Experimental

Cobalt(II)chloride hexahydrate [Co(II)Cl*6H2O], Gold(III) chloride trihydrate 49% [HAuCl4], Iron(III)acetylacetonate 97% [Fe(III)acac], Iron(III)chloride hexahydrate [Fe(III)Cl*6H2O], Oleic acid 90%, Phenylether 99%, Sodium citrate dihydrate 99% [Cit], Tannic acid [TA], Tetramethylammonium hydroxide pentahydrate [TMAOH], Triethylene glycol 99% [TrEG], and Tris(2-carboxyethyl)phosphine hydrochloride 98% [TCEP] were purchased from Sigma Aldrich.

Sodium oleate [NaOle] was purchased from TCI. Phosphonoacetic acid 98% [PsAA] was purchased from Alfa Aesar. All used solvents have the reagent grade. All chemicals were directly used without further purification. For the sample preparation with ELP as well as the buffer and salt solutions Milli-Q water was used. All plastic tools in the protein sample preparation as well as all buffer and salt solutions were cleaned in the autoclave before use.

3.2.2 Nanoparticle syntheses Gold nanoparticles (Au-NPs)

For 10 nm Au-NP synthesis a modified protocol of Slot et al.¹¹⁵ was used. A mixture A of 80 mL distilled water and 1 mL of a 1 percentage by weight [wt%] aqueous HAuCl4 solution was prepared. A mixture B of 4 mL of a 1 wt% Cit solution, 16 mL H2O and 50 L of a 1 wt% TA solution was prepared. Both mixtures A and B were separately heated to 60 °C and afterwards mixed at this temperature while stirring. The mixture was immediately heated to 100 °C and

A) 25°C B) 60°C

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kept at this temperature for 20 min. A red solution of Au-NPs was obtained and cooled down to room temperature. The NP solution was stored in the fridge and was stable for two months.

Cobalt ferrite and magnetite nanoparticles (CoFe2O4- and Fe3O4-NPs)

CoFe2O4-NPs were synthesized by the protocol of Cabrera¹¹⁶. Firstly, the metal oleate precursors were prepared by exchange of the chloride ionstowards oleate anions of sodium oleate. The obtained products are a red, wax-like Fe(III)Ole precursor and a purple, solid Co(II)Ole precursor.

Both precursors were kept under inert atmosphere, since the Co(II)Ole precursor showed air sensitivity indicated by a color change from purple to black after one week. Therefore, the precursors were added to the three-neck bottle in a glove box. Under the counterflow additions oleic acid and phenylether were added to the precursors. A mixture of 0.57 mmol of Fe(III)(Ole) and 0.29 mmol of Co(II)(Ole), 2.55 mmol oleic acid and 25 mL phenylether was heated to 50 °C under vacuum and kept at this temperature for 30 min. The reaction mixture was slowly heated to the reflux temperature of 260 °C with a heating rate of 4 °C/min and kept at this temperature for 3 h under argon atmosphere. The stirring speed was kept at a minimum level which still allows a sufficient mixing. After cooling, a mixture of 1:1 toluene:heptane was added to the reaction mixture. The nanoparticles were precipitated by addition of an excess amount of a 1:3 2-propanol:ethanol mixture and separated by centrifugation. The procedure was repeated three times. The black, solid product was kept under inert atmosphere and could be easily dispersed in toluene. The obtained product is 5.5 nm CoFe2O4-NPs. Afterwards, the oleic acid ligand was exchanged to phosphonoacetic acid to disperse them in water¹¹⁷. In the modified protocol the precipitation with hexane/ethanol was replaced by centrifugation of the solution at 40000 rpm.

The supernatant was discarded and precipitate was collected and redispersed in MeOH. The procedure was repeated three times.

Fe3O4-NPs were synthesized by the protocol of Wan¹¹⁸. 2 mmol Fe(III)acac and 60 mL TrEG were added into a three-neck round 100 mL bottomed flask equipped with condenser, magnetic stirrer, thermometer and heating mantle. Under continuous stirring the mixture was heated to 40 °C and kept at this temperature under vacuum for 15 min to remove water and volatile components in the solvent. The mixture was then flushed with argon and stirred under argon.

The mixture was slowly heated to 180 °C for 20 min. This temperature was kept for 30 min.

Finally, the mixture was quickly heated to the reflux temperature of 280 °C in 10 min and refluxed at this temperature for another 30 min. After cooling down to room temperature, the nanoparticles were precipitated by addition of a 1:1 acetone:ethyl acetate mixture. The mixture was also placed on a magnet. The black precipitate was collected and re-dispersed in water. The

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washing step was repeated three times. The 9 nm nanoparticles were dried under vacuum and kept in an inert atmosphere to avoid oxidation of the material.

3.2.3 Protein preparation

The sequence of the ELP structure with 20 PGVGV pentapeptide units and cysteine (C) modification is shown below. This structure is labeled as ELP-V20.

MGSSHHHHHHSSGLVPRGSHMAMGV-(PGVGV)20-PGCGG

For the second modification the MamJ peptide was incorporated into the ELP structure. The resulting ELP species contain 20 (ELP-V20-MamJ) and 40 (ELP-V40-MamJ) pentapeptide units.

The storage buffer (50 mM Tris, 500 mM NaCl, 150 mM Imidazol, 10% Glycerol) of the ELP proteins was removed with PD Spin Trap G-25 from GE Healthcare. The protein was dispersed in water. TCEP as a reduction agent to avoid the formation of disulfide bonds between the cysteines in ELP was added to the protein solution leading to a final concentration of 2 mM TCEP in the solution.

3.2.4 Sample preparation

For the sample preparation Milli-Q water was used. All plastic tools in the protein sample preparation were cleaned in the autoclave before use. The 10.7 nm Au-NP concentration was determined by measuring the absorbance of the plasmon peak at 520 nm and using the extinction coefficient of Au-NPs with the size of about 10 nm¹¹⁹. A Fe3O4-NP solution in water with a concentration of 0.3 M (based on the mass concentration of 0.5 mg/mL, diameter of 9 nm and a density of magnetite bulk material of 5.2 g/cm³) and a CoFe2O4-NP solution in water with a concentration of 2.9 M (based on the mass concentration of 1.0 mg/mL, diameter of 5.5 nm and bulk density of 5.3 g/cm³) were prepared. The protein concentration was calculated by gravimetric determination of ELP after lyophilization and using the molecular weight of 11664.5 g/mol. The general sample preparation was composed of the addition of the protein solution to the NP solution. The mixture was mixed without a previous incubation step. The mixture was shaken over 16 h at a speed of 700 rpm. The protein equivalent bases on the NP concentration. The molar ratio of protein to NP was kept at 5-10 to 1. The NP-ELP mixture was heated to 60 °C in an incubator with temperature controller from Eppendorf.

3.2.5 Analytical methods

The crystal structure of the NP sample was investigated by a P-XRD Bruker D-8 Advance. The IR spectra were collected by a Perkin-Elmer spectrometer with an ATR crystal. The absorption spectra at different temperatures were recorded on a UV-Vis Cary 50 Probe with a temperature

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controller from Varian. Transmission electron microscopy (TEM) images were collected using a Zeiss Libra120 TEM operated at 120 keV. For TEM a volume of 10 µl was transferred onto a glow discharged carbon coated copper grid. After 5 min the drop was removed by a filter paper. For the mean size determination the size of 300 NPs in the TEM image was investigated.