly fully dilyzed. In contrst, if lysozyme is dilyzed from ctin lysozyme complexes (Fig. 6), C lysozyme shows lrge decrese in the mount of dilyzed WT lysozyme, reflecting the strong sequestrtion of WT lysozyme y the ctin filments. On the other hnd, the much weker inding of mutnt lysozyme is confirmed y the wek increse of C lysozyme. In Fig. 6c, we normlize the dilysis results otined in the presence of ctin (Fig. 6) y those for lysozyme only (Fig. 6), which shows tht for WT lysozyme the degree of sequestrtion s mesured y the devition from homogeneous distriution is 17 times lrger thn tht for the mutnt lysozyme. The reduced dhesion of geneticlly engineered lysozyme to ctin results in n incresed vilility of lysozyme. To ssess whether this cn trnslte into incresed ntimicroil ctivity, it is necessry to evlute whether the structurl modifictions of chrge-reduced mutnt lysozyme hve not lted its nticteril ctivity. We perform cteril killing ssys on the PAO1 strin of P. eruginos, Grm-negtive cterium commonly found in CF irwy infections. Fig. 6d shows the verged results from series of 2 trils in which cteril survivl is monitored s function of incresing protein concentrtion. The killing efficiency for the 5e mutnt chrge-reduced lysozyme (Fig. 6d, gry line) is comprle to, leit slightly lower thn, tht of the WT 9e lysozyme (Fig. 6d, lck line). Moreover, t concentrtion of 1 g/ml, the 5e mutnt kills nerly ll of the cteri. These results suggest tht it is indeed possile to optimize the chrge distriution in ntimicroils to simultneously minimize dventitious inding to inflmmtory polymers nd mintin ntimicroil ctivity cgi doi pns Snders et l.

6 detector distnce ws 42 mm corresponding to q.3 Å 1. Grid scns were tken out center point with 1- m stepsizein oth the horizontl nd verticl directions. Equilirium dilysis ws performed in microdilyzers (Hrvrd Apprtus, Boston, MA) contining two 1- l chmers seprted y 5,-D cutoff cellulose cette memrne. The smple chmer contined protein solutions in 1 mm NCl/2 mm Tris uffer; the ssy chmer contined only the 1 mm NCl/2 mm Tris uffer. The protein solutions were either ctin lysozyme complexes (1.4 mg/ml ctin.72 mg/ml lysozyme) or lysozyme-only controls (.72 mg/ml). Protein solutions were llowed to dilyze ginst the NCl/Tris uffer for 6 dys to ensure complete dilysis. Protein concentrtions were mesured y using UV-visile spectroscopy t 28 nm. The ctericidl ctivities of WT nd mutnt lysozyme were tested y using microdilution killing ssy on P. eruginos strin PAO1. Bcteri were grown in ction-djusted Mueller Hinton roth from n overnight culture (1:1 dilution) to mid-log phse t 37 C, hrvested y centrifuging t 9,25 g for 1 min, nd resuspended in PBS (1 mm N 2 HPO 4 /1 mm NCl, ph 7.4). Bcteri were diluted so tht 1 5 cfu were present in finl volume of 15 l of the ssy uffer, PBS. Bcteri were incuted with lysozyme in sterile 96-well flt-ottom polypropylene dishes while shking for3ht37 C. After the incution 1. Wong GCL (26) Curr Opin Col Int Sci 11: Groserg AY, Nguyen TT, Shklovskii BI (22) Rev Mod Phys 74: Levin Y (22) Rep Prog Phys 65: Bloomfield VA (1996) Curr Opin Struct Biol 6: Gelrt WM, Bruinsm RF, Pincus PA, Prsegin VA (2) Phys Tody 53: Olver de l Cruz M, Belloni L, Delsnti M, Dliez JP, Spll O, Drifford M (1995) J Chem Phys 13: Angelini TE, Ling H, Wriggers W, Wong GCL (23) Proc Ntl Acd Sci USA 1: Angelini TE, Golestnin R, Coridn RH, Butler JC, Berud A, Krisch M, Sinn H, Schweizer KS, Wong GCL (26) Proc Ntl Acd Sci USA 13: Snders LK, Guáquet C, Angelini TE, Lee J-W, Slimmer SC, Luijten E, Wong GCL (25) Phys Rev Lett 95: Guáquet C, Snders LK, Wong GCL, Luijten E (26) Biophys J 9: Welsh MJ, Smith AE (1995) Sci Am 273(6): Vsconcellos CA, Allen PG, Wohl ME, Drzen JM, Jnmey PA, Stossel TP (1994) Science 263: Brndt T, Breitenstein S, von der Hrdt H, Tummler B (1995) Thorx 5: period, cteri were serilly diluted, dropped on Mueller Hinton gr pltes, nd incuted 18 h t 37 C, nd colonyforming units were determined y using plte-counting methods. We thnk Dr. Brin W. Mtthews (University of Oregon, Eugene, OR) for providing the T4 lysozyme plsmids nd Dr. John K. Sheehn nd Dr. C. Willim Dvis (University of North Crolin, Chpel Hill, NC) for providing humn mucins. We lso grtefully cknowledge Dr. Michel J. Welsh nd Dr. Prdeep K. Singh for insightful discussions nd Dr. Zhonghou Ci, Dr. Brry Li, Dr. Donld Dvidson, Jnice Dougls, Blise Bowles, Leslie Gy, Scott C. Slimmer, John C. Butler, Thoms E. Angelini, Je-Wook Lee, nd Evelyn Hung for technicl ssistnce. This mteril is sed on work supported y the Ntionl Institutes of Helth under Grnt 1R21DK (to G.C.L.W.), the Cystic Firosis Foundtion (G.C.L.W.), nd the Ntionl Science Foundtion under Grnts DMR (to E.L.), DMR (to G.C.L.W.), nd CTS (to E.L. nd G.C.L.W.) vi the WterCAMPWS Science nd Technology Center. Portions of this reserch were crried out t the Stnford Synchrotron Rdition Lortory nd t the Advnced Photon Source. The Stnford Synchrotron Rdition Lortory Structurl Moleculr Biology Progrm is supported y the U.S. Deprtment of Energy, Office of Biologicl nd Environmentl Reserch, nd y the Ntionl Institutes of Helth, Ntionl Center for Reserch Resources, Biomedicl Technology Progrm. Use of the Advnced Photon Source is supported y the U.S. Deprtment of Energy, Office of Bsic Energy Sciences, under Contrct DE-AC2-6CH Shk S, Cpon DJ, Hellmiss R, Mrsters SA, Bker CL (199) Proc Ntl Acd Sci USA 87: Sheils CA, Käs J, Trvssos W, Allen PG, Jnmey PA, Wohl ME, Stossel TP (1996) Am J Pthol 148: Felgentreff K, Beisswenger C, Griese M, Gulder T, Bringmnn G, Bls R (26) Peptides 27: Tng JX, Wen Q, Bennett A, Kim B, Sheils CA, Bucki R, Jnmey PA (25) Am J Physiol 289:L599 L Trvis SM, Singh PK, Welsh MJ (21) Curr Opin Immunol 13: Brogn TD, Ryley HC, Nele L, Yss J (1975) Thorx 3: Weiner DJ, Bucki R, Jnmey PA (23) Am J Respir Cell Mol Biol 28: Bucki R, Byfield FJ, Jnmey PA (27) Eur Respir J 29: Sun DP, Söderlind E, Bse WA, Woznik JA, Suer U, Mtthews BW (1991) J Mol Biol 221: Mtthews BW, Remington SJ (1974) Proc Ntl Acd Sci USA 71: Broughton-Hed VJ, Smith JR, Shur J, Shute JK (Septemer 9, 26) Pulm Phrmcol Ther, 1.116/j.pupt Angelini TE, Ling H, Wriggers W, Wong GCL (25) Eur Phys J B 16: Purdy KR, Brtles JR, Wong GCL (27) Phys Rev Lett 98:5815. APPLIED PHYSICAL SCIENCES Snders et l. PNAS Octoer 9, 27 vol. 14 no