Protein Handshake on the Nanoscale: How Albumin and Hemoglobin

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Protein Handshake on the Nanoscale: How Albumin and Hemoglobin Self-Assemble into Nanohybrid Fibers Christian Helbing, Tanja Deckert-Gaudig, Izabela FirkowskaBoden, Gang Wei, Volker Deckert, and Klaus D. Jandt ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b07196 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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Protein Handshake on the Nanoscale: How Albumin and Hemoglobin Self-Assemble into Nanohybrid Fibers Christian Helbing1, Tanja Deckert-Gaudig2, Izabela Firkowska-Boden1, Gang Wei3, Volker Deckert2,4, Klaus D. Jandt1,5,6* 1

Chair of Materials Science (CMS), Otto Schott Institute of Materials Research, Faculty of

Physics and Astronomy, Friedrich Schiller University Jena, Löbdergraben 32, 07743 Jena, Germany 2

Leibnitz Institute of Photonic Technology IPHT, Albert-Einstein-Strasse 9, 07745 Jena,

Germany 3

Faculty of Production Engineering, University of Bremen, Am Fallturm 1, 28359 Bremen,

Germany 4

Institute for Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University

Jena, Helmholtzweg 4, 07743 Jena, Germany 5

Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Humboldtstraße 10,

07743 Jena, Germany 6

Jena School for Microbial Communication (JSMC), Friedrich Schiller University, Jena,

Germany

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ABSTRACT Creating and establishing proof of hybrid protein nanofibers (hPNFs), i.e., PNFs that contain more than one protein, is a currently unsolved challenge in bioinspired materials science. Such hPNFs could serve as universal building blocks for the bottom-up preparation of functional materials with bespoke properties. Here, inspired by the protein assemblies occurring in nature, we introduce hPNFs created via a facile self-assembly route and composed of human serum albumin (HSA) and human hemoglobin (HGB) proteins. Our circular dichroism results shed light on the mechanism of the proteins self-assembly into hybrid nanofibers, which is driven by electrostatic/hydrophobic interactions between similar amino acid sequences (protein-handshake) exposed upon ethanol-triggered protein denaturation. Based on nanoscale characterization with tip-enhanced Raman spectroscopy (TERS) and immunogold labelling our results demonstrate the existence and heterogenic nature of the hPNFs and reveal the high HSA/HGB composition ratio, which is attributed to the fast self-assembling kinetics of HSA. The self-assembled hPNFs with a high aspect ratio of over 100 can potentially serve as biocompatible units to create larger bioactive structures, devices and sensors. KEYWORDS: serum albumin, hemoglobin, AFM, TERS, self-assembly, protein nanofibers

Natural and synthetic protein nanofibers (PNFs) are of major interest in nanoscience and biomedical engineering.1-10 On account of their excellent properties like large surface area, biocompatibility, tensile strength or chemical stability, PNFs are high-potential materials for numerous biological applications including tissue engineering, biosensors, and drug delivery.1, 57, 11-14

To expand the application range of the PNFs even further and to achieve specific 2 ACS Paragon Plus Environment

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therapeutic goals, PNFs with multifaceted biological and physical characteristics are required. Therefore, the fabrication of hybrid protein nanofibers (hPNFs) has recently received considerable attention, as the synergistic combination of protein functionalities will allow greater flexibility in the rational design of PNFs with, for example, tailored cell adhesion and superior mechanical properties. Towards this aim, different approaches have been employed to create hybrid protein nanofibers,

including

electrospinning,15-18

extrusion19,

20

or

template

synthesis.21-23

Electrospinning is the most commonly employed technique as it can produce scalable and long continuous fibers with diameters from 3 nm to a few microns.24 In previous works, the successful fabrication of collagen-elastin,17 silk-fibronectin15 or silk-collagen16 fibers by electrospinning has been shown. The incorporation of fibronectin into microscaled silk fibroin fibers has improved the hybrid fiber biocompatibility without sacrificing its mechanical integrity.15 The most frequently reported electrospun hybrid fibers, however, have diameters in the microscale and not in the nanoscale. Thus, the nanoscale advantage of a large surface to volume ratio, which is beneficial for surface induced processes, is not given. Additionally, the use of water soluble proteins, like plasma proteins, is challenging in electrospinning due to their poor stability during the spinning process. Therefore, the addition of a stabilizer is a commonly used method to obtain homogenous fibers dimensions. Another approach to create protein hybrid fibers is the extrusion process described by Underwood et al. as well as Raoufi et al.19, 20, 25 The advantage of this method is the use of native proteins from a buffer solution and the absence of an electric field. Yet, external stimuli, coagulation baths or shear stresses, which are required for the fiber formation, may have debilitating effects on the protein specific properties. Although the single protein hybrid fibers 3 ACS Paragon Plus Environment

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produced by this method have a diameter in the range of several tens of nanometers, the nanofibers almost immediately assemble to thicker bundles in the micrometer range.20 Therefore, with this approach also, the biomimetic nature of extruded hybrid fibers, in terms of their dimensional resemblance to protein fibers occurring in the extracellular matrix, has not been realized. Hybrid protein fibers have also been fabricated via co-polymerization of methacrylatefunctionalized polyvinyl alcohol and human serum albumin or by utilizing hydrophobic, highly unstable peptide templates to assist the self-assembly of various α-helical proteins into micrometer sized amyloid fibers.21 Conversely, chemical modification- and template-free protein self-assembly into a fiber structure can as well be driven by intrinsic properties of the protein and changes in the environmental conditions,12, elevated temperature or a pH change.12,

28

26-28

for example, by denaturants like ethanol,

As demonstrated by Wei et al., ethanol along with

acidic solution (pH = 2) induced the self-assembly of human plasma fibrinogen to homogenous PNFs with a diameter in the range of 10 – 20 nm.29, 30 Despite the simplicity of this self-assembly approach, the “handshake” between two different, yet unmodified plasma proteins, and their selfassembly into hybrid nanofibers has, to the best of our knowledge, not yet been reported. In this study, we address the challenge of combining two model proteins, human plasma albumin (HSA) and hemoglobin (HGB), into a single hPNF by using a self-assembly mechanism supported by ethanol-induced protein denaturation. In particular, we investigated the hybrid nature of the self-assembled nanofibers with tip-enhanced Raman Spectroscopy (TERS),31 which allows sample probing under optimal conditions with a spatial resolution below 1 nm.32 The surface sensitive TERS results are supported by complementary immunolabelling combined with a scanning transition electron microscope (STEM) and reveal the presence of HGB molecules on 4 ACS Paragon Plus Environment

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the hPNFs surface. Yet, the force spectroscopy data indicate that HGB molecules are incorporated within the HSA nanofibers rather than simply being a surface modification. The relatively low concentration of detected HGB in the hPNFs is explained by protein self-assembly kinetics assessed by circular dichroism (CD) spectroscopy. RESULTS AND DISCUSSION Fabrication of self-assembled hybrid protein fibers Inspired by the work of Juárez et al., who showed that HSAs form self-assembled PNFs at 50% ethanol at 65 °C, the self-assembly of HSA and HGB (weight ratio of 1/1) was induced by ethanol and MilliQ water solution 1/1 (v/v) at 65 °C.33 HSA was chosen as one of the model components based on the wealth of knowledge of its self-assembly mechanism.33, 34 In addition, it is known that the incorporation of various molecules may promote or inhibit the HSA fiber formation.35, 36 As for HGB, the formation of self-assembled hybrid peptide-HGB fibers has been reported.21 In order to test the influence of HGB on the fiber formation, the HSA-HGB solution after different assembly periods was deposited onto plasma cleaned silicon wafers and characterized with atomic force microscopy (AFM). Figure 1 and Figure S1 (Supporting information (SI)) show typical HSA-HGB assemblies and their time-dependent polymorphism, i.e., protofibrils, ribbon structures and twisted fibers. A similar kind of time dependent polymorphism of pure HSA nanofibers at a pH of 2 has been reported and discussed in previous studies.33,

34

In the

present study, the initial state of the fiber formation involves protofibrils formation, which takes place within the first two hours (Figure 1a). The protofibrils were found to be approximately 1 nm high and a few hundred nm long. 5 ACS Paragon Plus Environment

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Figure 1. (a-c) AFM height images of HSA-HGB assemblies (top) and their schematic representation (bottom) after different time periods (2 h, 24 h and 7 days). (100 µg/ml HSA/HGB 1/1 (w/w) 50% ethanol and 65 °C).

The intermediate state (6 – 24 hours) involves single fiber formation and their preferential side-by-side alignment to ribbon structures, as shown schematically in Figure 1b. According to the literature, fiber assembly to ribbons is driven by hydrophobic and electrostatic interactions between individual protein fibers.37 Also in our system, similar interactions between single nanofibers are possible due to ethanol induced exposition of hydrophobic side chains and polar groups.33, 38 The characteristic length of the ribbon structure ranged from 611 to 985 nm after 6 and 24 hours, respectively. Interestingly, the width of single nanofibers was found to be 22 ± 4 nm, which is approximately twice the width reported in the literature for pure HSA fibers under similar growth conditions.33 This discrepancy is the first indication that the HSA fiber formation is influenced by the second protein, and points toward proteins hybrid formation.


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Extending the assembly time to several days caused the ribbon structures to evolve into twisted nanofibers, with a width of 59 ± 10 nm and a length up to 6 µm (Figure 1c). The aspect ratio increases with longer assembly times up to 100 after 7 days. Twisted fiber structures have been observed for various proteins, including HSA, insulin and albumin.39-41 For twisted insulin nanofibers it has been suggested that the formation is predominantly influenced by the carboxylrich regions on the surface and takes place by surface template association.41 A similar mechanism could be responsible for the formation of both twisted and flat fibrils observed in our HSA-HGB system. Tip-enhanced Raman spectroscopy of hPNFs

Figure 2. (a) AFM topography image of a single HSA-HGB fiber. The white line represents the TERS measurement area and the two points where the spectra in (b) were taken. (b) Two representative TERS spectra with and without characteristic HGB bands. For a better comparison, the intensity of spectrum 1 was lowered. In the shown spectra, corresponding bands for porphyrin (660 cm-1, 749 cm-1, 1128 cm-1, 1312 cm-1, 1545 cm-1) are marked blue and for Fe2+ (1355 cm-1) and Fe3+ (1378 cm-1) are marked red (excitation at 532 nm, incident laser power 360 µW, acquisition time: 1 s). The band around 1640 to 1679 cm-1 corresponds to


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the amide I mode of the peptide bonds of the proteins. Note, the amide I band can be suppressed, most likely due to the protein backbone, large amino acid side chains or protein orientation.42,

43

(c) A schematic

representation of an HGB molecule with two heme groups containing porphyrin rings and the iron cation of the heme group.

As suggested by existing studies on hybrid protein systems, the validation of the hybrid fiber composition remains a major challenge.15, 20, 21 The nanoscale dimensions of both proteins and PNFs require a noninvasive, yet highly sensitive tool with a high spatial resolution to verify the hybrid nature of PNFs. The combination of AFM and Raman spectroscopy, known as TERS, has proven to be useful for small biomolecule characterizations, e.g. proteins.41, 44-47 The application of TERS in our PNFs system is possible due to Raman active vibrational modes of the four heme-group (porphyrin ring with iron, Fe, ion) present in the HGB structure (see Figure 2b and Figure S2). The heme resonance Raman spectra are dominated by the bands at 660 cm-1, 749 cm1

, 1128 cm-1, 1312 cm-1, 1545 cm-1 (Table S1, SI).48-50, including bands related to iron vibrations

at 1355 cm-1 (Fe2+) and 1378 cm-1 (Fe3+).50 (Table S1, SI). HSA belongs to the protein family which lacks a heme group,51, 52 and thus its Raman spectrum can be distinguished from HGB when directly compared. The detectability of heme-containing proteins by Raman spectroscopy and TERS has been reported earlier.50,53 For example, the obtained structural information allowed to identify oxidation processes on a crystal surface50 or an electron transfer.54 We used TERS to collect spatially resolved Raman spectra over several nm to determine the presence of HGB in synthesized PNFs, pointing to HSA-HGB hybrid formation. Figure 2b shows two representative TERS spectra obtained on a single HSA-HGB hPNFs. Spectrum 1, collected at position 1, shows a characteristic HGB fingerprint, namely its porphyrin (marked in blue) and iron ion bands (red). In contrast, HGB-related bands are not observed in spectrum 2, which indicates the absence of a heme group and thus indicating HSA. Further, the difference in the 8 ACS Paragon Plus Environment

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intensity between the recorded spectra can be related to the photo-enhancing property of the heme group.55 Overall, in 4 % of the analyzed spectra (n = 526), both, all porphyrin bands with and without iron bands as well as four of five porphyrin bands in combination with the iron bands have successfully been assigned. Considering the denatured and thus unfolded structure of the HGB (the native diameter of an HGB molecule is around 5 nm56) in the nanofiber (as indicated by the change in the secondary structure, see Figure S4, SI), groups should be observed every 6 to 10 nm. Consequently, by a step width of 2.0 - 2.5 nm, during the TERS measurements, the characteristic bands should appear at least in 20 - 25 % of the spectra, assuming that the protein fiber consists of HGB only. In HSA-HGB hPNFs, only one in ten spectra should be heme related. In view of the above considerations, the low percentage can indeed be a reliable indication of successful HSA-HGB hybrid formation. A detailed analysis of the appearance of the heme groups along the TERS scan line, as shown in Fig. S2, implies a nonhomogeneous (non-alternating) distribution of the HGB in the hPNFs. The continuous appearance/disappearance of heme groups along TERS scan line may be further explained by a misalignment of the HGB with respect to the scan line. It is important to note that TERS is a surface sensitive spectroscopy method, which allows the detection of Raman scattering from a nanoscopic volume. Despite the resonance amplification of the heme moiety by excitation at 532 nm,57 information about the inner composition of the PNF cannot be reliably accessed. Nonetheless, TERS data demonstrate that (i) a certain amount of the HGB has been incorporated into the hPNFs, and (ii) the HSA is the major component of the selfassembled hPNFs. Yet, the higher concentration of HGB in the core of the hPNFs cannot be ruled out. To show additionally that the appearance of the porphyrin bands corresponds to the presence of HGB, we characterized self-assembled HSA fibers prepared under identical 9 ACS Paragon Plus Environment

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conditions with TERS. (Figure S3) In these spectra no porphyrin bands were observed simultaneously due to the absence of heme groups in the HSA molecule. An interesting feature of the HGB-related TERS spectra is the appearance of both ferric and ferrous oxidation bands at 1378 and 1355 cm-1, respectively. A similar observation has been reported by Wood et al. and Böhme et al.,50, 57 and explained as the constant exchange of the oxygen molecules with the HGB molecules or possible oxidation of the heme species in the vicinity of the silver tip. The authors also showed that the existence of both oxidation bands could not be detected with a conventional Raman spectroscopy, which highlights the sensitivity of the TERS approach to biological/oxidation events occurring at the nanoscale. Assuming the “oxygen exchange” explanation, one could speculate that the biological function of HGB, such as energy transfer, has not been altered through the self-assembly process. Yet this hypothesis awaits further investigation. Immunogold labelling and atomic force spectroscopy of hPNFs


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Figure 3. (a) Schematic depiction of hPNFs labelling with AuNPs. (b) Representative STEM image of hPNFs immunolabeled with antibodies conjugated with AuNPs. Particles with a diameter below 12 nm – assigned to HSA – are marked orange, whereas nanoparticles with a diameter above 13 nm – assigned to HGB – are marked purple. Note, NPs aggregation was not observed.

To verify our TERS results and examine closely if the presence of both proteins in the selfassembled hPNFs can be visualized, an immunolabelling-based method was used. A similar method has been used to investigate the composition of the extruded blend of collagen and fibrinogen.20 Here, however, instead of fluorescence markers, we used secondary antibodies conjugated to gold nanoparticles (AuNPs) of different diameters. This approach has the advantage of identifying single protein molecules on the PNF surface with a STEM. Accordingly, antibody conjugated-AuNPs of 15 and 10 nm were used to identify HGB and HSA, respectively. Figure 3b shows representative STEM images of immunogold labelled PNFs on which differently sized AuNPs can be easily localized. The partial coverage of hPNFs with AuNPs is related to the general low labelling efficiency, which is about 10-15%.58 Nevertheless, immunogold labelling is usually considered as adequate for quantitation purposes.59 As exemplarily depicted in the insert, the majority of the AuNPs indicate the HSA protein (marked in orange). Note, the AuNPs larger than 13 nm, and thus assigned to the HGB, were marked in purple. In general, the majority (90 %) of the analysed AuNPs (n = 281 at 16 areas of 5 different fibers) had a diameter below 12 nm, which clearly shows that the PNFs consist predominantly of HSA. In contrast, only 7 % of the AuNPs had a diameter above 13 nm, which corresponds to the HGB. The remaining 3 % of AuNPs had diameters between 12 and 13 nm and could not be unambiguously assigned to either one protein. In analogy to TERS, immunolabelling is a surface sensitive method, which provides no information about the core of the PNFs. Yet, it


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complements the above discussed TERS results, and strongly implies that the dual protein system can self-assemble to hPNFs. To learn whether it is possible to improve the composition of HGB in the hPNFs, we performed self-assembly of HSA and HGB at 1/2 ratio. Unfortunately, an increased concentration of HGB protein prevented PNF formation and resulted in globular structures. An analogous behavior was described by Pandey et al., who showed the inhibition of HSA fibrillation in the presence of naturally occurring sugars, such as fructose, at a higher concentration. 35 An indirect way to obtain information on the HGB presence in the fiber volume is to assess the mechanical integrity of hPNFs. Based on the fibers’ nanoscopic dimensions, we used AFM force spectroscopy (Figure S5, SI) to acquire force data of AFM-tips covalently functionalized with PNFs, which can be interpreted in two ways: (i) as a force necessary to pull off the fiber from the surface or (ii) force required to rupture the fiber. Direct comparison of pure HSA fibers and hPNFs showed a significant difference in the measured force, 158.4 ± 46.1 nN (n = 41), and 38.79 ± 9.67 nN (n= 47), respectively. Considering the measured force to be a pull-off force, the difference may be explained by the presence of HGB molecules on the surface of hPNFs, resulting in reduced interaction between the fiber and functionalized surface. However, the force reduced by approximately 25 % cannot be solely attributed to the 5% of the surface-incorporated HGB (based on TERS data). Instead, we support the hypothesis that the difference is likely due to the presence of HGB within the hPNFs, which disrupts the mechanical properties and leads to the hPNFs rupture. Although the methodological barrier does not allow distinguishing between the measured force types, both scenarios strongly support the hybrid nature of the self-assembled PNFs. 12 ACS Paragon Plus Environment

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Assembly kinetics and mechanism of hPNFs The logic behind the low concentration of detected HBG in our hPNFs can be assumed to be due to (i) the structural differences of the used proteins and/or (ii) the different formation kinetics. It is known that protein self-assembly to a nanofiber structure is induced by a protein specific conformational transition driven by changes in the environment. The conformational transition, on the other hand, influences the formation kinetics of the resulting structures.12, 30, 6062

Therefore, insight into the proteins' conformational changes and kinetics will help to

understand the hPNFs formation mechanism. A suitable method to study in situ structural changes and PNF-formation/protein aggregation is CD spectroscopy.37 Accordingly, we employed CD to investigate the environmental influence on the structural changes in our HSAHGB system (Figure S4, SI). The CD results showed a change in the secondary structure of the protein mixture after adding 50% ethanol and heating up to 65 °C, in contrast to the measurements performed in water at 37 °C (Figure S4, SI). Further, the CD spectrum of the protein mixture revealed the increased content of β-sheet structures and decrease in the, still dominant, helical structure (for further information see the CD Spectroscopy section in the SI). Even though the exact content of the secondary structure can be determined for only the single protein system, one can assume that the HSA-HGB system is dominated by the helical structure based on the analysis of the secondary structure of the individual proteins. Note, both proteins revealed helical structures under fiber formation condition (Figure S4, SI). Further, the protein structures, especially of HGB, were destabilized by the addition of ethanol due to a regulation of hydrogen bonds and attractive hydrophobic interactions as shown by Juárez et al..33 This leads to protein aggregation and may facilitate interaction between HSA and HGB molecules.


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Figure 4. (a-c) 24 hours CD measurements of 2 µM HSA-HGB, HSA, and HGB, respectively, under fiber formation conditions (50 % ethanol at 65 °C).

In terms of the hPNFs formation kinetics, hourly CD measurements over a period of 24 hours were performed for the HSA-HGB mixture and both single proteins. The minor CD signal shifts during the first hours of the HSA-HGB fiber formation (1 – 10 hours, red and blue lines Figure 4a) can be assigned to the first steps of PNF formation, i.e., the nucleation phase. During this step, the secondary structure transformation leads to an activation of the HSA and HGB molecules and enables the later hPNF formation. This nucleation process mediates the interactions between molecules, which initiate the second PNF formation step, the self-assembly to first agglomerates and the formation of protofibrils. A strong decrease in the signal starts after 10 hours, step three, corresponding to the formation of larger structures, which continue to grow for another 3 hours 33 (11 – 13 hours, green lines Figure 4a). Upon prolonged self-assembly time the signal continues to slowly decrease and plateaus after 24 hours. This corresponds to the elongation process of the previously formed protofibrils and their assembly to ribbon structures, as shown in Figure 1. These CD measurements demonstrate that the hPNF formation starts with 14 ACS Paragon Plus Environment

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a nucleation process, which leads to the protofibrils assembly followed by nanofiber formation. A similar PNF formation mechanism is described in the literature for amyloid PNFs.12 In comparison to the HSA-HGB hourly CD scans, the 24 hours HSA spectra show a similar behavior (Figure 4b). Yet, the strong decrease of the signal after 6 hours together with a promptly reached plateau stage, indicate a faster self-assembly kinetic when compared to the HSA-HGB system. Similar changes in the CD signal were described in the literature for the selfassembly of HSA.37 HGB, on the other hand, shows a very constant and slow signal decrease over the entire time range, and thus no fiber formation (Figure 4c). Corroborating the earlier TERS and immunolabelling results, the observed mismatch in the self-assembly kinetics of individual proteins as well as a reduced kinetic of HSA-HGB (when comparing to pure HSA) indicate the interaction between the proteins and their self-assembly to hybrid fibers. More important, the observed differences in the protein self-assembly kinetics can explain the low content of HGB in the hPNFs. Based on the above discussed results, we propose the following self-assembly mechanism of hPNFs.


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Figure 5. The formation mechanism of HSA-HGB hPNFs consists of five steps, whereas each step is supported by the data from at least one experimental method.

In the model schematically presented in Figure 5, we assume that the denaturation-induced “activation” of protein molecules is required for nucleation (Figure 5 step 1). These activated proteins interact and form protofibrils, whereby protofibrils are mainly composed of HSA molecules on account of the fast protein self-assembly kinetics. The formation of homogenous HSA protofibrils subsequently leads to a depletion of activated HSA molecules in the surrounding of the protofibrils, which increases the possibility for HGB to be incorporated into the protofibrils. Rational interactions between HSA and HGB molecules can be explained by the presence of distinguished regions in the primary structure with similar amino acid sequences (shown in the SI), which facilitates the HSA and HGB interaction by electrostatic and/or hydrophobic forces. We assume that these particular regions are exposed upon ethanol-triggered denaturation. Although the verification of the exact amino acids sequences exposed by the proteins, and thus the nature of the potential interactions is not possible, we assume hydrophobic 16 ACS Paragon Plus Environment

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interactions. This assumption is supported by the work of Ridgley et al. who showed that hydrophobic interactions between highly α-helical proteins, like HGB, and template peptide are essential for their self-assembly into amyloid fibers.21 In addition, the fact that the majority of amino acids in HSA and HGB are hydrophobic (based on their residues), 42 % and 54 %, respectively, indicates negligible contribution of hydrogen bonding and electrostatic interactions. (Table S2, SI) Furthermore, we assume that the protofibrils are formed by end-to-end connected molecules, based on protofibril width, similar to the dimensions of a native HSA molecule (~ 14 nm).63 This statement is supported by the differences observed in our force spectroscopy measurements. With increasing self-assembly time the protofibrils grow to longer hybrid nanofibers followed by their assembly to ribbon structures and finally hybrid twisted nanofibers (see Figure 1 and 5). CONCLUSION This study shows that it is possible to create hPNFs composed of two plasma proteins using a facile self-assembly approach supported by ethanol-induced protein denaturation. The similarity in the amino acid sequences, in both HSA and HGB proteins, exposed upon conformational changes and their hydrophobic interactions are recognized as the driving force for the selfassembly mechanism of the hPNFs. The challenge of establishing the proof of hPNFs heterogenic nature can be overcome with nanospectroscopic techniques, like TERS and AFM force spectroscopy. We believe that this self-assembly approach can successfully be applied to other plasma proteins assuming that the same amino acid sequences can be found in both proteins upon conformational changes. For example, the fibrinogen and fibronectin system is expected to form 17 ACS Paragon Plus Environment

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hPNFs due to the similarity in the primary structure and the existence of binding sites for fibrin in fibronectin. Such hPNFs can be for example utilized as universal building blocks for the bottom up preparation of larger bioactive structures or, on account of their high surface area, can be used as bioactive devices and sensors. METHODS Preparation of hPNFs To create self-assembled HSA-HGB PNFs, stock solutions of both proteins were prepared in MilliQ water and diluted to 100 µg/ml. Afterwards, the protein solutions were mixed in a volume ratio of 1/1 and concentrated ethanol was subsequently added to achieve a concentration of 50 %. (The self-assembly of HSA (66.5 kDa) and HGB (65kDa) was tested for various w/w ratios, namely 1/0, 1/1, 2/1, 0/1, and 1/2. Only the first three ratios resulted in fibers formation.) The final HSA-HGB solution was stored in an oven at 65 °C for different time ranges. After assembly times of 2, 6, 10, 12, 18, and 24 hours and 2 to 7 days, 10 µL of each protein solution was deposited onto a plasma cleaned silicon wafer for AFM characterization and cleaned cover glasses for TERS measurements. Characterization of PNF To characterize the morphology of self-assembled PNFs, the substrates were investigated by tapping mode AFM. AFM measurements in air were performed by using a Dimension 3100 and a MultiMode (both from Digital Instruments, Vecco, Santa Barbara, CA) equipped with a Nanoscope IV controller. Measurements were performed at room temperature by using standard tapping mode silicon cantilevers from Bruker (model RTESP, Vecco, Santa Barbara, CA) with a


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resonance frequency in the range of 315−364 kHz in air, a spring constant in the range of 20−80 N/m, and a typical tip radius of less than 10 nm (typical 7 nm). Circular Dichroism Spectroscopy Ultraviolet CD spectra were obtained using a Jasco CD-Spectrometer J-715 with a 1 mm path length cuvette (Hellma Analytics, Germany). The previously prepared stock solution was diluted to concentrations of 2 µM for HSA and HGB. Spectra of solutions with and without protein were recorded between 195 and 300 nm at 65°C. For the measurement of the combination, 2 µM of HSA and 2 µM of HGB were mixed in a volume ratio of 1/1. For 24 hours measurements the prepared HSA, HGB and HSA-HGB solutions were heated up to 65 °C. After a constant temperature was reached, every hour one spectrum was collected. Tip Enhanced Raman Spectroscopy TERS spectra were acquired on a Nanowizard III (JPK Instruments AG, Berlin, Germany) mounted on an inverted microscope (Olympus IX70, Hamburg, Germany) equipped with a confocal spectrometer (SP2750A, Acton Advanced, Princeton Instruments, Roper Scientific, USA) and a CCD camera (Pixis 256, Princeton Instruments, Roper Scientific, USA). The following objective conditions were used: oil immersion, 60x, NA = 1.45 (Olympus, Hamburg, Germany). TERS tips were prepared by evaporating 25 nm silver on commercially available AFM cantilevers (Tap190Al-G, Budget Sensors, Germany), which were stored under argon atmosphere until used. The samples were illuminated with λ = 532 nm at P = 360 µW for HSAHGB hPNFs and P = 720 µW for pure HSA-PNFs. The acquisition times were, tacq = 1 s. 1700 spectra were collected from the fiber surface of 8 different HSA-HGB hPNFs and 1200 spectra from the fiber surface of 6 different HSA PNFs with a lateral distance step that varied from 1 to 19 ACS Paragon Plus Environment

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5 nm. The minimum step that resulted in the appearance of different modes in the collected spectra was 2 nm, which was estimated to be at least the spatial resolution of the TERS measurement in this work. Force Spectroscopy The silicon AFM tips (DNP S10, Bruker, Santa Barbara, CA, USA) and silicon substrates were modified to covalently bind the hPNFs, as described in the following: AFM probes and substrates were cleaned for 30 minutes with freshly prepared Piranha solution (H2SO4: 30% H2O2, 7:3, v/v) then washed with large amounts of DI water (3x) and ethanol (99%) (2x). The cleaned AFM probes and substrates were immersed into a mixed solution of 3-aminopropyl triethoxysilane

(3-aminopropyl

trimethoxysilane)

(APTES)

and

thiethoxychlorosilane

(Triethoxy-chlorosilane) (TTCS) (1% in toluene, APTES:TTCS, 1:4, v/v) for 15 minutes. Then washed with DI water (2x) and ethanol (99%) (1x) several times (3x). Afterwards, the AFM probes and substrates were transferred into PEG-NHS ester disulfide (0.1 mg/mL) for 1 hour to bind the PEG-NHS ester disulfide onto the AFM probe and substrate. At the last step, the AFM probes were immersed into a hPNF solution for 30 minutes to promote the binding of hPNF onto the probes. The hPNF-modified AFM probes were washed with a large amount of DI water to remove noncovalently adsorbed hPNF. The single force-distance curves of hPNFs were measured with a Catalyst AFM interfaced with a Nanoscope VIII controller (Bruker, Santa Barbara, CA, USA) in water. The DNP-S10 (Bruker, Santa Barbara, CA, USA), a probe with four different cantilevers, was used as tip for modification. To measure the force, only the tips B and D with spring constants of 0.12 N/m and 0.06 N/m, respectively, were used. To ensure binding of the hPNFs on the silicon substrate, we 20 ACS Paragon Plus Environment

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used a surface delay of 30 s. The pull-off-/ rupture-force was determined as maximum force, which was needed to retract the modified tip from the substrate. Immunogold labelling A 10 µl drop of the prepared HSA-HGB PNF solution was placed on a parafilm and a clean carbon-film coated Cu-TEM grid was placed in the solution for 30 s. Subsequently the grid was transferred twice for 5 minutes into 0.1% gelatin from cold water fish skin (Sigma-Aldrich, Schnelldorf, Germany) in Tris-buffered saline (TBS) (pH = 7.6) as blocking step. The TEM grids were placed in the primary antibody solution for 1 hour at room temperature. To label the proteins antibodies from a rabbit against HGB (sc-21005 from Santa Cruz, Dallas, TX, USA) and antibodies from a sheep against HSA (ab8940, from Abcam, Cambridge, United Kingdom) were used. After further washing steps with 0.1% gelatin in TBS for 5 minutes the grids were placed in a secondary antibody solution in 0.1% gelatin in TBS for 30 minutes at room temperature. For HGB we employed anti-rabbit secondary antibodies conjugated with AuNPs of a diameter of 15 nm (EM.GAR15 from BBI Soultions, Cardiff, United Kingdom) and for HSA anti-sheep secondary antibodies conjugated with AuNPs of a diameter of 10 nm (EM.DAS10 from BBI Soultions, Cardiff, United Kingdom). The immunogold labled samples were washed four more times with 0.1% gelatin in TBS for 5 minutes and two more times in distilled water. The immunolabeled fibers were characterized by scanning transmission electron microscopy with an AURIGA 60 CrossBeam® Workstation (Carl Zeiss AG, Oberkochen, Germany) and the gold nanoparticles’ diameters along the hPNFs were analysed. ASSOCIATED CONTENT Supporting Information. 21 ACS Paragon Plus Environment

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Supporting Information Available: AFM images, secondary structure of HSA-HGB, HSA and HGB, AFM force spectroscopy, details to the amino acid sequences. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author ** [email protected] (KDJ) Author Contributions The manuscript was written by contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (DFG) project: “Novel functional materials based on self-assembled protein nanofibers: creating and understanding nanofibers”, AOBJ: 609403. ACKNOWLEDGMENT Further, we gratefully thank the Biomolecular NMR Spectroscopy Group of Dr. Görlach from the Fritz Lipmann Institute for performing the CD-spectroscopy measurements and Dr. C. Lüdecke from the Chair of Materials Science and Dr. M. M. L. Arras from Oak Ridge National Laboratory for their help during the preparation of this manuscript. Further, we gratefully acknowledge M. Scholl and K. Scheuer from the Chair of Materials Science for their help by analyzing the STEM and AFM-Force Spectroscopy data.

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TOC


Protein Handshake on the Nanoscale: How Albumin and Hemoglobin

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