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1 Supprting infrmatin Prteins Human serum albumin frm Sigma (378, fatty acid free, 99% pure) was purified frm dimer and cntaminating prteins using gel filtratin n a 00 x 3.4 cm Sephadex G50 clumn in ammnium acetate buffer, ph 6.5 (75 mg prtein in 0 ml buffer was applied at a time). Fractins cntaining HS mnmer were pled, lyphilized and desalted by gel filtratin n a G5 Sephadex superfine clumn in Millipre water. nalytical gel filtratin shwed that after this prcedure mre than 99% f the prtein was in the mnmeric frm. Human calmdulin was expressed frm a Pet3a plasmid in E. cli Bl1 De3 PlysS Star, and purified using heat treatment, DEE cellulse anin exchange chrmatgraphy, hydrphbic interactin chrmatgraphy n a phenyl sepharse clumn, gel filtratin and DEE sephacel anin exchange chrmatgraphy, as described in reference 1. Human α-lactalbumin was purified frm human milk using ammnium sulphate precipitatin, hydrphbic interactin chrmatgraphy n a phenyl sepharse clumn, and dialysed, as described in reference. Human carbnic anhydrase II ( pseud wildtype with the C06S mutatin) was expressed frm the pcpwt plasmid (a kind gift frm Prfessr Nalle Jnssn, Linköping University) in E. cli Bl1 De3 PlysS Star. The cells were harvested by centrifugatin at 6000 g fr 5 minutes and the pellet was frzen. The prtein was purified using in exchange chrmatgraphy and gel filtratin as fllws. The cell pellet was suspended in buffer (10 mm Tris/HCl, ph 7.5 with 1 mm EDT; al vlume 00 ml fr pellet frm a 5.4 litre culture), snicated n ice, pured int an equal vlume f biling buffer, heated t 85 C, and then rapidly cled n ice. Precipitated E. cli prtein were remved by centrifugatin at rpm fr 15 minutes, and the supernatant was pumped nt a (3.4 x 5 cm) DEE cellulse anin exchange clumn and eluted using a NaCl gradient frm 0 t 0. M in buffer. Carbnic anhydrase II fractins were

2 lyphlized, disslved in 5 ml H O, and applied t a 3.4 x 180 cm sephadex G50 superfine gel filtratin clumn. The clumn was packed and perated in 50 mm ammnium acetate buffer, ph 6.5. The pure carbnic anhydrase II fractins were pled and dialyzed against Millipre water. Prtein G B1 was expressed in E. cli Bl1 De3 PlysS Star frm a synthetic gene clned int PetSac plasmid, and purifed using anin exchange chrmatgraphy (DEE cellulse clumn) and gel filtratin (3.4 x 180 cm sephadex G50 superfine clumn) as described in reference 3. Lyszyme frm chicken egg white was frm Sigma and purified using in exchange chrmatgraphy and gel filtratin. Ovalbumin and Fibringen were purchased frm Sigma and used withut further purificatin. The purity f prteins was cnfirmed by SDS PGE and agarse gel electrphresis, iselectric fcussing and 1 H NMR spectrscpy. Oleic acid was frm Sigma (O1008, 99% pure). Nanparticles. N-is-prpylacrylamide-c-N-tert-butylacrylamide (NIPM:BM) cplymer particles f diameters frm nm and with three different ratis f the c-mnmers (85:15, 65:35 and 50:50 NIPM:BM) were synthesized in SDS micelles as described previusly 4 althugh higher SDS cncentratins were used in the present wrk, resulting in similarly sized particles. The prcedure fr the synthesis was as fllws:.8 g mnmers (in the apprpriate wt/wt rati), and 0.8 g crsslinker (N,Nmethylenebisacrylamide) was disslved in 190mL MilliQ water with 0.8, 0.575, 0.3, 0.04 and 0.03 g SDS fr the 70, 10, 00, 400 and 700 nm particles respectively and degassed by bubbling with N fr 30 minutes. Plymerisatin was induced by adding 0.095g ammnium persulfate initiatr in 10 ml MilliQ water and heating at 70 C fr 4 hurs 5. Particles were extensively dialysed against MilliQ water fr several weeks, changing the water daily, until n traces f mnmers, crsslinker, initiatr r SDS culd

3 be detected by prtn NMR (spectra were acquired in D O using a 500 MHz Varian Inva spectrmeter). Particles were freeze-dried and stred in the fridge until used. Quantum dts. 16 nm hydrphilic plymer cated quantum dts (7) were prvided by W. Parak, Ludwig Maximilians University, München. Isthermal titratin calrimetry. HS was titrated frm a 38 µm stck int nanparticle slutin in 10 mm Hepes/NaOH buffer, ph 7.5 with 150 mm NaCl and 1 mm EDT at 5 C. The reactin cell cntained 1mg/ml f the nanparticles, which crrespnds t 9.3, 1.84, 0.4, 0.05 and nm fr the 70, 10, 00, 400 and 700 nm particles respectively. The first prtein injectin was 1 µl fllwed by 59 injectins f 5 µl. The prtein cncentratin in the first HS stck was determined by amin acid analysis after acid hydrlysis (analysis purchased frm BMC Uppsala). The cncentratin in subsequent stck slutins was determined by UV-absrbance at 80 nm using the extinctin cefficient btained frm the first stck. ITC data were als btained fr titratin f prteins int slutins f quantum dts (16 nm diameter) in 10 mm Hepes/NaOH, 0.15 M NaCl, 1 mm EDT, ph 7.5. HS (38 µm) was titrated int an 800 nm quantum dt slutin and α-lactalbumin (30 µm) was titrated int a 500 nm slutin f quantum dts. The first prtein injectin was 1 µl fllwed by 39 injectins f 5 µl. The stck slutin fr α-lactalbumin was prepared frm Ca -depleted prtein. Data analysis. Data were fitted using the sftware ORIGIN (Micrcal, Nrthhamptn, M), assuming a single set f identical sites binding istherm with assciatin cnstant, stichimetry, and H as variable parameters. The equilibrium assciatin cnstant, K, fr adsrptin f HS t a nanparticle (np) with N equal and independent sites is: K [ nps HS] [ nps] = (1)

4 where nps are HS-binding sites n nanparticles. The degree f saturatin ges frm 0-1 fr each site and is defined as: f sat K = () 1 K Using that [HS]=[HS] N* [nps HS] and [np]=[np] [nps HS], f sat can be expressed as: f sat = N[ np] NK [ np] 1 N[ np] K N[ np] 1 4 N [ np] (3) The heat absrbed r released by the adsrptin f the prtein t the particles in an ITC experiment is: Q [ np] f sat = H nhsbund = H Vcellccmplex = H Vcell N (4) where n HS bund is the number f mles f adsrbed HS, H is the enthalpy change upn binding (J/ml HS) and V cell is the cell vlume. Cmbining eq. 3 and 4 yields: V Q = cell [ np] N H 1 N[ np] K N[ np] 1 N[ np] K N[ np] 4 N [ np] (5) Taking int accunt the displaced vlume, the change in heat cntent frm injectin i-1 t i is

5 dv Q( i) = Q( i) Q( i 1) V 0 i Q( i) Q( i 1) (6) where Q i is the heat cntent after injectin i, V 0 is the initial cell vlume and dv i is the injected vlume. By an iterative fitting prcedure, using standard Marquardt methds, N, K and H can be btained frm eq. 5 and 6. Thil-linked Nanpartices. 50:50 NIPM:BM:acrylic acid cplymer nanparticles f 70 nm diameter were synthesized as abve, with the additin f apprpriate amunts f acrylic acid t btain particles with n average less than ne carbxyl grup n the particle surface. crylic acid was distilled under reduced pressure befre use t remve stabilizers. stck slutin f 1 mg/ml acrylic acid was prepared, and 10 µl f this slutin was added t the mnmer slutin (190mL). Reactin prceeded at 70 C fr 4 hurs fllwed by dialysis against MilliQ water fr a cuple f weeks. The cvalent attachment f hmcysteine t the acrylic acid grups invlves the frmatin f amide bnds between the primary amin grup f the amin acid and carbxylic acid 6. Briefly, 50 ml f the particle slutin (after dialysis) was adjusted t ph 5 by small amunts f 5 M NaOH. 1-ethyl-3-(3-dimethylaminprpyl) carbdiimide hydrchlride was added t a final cncentratin f 150 mm t activate the carbxylic acid mieties. fter 1 hur f incubatin with stirring at 4 C, 0.4 g hmcysteine was added and the ph was readjusted t 5. The reactin mixture was incubated fr 5 h at rm temperature under stirring, dialysed extensively against MilliQ water t ensure that n residual chemicals remained, and freeze-dried. Cnjugatin f nanparticles t gld surfaces fr SPR studies. The SI u kit (BIcre B, Uppsala) was used fr sensr chip preparatin. Thil-linked nanparticles were disslved at 0. mg/ml in 0 mm sdium phsphate buffer, 100 mm NaCl, ph 7.5) n ice and 10 µl was applied t a 10 x 10 mm gld surface fr fur hurs r ver night, befre the surface was rinsed with H O, dried and assembled in a sensrchip cassette. The change in respnse units after cupling f the nanparticles t gld reveals the amunt f

6 immbilized nanparticles. densely packed layer f 70 nm particles yields 35 ng/mm and the increase in respnse btained (6 ng/mm ) crrespnds t 74 % f this number. Surface Plasmn Resnance (SPR) experiments. SPR studies f prtein assciating t the nanparticles were perfrmed using a BIcre 3000 instrument (BIcre B, Uppsala). The flw buffer cntained 10 mm Tris/HCl ph 7.4 with 3 mm EDT, 150 mm NaCl and 0.005% Tween0, and was filtered (0. µm filter) and degassed fr 30 minutes. The sensrchip surface with attached particles was washed fr 5 hurs at a flw rate f µl/min and then equilibrated at 10 µl/min fr 30 minutes until the baseline was stable. Each prtein was disslved at 5-0 µm cncentratin in the flw buffer and injected ver the sensrchip surface fr 30 minutes t study the assciatin kinetics, fllwed by buffer flw fr 5-4 hurs at 10 µl/min t btain a stable baseline befre the next injectin f prtein. 1. Walterssn, Y.; Linse, S.; Brdin, P.; Grundstrm, T., Bichemistry 1993, 3, (31), Svenssn, M.; Hakanssn,.; Mssberg,. K.; Linse, S.; Svanbrg, C., Prceedings f the Natinal cademy f Sciences f the United States f merica 000, 97, (8), Lindman, S.; Xue, W.-F.; Szczepankiewicz, O.; Bauer, M. C.; Nilssn, H.; Linse, S., Biphys. J. 006, 90, (8), Wu, X.; Peltn, R.; Hamielec,.; Wds, D.; McPhee, W., Cllid Plym. Sci. 1994, 7, Lynch, I.; Miller, I.; Gallagher, W. M.; Dawsn, K.., Jurnal f Physical Chemistry B 006, 110, (30), Bernkp-Schnurch,.; Leitner, V.; Mser, V., Drug Develpment and Industrial Pharmacy 004, 30, (1), Liedl, T.; Keller, S.; Simmel, F. C.; Radler, J. O.; Parak, W. J., Small 005, 1, (10),