Biomolecules

Article The Kinetics of Amyloid Fibril Formation by de Novo Protein Albebetin and Its Mutant Variants

Vitalii Balobanov 1,*, Rita Chertkova 2,3,*, Anna Egorova 1, Dmitry Dolgikh 2,4, Valentina Bychkova 1 and Mikhail Kirpichnikov 2,4

1 Institute of Protein Research, Pushchino, Moscow 142290, Russia; [email protected] (A.E.); [email protected] (V.B.) 2 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho–Maklaya st. 16/10, Moscow 117997, Russia; [email protected] (D.D.); [email protected] (M.K.) 3 Faculty of Biology and Biotechnologies, National Research University Higher School of Economics, Moscow 117312, Russia 4 Biology Department, Lomonosov Moscow State University, Leninskie gory, 1/12, Moscow 119899, Russia * Correspondence: [email protected] (V.B.); [email protected] (R.C.)

Received: 29 November 2019; Accepted: 3 February 2020; Published: 5 February 2020

Abstract: Engineering of amyloid structures is one of the new perspective areas of protein engineering. Studying the process of amyloid formation can help find ways to manage it in the interests of medicine and biotechnology. One of the promising candidates for the structural basis of artificial functional amyloid fibrils is albebetin (ABB), an artificial protein engineered under the leadership of O.B. Ptitsyn. Various aspects of the amyloid formation of this protein and some methods for controlling this process are investigated in this paper. Four stages of amyloid fibrils formation by this protein from the first non-fibrillar aggregates to mature fibrils and large micron-sized complexes have been described in detail. Dependence of albebetin amyloids formation on external conditions and some mutations also have been described. The introduction of similar point mutations in the two structurally identical α-β-β motifs of ABB lead to different amiloidogenesis kinetics. The inhibitory effect of a disulfide bond and high pH on amyloid fibrils formation, that can be used to control this process, was shown. The results of this work are a good basis for the further design and use of ABB-based amyloid constructs.

Keywords: amyloidogenesis; amyloid ﬁbrils; cross-beta structure; albebetin; de novo proteins

1. Introduction Protein misfolding and aggregation, when monomeric protein molecules turn into amyloid fibrils, is one of the factors causing severe disorders, including neurodegenerative diseases [1]. Amyloids are fibrils, usually unbranched, consisting of protein monomers linked by hydrogen bonds between β-strands of an intermolecular β-sheet; they lie perpendicularly to the axis of the fibril. This structure is known as the cross-beta structure and is considered a universal sign of amyloid fibrils [2,3]. It is important to mention that there is evidence of amyloid formation by proteins under conditions associated not only with pathology, but also with the implementation of the key biological functions of these proteins [4–7]. There are proteins exhibiting functional activity only while having an amyloid configuration [8–10]. Numerous facts demonstrate that amyloidogenesis is a fundamental property of the polypeptide chain, which allows one to consider the amyloid structures as variants of protein quaternary structure. The formation of amyloid structures is typically mediated by protein transformation due to certain factors to an intermediate state which properties are similar to those of a molten globule (i.e., the presence of a secondary structure but no dense packing of side groups) [11]. Such states, as a rule, have greater mobility of the polypeptide chain and increased hydrophobicity [12].

Biomolecules 2020, 10, 241; doi:10.3390/biom10020241 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 241 2 of 16

This, as many authors suppose, leads to increased aggregation ability compared to the native state. This comparison, however, is not applicable to natively unfolded and highly destabilized proteins, since they initially do not have a native state, which eliminates the need to cross the free-energy barrier between the native and intermediate states. Albebetin (ABB), a de novo designed protein with a predesigned spatial structure the topology of which is not found in natural proteins, has the following properties [13]. Albebetin is an ~8 kDa polypeptide chain consisting of 73 amino acid residue (AAR). It has a predesigned secondary structure (two identical α-β-β repeat motifs), but does not exhibit dense packing of side groups and exists in the state close to that of a molten globule [13]. The absence of tight packaging makes structural and thermodynamic studies of this protein difficult. For the same reason, it is difficult to use computational methods for calculating the mobility of the protein chain and the solvent accessible surface area. In our work, we will use the theoretically predicted structures. Albebetin was used in our previous works to illustrate the fundamental possibility to utilize de novo designed proteins as efficient carriers of biologically active peptide sequences. Thus, an artificial protein albeferon [14], which is structurally identical to albebetin with the inserted active fragment of human interferon α2 (IFN-α2), retained the predesigned structure of the original albebetin [15], exhibited high biological activity (comparable to that of IFN-α2)[16], and low immunogenicity [17]. Next, a number of artificial proteins were designed based on albebetin and albeferon; these proteins carried not only functional peptide fragments of natural IFN-α2 [18], but also of differentiation factor HLDF [19] and of human insulin, exhibiting the respective biological activities and relatively low immunogenicity [20,21]. Earlier, we demonstrated that the de novo protein ABB and its derivatives carrying biologically active fragments of natural proteins can form fibrils at physiological pH [22–24]. The amyloid nature of these aggregates was verified by analyzing far-UV circular dichroism (CD) spectra and the aggregate morphology by atomic force microscopy (AFM). It was shown that addition of short peptides (the octapeptide of human INF-α2 or the hexapeptide HLDF-6 of the differentiation factor of human promyelocytic leukemia cell line HL-60 (HLDF)) to the N-terminal end of ABB significantly enhances the amyloidogenic properties of the protein and increases the rate of amyloid fibrils formation. A similar effect was also observed in the presence of the free hexapeptide HLDF-6 in ABB solution [22]. Higher ionic strength of the solution also accelerates the formation of ABB amyloids, although to a lesser extent than adding the peptides [23]. Hence, the artificial protein ABB and its modified variants can be used as a convenient model to study the mechanisms of fibril formation and the cross-beta core structure of amyloid fibrils. Mutations are often the significant reason of why a normally functioning protein becomes prone to amyloid fibril formation [1]. In this connection, it would be interesting to model such amino acid substitutions in the ABB that could change the kinetics of amyloid formation in either side. For this reason, a thorough study of the amyloidogenic properties of original ABB and its mutant variants carrying substitutions that enhance/attenuate the amyloid formation ability of the protein is relevant, as well as investigating the influence of external factors on this process. These studies can shed light on the molecular mechanisms underlying amyloidogenesis, which in the long run will make it possible to use amyloid structures to design novel biomaterials having a broad scope of application, as well as potential therapeutic agents capable of inhibiting or accelerating the amyloid formation. In this study, the amyloid fibril formation by the de novo protein ABB is scrutinized using a number of spectral and microscopic methods. The effect of some point mutations on this process is evaluated. In addition, we discuss the prospects for the use of ABB as an amyloid carrier of functional domains and the possibility of using the detected effects to control amyloid formation in practical applications. Biomolecules 2020, 10, 241 3 of 16

2. Materials and Methods

2.1. Constructing Mutant ABB Genes The mutant genes of artificial protein ABB were obtained using a PCR series. The protein ABB consists of two identical fragments of amino acid sequence. It is clear that the two perfect repeats in the ABB sequence impede the introduction of mutations by PCR. We suggested using a two-stage process to obtain the mutant ABB genes. At the first stage, two PCR reactions were performed as follows: the 5’-end and 3’-modified primers were employed for the one PCR experiment, while the 5’-modified and 3’-end primers were used for another PCR experiment. Each reaction yielded two products, since the mutant primers bind to the gene regions carrying codons for both mutations (His30 and His65). The amplification reactions were conducted using the ABB gene within a pET-32 LIC plasmid vector (Novagen, Kenilworth, NJ, USA) as a template. The DNA fragments obtained in these PCR experiments, which presumably carried the desired mutations and whose molecular weight matched the calculated one, were used to produce full-length fragments carrying the desired mutations by PCR-2. At the second stage, the products of PCR-1 and PCR-2 reactions carrying a H30F point substitution were used for PCR-3, and the products carrying a H65F point substitution were employed for PCR-4. The DNA fragments carrying the mutations were completed, and the resulting full-length fragments with H30F and H65F substitutions were used as templates in the next amplification cycles. Since we have demonstrated that the disulfide bond plays a significant role in amyloid formation, it was interesting to synthesize and study the albebetin variant carrying a double mutation at Cys residues. The gene of the mutant variant ABB(C8S/C43S) was constructed and synthesized according to the scheme described above. The nucleotide sequences of mutant genes in the plasmid DNA were determined on an ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). Full-length DNA fragments were cloned into the plasmid vector pET-32-ABB LIC via the ligation reaction. The reaction medium (10 µL) contained the following components: buffer for T4 DNA ligase (Fermentas, Vilnius Lithuania), T4 DNA ligase, an insert—gene of ABB or its mutant, and plasmid vector pET-32 LIC/BamHI/Hind III. The reaction was carried out at 16 ◦C during 1 h.

2.2. Gene Expression; Isolation and Purification of Recombinant Proteins The plasmid vector pET-32-ABB LIC allows one to express the target protein gene fused with the trx gene of thioredoxin within a hybrid protein (TRX-ABB). The hybrid protein also carries the 6 His × sequence to ensure rapid and efficient purification of the product using metal chelate chromatography. The expression level of hybrid protein in this construct was appreciably high, making it possible to quantitatively produce the target proteins. The expression of mutant ABB genes was performed in E. coli strain BL-21 DE3. The cells transformed with the plasmid carrying the gene encoding the synthetic protein were cultured in Terrific Broth liquid growth medium supplemented with ampicillin as in [18,19] to a final concentration of 100 µg/mL at 37 ◦C under vigorous stirring (250 rpm) for 22–24 h. Once expression had been completed, E. coli cells were precipitated by centrifugation at 6000 g and 4 ◦C for 15 min. The resulting cell biomass was resuspended in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.02 M β-mercaptoethanol; MgCl2 (to a final concentration of 5 mM) and DNase I (Sigma-Aldrich, St. Louis, MO, USA) (50 µg per g of cells) were added. The cell suspension was homogenized on a French press (Spectronic Instruments, Inc., Irvine, CA, USA). The water-insoluble fraction of the homogenate was precipitated by centrifugation at 30,000 rpm for 30 min; approximately 90% of hybrid protein was present in the water-soluble fraction. The hybrid protein was isolated from the cell lysate by ion-exchange chromatography on a DEAE-cellulose column (Sigma-Aldrich, St. Louis, MO, USA) in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl. The hybrid protein was eluted with 50–500 mM NaCl gradient in the same buffer as in [18,19]. Next, the hybrid protein was purified on a column packed with nickel-nitrilotriacetic acid (Ni-NTA) metal chelate resin (Qiagen, Hilden, Germany) equilibrated with 50 mM Tris-HCl buffer, pH Biomolecules 2020, 10, 241 4 of 16

8.0, containing 300 mM NaCl. The hybrid protein was eluted with 20–250 mM imidazole gradient. The purified hybrid protein was concentrated on the DEAE-cellulose column until a concentration of at least 10 mg/mL. The hybrid protein was subjected to enzymatic cleavage using factor Xa protease (Sigma-Aldrich, St. Louis, MO, USA) in 20 mM Tris-HCl buffer, pH 8.0, containing 100 mM NaCl and 5 mM CaCl2, at room temperature for 24 h at a 1:2000 molar ratio between the enzyme and the protein. The reaction mixture was applied to the column packed with Ni-NTA in the respective buffer; the target albebetin was not bound to the sorbent and was eluted from the column immediately after the void volume mark as in [18,19].

2.3. Protein Purity Analisis All stages of protein isolation and puriﬁcation were controlled by denaturing gel electrophoresis in 12% Tris-Tricine PAGE gel with 1% SDS [25] and tris(2-carboxyethyl)phosphin hydrochloride (TCEP), a disulﬁde bound reducing agent. Gel electrophoresis under nondenaturing conditions in 9% PAGE gel was used to evaluate the homogeneity of the resulting protein under native conditions and to verify whether there were any aggregates in the solution.

2.4. Absorption Spectroscopy Absorption spectra were recorded on a Cary-100Bio spectrophotometer (Varian, Palo Alto, CA, 1 cm USA). Protein concentrations were determined using the calculated extinction coeﬃcients A 1 mg/mL 1 cm = 15 at λ = 220 nm for ABB and its mutant and A 1 mg/mL = 0.6 at λ = 280 nm for hybrid proteins.

2.5. Fluorescence Spectroscopy Thioflavin T (ThT) (Sigma, St. Louis, MO, USA) was used as a cross-beta specific fluorescent dye. The final concentration of Thioflavin T was 0.1 µM. The fluorescence measurements were made on a Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, CA, USA) in quartz cells 3 3 mm. × Continuous monitoring was performed at an excitation wavelength of 455 nm; the emission was recorded at 480 nm. When collecting the samples after certain time intervals, the fluorescence spectra were recorded at 448–600 nm at an excitation wavelength of 455 nm. When studying the kinetics of amyloid aggregation of hybrid protein, the intrinsic fluorescence of tryptophan residues was also recorded at 300–450 nm, at an excitation wavelength of 280 nm.

2.6. Light Scattering The changes of light scattering intensity during aggregate growth were recorded by continuous monitoring in a cell. Light scattering was measured simultaneously with recording the ﬂuorescence intensity of ThT for the same samples at an excitation wavelength of 455 nm; the recording wavelength was 449 nm.

2.7. CD Spectroscopy The far-ultraviolet (UV) CD spectra were recorded on a Chiroscan spectropolarimeter (Applied Photophysics Ltd, London, UK) in a cell with an optical path length of 3 mm in the wavelength range of 190–250 nm. Molar ellipticity [Θ] was calculated taking into account the concentration of the protein, its amino acid composition and the dimension of the cuvette [26].

2.8. Electron Microscopy To prepare the specimens, the protein solution after the incubation was diluted to 0.1 mg/mL. The specimens were adsorbed onto a carbon ﬁlm and subjected to negative staining with 1% aqueous solution of uranyl acetate. Electron microimages under magniﬁcation 220,000 and 160,000 were recorded on a JEM100C electron microscope (JEOL, Tokyo, Japan). Biomolecules 2020, 10, 241 5 of 16

2.9. Atomic Force Microscopy A solution of the incubated specimen was diluted to a concentration of 0.1 mg/mL. A specimen droplet containing the aggregates was applied onto the cleaved mica surface. After 5-min incubation, the specimen was washed with water and allowed to dry at room temperature. Silicon probes with the tip curvature radius of ~10 nm (NT-MDT, Moscow, Russia) were used for the semi-contact mode. Their resonance frequency was varied for different probes within 60–120 kHz. The specimen was scanned at a resolution of 256 256 or 512 512 points. × × 2.10. Confocal Fluorescence Microscopy The size and morphology of the resulting aggregates were evaluated using a Leica TCS SPE confocal fluorescent microscope. After incubation, the protein solution was diluted with Prolong Gold anti-fade solution (Invitrogen, Waltham, MA, USA) at a 1:1 ratio. The specimens (5 µL) were applied onto the microscope slides and covered with a cover slip slide. Thioflavin dye fluorescence was laser-excited at 405 nm. Confocal fluorescence microscopy images were recorded in a wavelength range of 450–600 nm. Serial confocal images were obtained at a 0.5 µm scan step between the focal planes.

2.11. Error Calculation Two types of errors were used in the work. The first is the error of the measuring instruments. This indicator is a characteristic of devices and was determined when baselines were measured. It was used to compare data obtained in the same experiment with different instruments. The second type of error reflects the relative standard deviation from the mean value upon repeated repetition of the experiment. This error was taken into account when comparing data for various mutant forms of ABB or when assessing the impact of conditions.

3. Results and Discussion

3.1. Studying the Artificial Protein ABB Amyloid Formation The experimental investigation of amyloid formation kinetics of remains a nontrivial problem. Amyloid formation by proteins depends on a number of stochastic factors, which often cannot be taken into account [27]. As a result, there are significant dispersions in the process rate and observed signal amplitude when the experiments are repeated. The aforementioned dispersion makes it impossible to compare the data obtained using different methods in different experimental runs. In order to solve this problem, we simultaneously conducted the measurements using several methods at the one and the same sample. In this case, we can take into account only the errors of the instruments, which are incomparably smaller than deviation upon repetition of the experiment. Protein solutions were incubated in a thermostated cell at 45 ◦C in a 20 mM Tris-HCl buffer, pH 7.4, containing 200 mM NaCl, at protein concentration of 10 mg/mL, in the presence of tris(2-carboxyethyl)phosphin (TCEP), a disulfide bonds reducing agent. The thioflavin T (ThT) dye fluorescence and the intensity of scattered light were continuously measured during incubation. Samples were collected from the cell after certain intervals to record the circular dichroism spectra and conduct other measurements. The morphology of the aggregates was studied by fluorescence microscopy, electron microscopy, and atomic force microscopy. The monomer content and the amount of nascent aggregates were estimated by electrophoresis under nondenaturing conditions.

3.1.1. Fluorescence Spectroscopy and Light Scattering The kinetics of amyloid formation has been studied by continuously recording the fluorescence intensity of ThT dye added to the protein solution. In order to evaluate the rate at which the size of nascent amyloids increased, the light scattering spectra were recorded simultaneously with the fluorescence (Figure1). The results demonstrate that the intensity of ThT fluorescence increased upon Biomolecules 2020, 10, 241 6 of 16

nascent amyloids increased, the light scattering spectra were recorded simultaneously with the Biomolecules 2020, 10, 241 6 of 16 fluorescence (Figure 1). The results demonstrate that the intensity of ThT fluorescence increased upon incubation of the sample due to the emergence of the cross-beta structure. The intensity of incubationscattered light of the was sample simultaneously due to the emergenceincreased, ofindi thecating cross-beta that larger structure. aggregates The intensity were formed. of scattered The light wasscattering simultaneously and fluorescence increased, curves indicating are S-shap that largered. The aggregates slight weredifference formed. in the The curve light scattering shape is andpossibly fluorescence related to curves aggregate are S-shaped. formation The and slight accumula differencetion, followed in the curve by their shape conversion is possibly to related the form to aggregatethat specifically formation binds and ThT. accumulation, A short, relatively followed to by the their time conversion of growth, to the lag-period form that (~10 specifically min vs.binds ~150 ThT.min) Atestifies short, relatively[28] a linear to the (without time of growth,branching lag-period or fragmentation) (~10 min vs. growth ~150 min) of testifiesthe amyloid [28] a and linear a (withoutrelatively branching large (~10 ormonomers) fragmentation) size of growth the fibril of nucleus. the amyloid It is andimportant a relatively to note large that (~10 upon monomers) repetition sizea sufficiently of the fibril large nucleus. distribution It is important mentioned to above note that is found. upon repetition a sufficiently large distribution mentioned above is found.

Figure 1.1. TimeTime dependencesdependences ofof the the intensity intensity of of thioflavin thioflavin T (ThT)T (ThT) fluorescence fluorescence and and light light scattering scattering on amyloidon amyloid formation formation by albebetin by albebetin (ABB) (ABB) in the 20in mMthe 20 Tris-HCl mM Tris-HCl buffer (pH buffer 7.4) at(pH 45 ◦7.4)C in at the 45 presence °C in the of 200presence mM NaCl.of 200 ThemM thickness NaCl. The of thickness the solid linesof the corresponds solid lines tocorresponds the instrumental to the instrumental errors; the hatching errors; showsthe hatching the dispersion shows the during dispersion repeated during repetition repeated of therepetition experiment. of the experiment. 3.1.2. Far-UV Circular Dichroism 3.1.2. Far-UV Circular Dichroism Amyloid formation is accompanied by significant changes in the β-structure content. This makes Amyloid formation is accompanied by significant changes in the β-structure content. This it possible to use CD spectroscopy to study the amyloidogenesis processes. The Far-UV CD spectra of makes it possible to use CD spectroscopy to study the amyloidogenesis processes. The Far-UV CD the specimens were recorded prior to incubation, as well as at 20 and 270 min after the beginning of spectra of the specimens were recorded prior to incubation, as well as at 20 and 270 min after the the incubation (Figure2b). The shape of the CD spectra demonstrates that the secondary structure of beginning of the incubation (Figure 2b). The shape of the CD spectra demonstrates that the the ABB protein has changed severely. secondary structure of the ABB protein has changed severely. One can easily see that the most significant changes take place at wavelengths of 220 and 205 nm. One can easily see that the most significant changes take place at wavelengths of 220 and 205 The time dependences of the ellipticity Θ at 205 and 220 nm allowed us to infer that the negative nm. The time dependences of the ellipticity Θ at 205 and 220 nm allowed us to infer that the ellipticity was initially slightly increased in both cases and then decreased. The time dependence of negative ellipticity was initially slightly increased in both cases and then decreased. The time the ratio of the ellipticities at these wavelengths (Figure2a) allows one to estimate the changes in the dependence of the ratio of the ellipticities at these wavelengths (Figure 2a) allows one to estimate shape of the CD spectra over time. the changes in the shape of the CD spectra over time. An analysis of the spectral data infers that the shape of the CD spectra is significantly altered in An analysis of the spectral data infers that the shape of the CD spectra is significantly altered the beginning of aggregation (related to the increasing ellipticity at 205 nm); however, there are only in the beginning of aggregation (related to the increasing ellipticity at 205 nm); however, there are minor changes in the intensity of ThT fluorescence. This fact indicates that intermediate aggregates the only minor changes in the intensity of ThT fluorescence. This fact indicates that intermediate secondary structure of which differs from that of the monomeric form of the protein have accumulated, aggregates the secondary structure of which differs from that of the monomeric form of the protein but have not bound ThT yet. Further changes observed using these two methods are also asynchronous: have accumulated, but have not bound ThT yet. Further changes observed using these two methods the shape of the CD spectra changes at a slower rate than the rate at which the intensity of ThT are also asynchronous: the shape of the CD spectra changes at a slower rate than the rate at which fluorescence increases. This behavior demonstrates that intermediate structures (protofibrils) have the intensity of ThT fluorescence increases. This behavior demonstrates that intermediate structures been accumulated: they have already bound the dye ThT, but their secondary structure does not match (protofibrils) have been accumulated: they have already bound the dye ThT, but their secondary that of the final aggregates yet. In all likelihood, fibril maturation and formation of the final secondary structure does not match that of the final aggregates yet. In all likelihood, fibril maturation and structure occur at the final stage. formation of the final secondary structure occur at the final stage. Biomolecules 2020, 10, 241 7 of 16

Biomolecules 2020, 10, 241 7 of 16 Biomolecules 2020, 10, 241 7 of 16

Figure 2. (a) Time dependences of the ratio of ellipticities, Θ2205/Θ205, and the intensity of ThT fluorescence recorded when studying the kinetics of amyloid formation by ABB. Instrumental errors correspond to the size of the characters and the thickness of the line. (b) The far-UV circular dichroism (CD) spectra of ABB measured before incubation (black), after 10-min incubation (red), Figure 2. (a) Time dependences of the ratio of ellipticities, Θ /Θ , and the intensity of ThT 20-min incubation (green), and 450-min incubation (blue) at 45 °C2205 in the205 20 mM Tris-HCl buffer (pH ﬂuorescence recorded when studying the kinetics of amyloid formation by ABB. Instrumental errors 7.4) inFigure the presence 2. (a) Time of 200 dependences mM NaCl. of the ratio of ellipticities, Θ2205/Θ205, and the intensity of ThT correspondfluorescence to the recorded size of the when characters studyingand the kinetics the thickness of amyloid of the formation line. (b )by The AB far-UVB. Instrumental circular errors dichroism 3.1.3.(CD) Electron spectracorrespond Microscopy of ABB to the measured size of beforethe characte incubationrs and (black), the thickness after 10-min of the incubation line. (b) The (red), far-UV 20-min circular incubation (green),dichroism and 450-min (CD) spectra incubation of ABB (blue) measured at 45 ◦ Cbefore in the in 20cubation mM Tris-HCl (black), after buﬀer 10-min (pH7.4) incubation in the presence (red), of Electron200 mM20-min NaCl. microscopy incubation (green), was employed and 450-mi nto incubation characterize (blue) theat 45 size °C in and the 20to mM study Tris-HCl the morphology buffer (pH of the resulting 7.4)aggregates. in the presence Electron of 200 microimagesmM NaCl. of ABB were recorded after incubating it at 45 °C for 10, 30,3.1.3. and Electron 300 min Microscopy (Figure 3). 3.1.3. Electron Microscopy OneElectron can microscopysee in the images was employed that small-sized to characterize globular the aggregates size and toare study detected the morphologyat the initial ofstage the ofresulting amyloid aggregates.Electron formation microscopy Electron (Figure was microimages3a). employed Next, extended to of characterize ABB fi werebrils recordedthe are size formed and after to (Figure study incubating the 3b). morphology Their it at 45 size◦C of forincreases the 10, 30, overand 300time,resulting min and (Figure aggregates. a fibril3). network Electron is microimages eventually formedof ABB were (Figure recorded 3c). after incubating it at 45 °C for 10, 30, and 300 min (Figure 3). One can see in the images that small-sized globular aggregates are detected at the initial stage of amyloid formation (Figure 3a). Next, extended fibrils are formed (Figure 3b). Their size increases over time, and a fibril network is eventually formed (Figure 3c).

Figure 3.3. ElectronElectron microimages microimages of amyloidof amyloid structures structures formed formed by ABB by recordedABB recorded using negative using negative staining with uranyl acetate after incubation at 45 C for (a) 10 min, (b) 30 min, and (c) 300 min. staining with uranyl acetate after incubation◦ at 45 °C for (a) 10 min, (b) 30 min, and (c) 300 min. OneFigure can see 3. inElectron the images microimages that small-sized of amyloid structures globular formed aggregates by ABB are recorded detected using at the negative initial stage of 3.1.4. Atomic Force Microscopy amyloid formationstaining with (Figure uranyl 3acetatea). Next, after extendedincubation at fibrils 45 °C arefor ( formeda) 10 min, (Figure (b) 30 min,3b). and Their (c) 300 size min. increases over According to the AFM data, ABB forms conventional amyloid fibrils in addition to small-sized time,3.1.4. and aAtomic fibril networkForce Microscopy is eventually formed (Figure3c). globular aggregates. The kinetics of aggregate morphology changes has been studied by AFM. Figure3.1.4. Atomic 4 showsAccording Force the imagesto Microscopy the AFM of amyloid data, ABB structures forms conventional formed by amyloid ABB observed fibrils in addition after different to small-sized incubation periods.globular Again, aggregates. it is seen The that kinetics small aggregatesof aggregate are morphology formed at changes short incubationhas been studied periods by (5–30 AFM. min), AccordingFigure 4 shows to thethe AFMimages data, of amyloid ABB forms structures conventional formed by amyloidABB observed fibrils after in additiondifferent incubation to small-sized followed by formation of extended fibrils (at 1–2 h), which eventually grow larger to form a fibril globularperiods. aggregates. Again, it The is seen kinetics that small of aggregate aggregates morphology are formed changes at short hasincubation been studied periods by (5–30 AFM. min), Figure 4 showsfollowed the images by formation of amyloid of extended structures fibrils formed (at 1–2 by h), ABB which observed eventually after grow diff erentlarger incubationto form a fibril periods. Again, it is seen that small aggregates are formed at short incubation periods (5–30 min), followed by formation of extended fibrils (at 1–2 h), which eventually grow larger to form a fibril network (at 3–6 h). After incubation for 6 h, the fibril network becomes very dense. Both individual fibrils and bundles consisting of several thin fibrils can be detected in it. Biomolecules 2020, 10, 241 8 of 16 network (at 3–6 h). After incubation for 6 h, the fibril network becomes very dense. Both individual Biomolecules 2020, 10, 241 8 of 16 fibrils and bundles consisting of several thin fibrils can be detected in it.

Figure 4. Atomic force microscopy (AFM) images of the amyloid structures formed by ABB upon Figure 4. Atomic force microscopy (AFM) images of the amyloid structures formed by ABB upon 5 5 min-6 h (a–f) incubation in 20 mM Tris-HCl buﬀer (pH 7.4) in the presence of 200 mM NaCl at 45 ◦C. min-6 h (a–f) incubation in 20 mM Tris-HCl buffer (pH 7.4) in the presence of 200 mM NaCl at 45 3.1.5.°C. Confocal Fluorescence Microscopy Fluorescence microscopy has a lower resolution compared to AFM and electron microscopy. Although 3.1.5. Confocal Fluorescence Microscopy it does not see oligomers at all, it allows one to examine larger fields and larger structures. Furthermore, this examinationFluorescence technique microscopy does has not a involve lower sampleresolution sorption compared onto the to surface,AFM and which electron reduces microscopy. the risk of Althoughartifacts. The it does kinetics not of see amyloid oligomers formation at all, by ABBit allows observed one byto fluorescenceexamine larger microscopy fields isand shown larger in structures.Figure5. One Furthermore, can see that fibrilsthis examination start aggregating techni intoque clusters does not after involve incubation sample for 30sorption min. The onto size the of surface,these clusters which then reduces increases, the risk and largeof artifacts. bundles The are kinetics formed. Afterof amyl 6 hoid incubation, formation these by bundlesABB observed become by as fluorescencelong as several microscopy hundred micrometers. is shown in DetailedFigure 5. examination One can see of that the bundlesfibrils start reveals aggregating a longitudinal into clusters packing afterof fibrils. incubation This means for 30 that min. fibrils The are size oriented of these along clus theters majorthen increases, axis in this and bundle. large bundles are formed. After 6 h incubation, these bundles become as long as several hundred micrometers. Detailed examination of the bundles reveals a longitudinal packing of fibrils. This means that fibrils are oriented along the major axis in this bundle. Biomolecules 2020, 10, 241 9 of 16 Biomolecules 2020, 10, 241 9 of 16

Figure 5. FluorescenceFigure 5. Fluorescence microscopy microscopy images images of theof the amyloid amyloid structures formed formed by ABB by ABBupon 5 upon min-15 5 min-15 h (a–f) incubationh (a–f) inincubation 20 mM in Tris-HCl 20 mM Tris-HCl buﬀer buffer (pH (p 7.4)H 7.4) containing containing 200200 mM mM NaCl NaCl at 45°C. at 45 ◦C.

3.2. The Eff3.2.ect The of External Effect of External Factors Factors on Amyloid on Amyloid Formation Formation Such external factors as pH and the presence of agents breaking disulfide bonds were found to Such external factors as pH and the presence of agents breaking disulfide bonds were found to be be most significant. most significant.Variation in pH within the range from pH 7 to pH 5 (Figure 6a) had the most substantial Variationinfluence in pH on withinamyloidthe formation, range whose from pHefficiency 7 topH was 5the (Figure highest6 a)at pH had 5.5 the (this most corresponds substantial to the influence on amyloidtitration formation, of side groups whose of e Hisfficiency residues). was In an the ABB highest molecule, at pHthese 5.5 residues (this are corresponds located at positions to the titration 30 and 65 in identical structural elements (namely, in the internal β-strands of ABB molecule) and of side groups of His residues). In an ABB molecule, these residues are located at positions 30 are solvent-exposed (Figure 7). It was found by analyzing the amino acid sequence of albebetin that and 65 in identicalintroduction structural of mutations elements at these (namely,residues (substitution in the internal of Hisβ residue-strands for ofthe ABB uncharged molecule) Phe and are solvent-exposedresidue) (Figuremay give7 rise). Itto wasregions found characterized by analyzing by high expected the amino contact acid density sequence [29] in addition of albebetin to that introductionchanges of mutations in charge at thesedistribution, residues which (substitution would contribute of His residueto the formation for the uncharged of additional Phe residue) amyloidogenic domains. may give rise to regions characterized by high expected contact density [29] in addition to changes in charge distribution, which would contribute to the formation of additional amyloidogenic domains. Biomolecules 2020, 10, 241 10 of 16

Figure 6. (Figurea) The 6. e ﬀ(aect) The of effect pH onof pH the on rate the ofrate amyloid of amyloid formation formation ofof ABB.ABB. The The thickness thickness of the of thesolid solid lines correspondslines to corresponds the instrumental to the instrume errors;ntal the hatchingerrors; the showshatching the shows dispersion the dispersion during during repeated repeated repetition of the experiment.repetition (b of) Thethe experiment. CD spectra (b) ofThe albebetin CD spectra at of pH albebetin 5.5 in at the pH presence5.5 in the presence and in theand in absence the of the disulﬁde bondabsence reducing of the disulfide agent bo TCEP.nd reducing agent TCEP.

Figure 7. The spatial structure of ABB predicted using the online service robetta.org [30]. (a) Surface distribution of polar and charged atoms. (b) Location of residues H30 and H65. (c) Location of residues C8 and C43.

The disulfide bond in albebetin can be formed between Cys residues at positions 8 and 43, which are symmetrically located in identical structural elements of the ABB molecule (as it is in the case for His30 and His65 residues). This bond`s formation increases the content of the secondary ABB structure (see Figure 6b). We conducted experiments to study kinetics of amyloid formation by ABB in the presence of TCEP, a disulfide bonds reducing agent (Figure 6b). Furthermore, it was demonstrated that in the absence of the reducing agent TCEP, the protein containing the disulfide bond forms amyloid fibrils at a much lower rate (several weeks rather than days; data not shown), which is consistent with the findings obtained using non-reduced ABB protein [22,23]. The results of our study may indicate that the conformational mobility of the polypeptide chain is very important for efficient amyloid formation and the disulfide bond plays a crucial role as one of the factors stabilizing the non-amyloid form of the protein. The removal of one of three disulfide bonds in an insulin molecule is known to destabilize the native state and enhance amyloidogenesis [31], while the insertion of the fourth non-canonical S-S bond between the Cys-substituted residues of chains A and B prevents the formation of amyloid structures [32]. However, it should be mentioned that the role of disulfide bond in amyloid fibril formation is not always interpreted using the paradigm of native state stabilization. Thus, it has been demonstrated for the highly amyloidogenic two-chain H-fragment of insulin that the presence of a disulfide bond plays a decisive role for its amyloidogenicity: association of H-fragment monomers to fibrils decreased abruptly in the presence of an agent reducing disulfide bonds [33]. In a similar Biomolecules 2020, 10, 241 10 of 16

Figure 6. (a) The effect of pH on the rate of amyloid formation of ABB. The thickness of the solid lines corresponds to the instrumental errors; the hatching shows the dispersion during repeated

Biomoleculesrepetition2020 of, 10 the, 241 experiment. (b) The CD spectra of albebetin at pH 5.5 in the presence and in the10 of 16 absence of the disulfide bond reducing agent TCEP.

FigureFigure 7. 7. TheThe spatial spatial structure structure of of ABB ABB predicted predicted using using the the online online service service robetta.org robetta.org [30]. [30]. (a (a) )Surface Surface distributiondistribution of of polar andand charged charged atoms. atoms. (b )(b Location) Location of residuesof residues H30 andH30 H65.and (H65.c) Location (c) Location of residues of residuesC8 and C43.C8 and C43.

TheThe disulfide disulfide bond inin albebetinalbebetin can can be be formed formed between between Cys Cys residues residues at positions at positions 8 and 8 43,and which 43, whichare symmetrically are symmetrically located located in identical in identical structural structural elements elements of the ABBof the molecule ABB molecule (as it is (as in theit is casein the for caseHis30 for and His30 His65 and residues). His65 residues). This bond‘s This formation bond`s increasesformation the increases content ofthe the content secondary of the ABB secondary structure ABB(see structure Figure6b). (see We Figure conducted 6b). We experiments conducted experi to studyments kinetics to study of amyloid kinetics formationof amyloid by formation ABB in theby ABBpresence in the of presence TCEP, a disulfide of TCEP, bonds a disulfide reducing bonds agent reducing (Figure6b). agent Furthermore, (Figure 6b). it was Furthermore, demonstrated it was that demonstratedin the absence that of the in reducingthe absence agent of the TCEP, reducing the protein agent containing TCEP, the theprotein disulfide containing bond forms the disulfide amyloid bondfibrils forms at a much amyloid lower fibrils rate at (several a much weeks lower rather rate than(several days; weeks data rather not shown), than days; which data is consistent not shown), with whichthe findings is consistent obtained with using the non-reducedfindings obtained ABB proteinusing non-reduced [22,23]. The ABB results prot ofein our [22,23]. study mayThe indicateresults ofthat our the study conformational may indicate mobility that the of theconformational polypeptide chainmobility is veryof the important polypeptide for effi chaincient amyloidis very importantformation for and efficient the disulfide amyloid bond formation plays a crucial and the role disulfide as one ofbond the factorsplays a stabilizing crucial role the as non-amyloid one of the factorsform ofstabilizing the protein. the non-amyloid The removal form of one of of the three prot disulfideein. The removal bonds in of an one insulin of three molecule disulfide is bonds known into an destabilize insulin molecule the native is known state and to enhancedestabilize amyloidogenesis the native state [ 31and], whileenhance the amyloidogenesis insertion of the fourth [31], whilenon-canonical the insertion S-S bond of the between fourth thenon-canonical Cys-substituted S-S residuesbond between of chains the A Cys-substituted and B prevents the residues formation of chainsof amyloid A and structures B prevents [32 the]. formation of amyloid structures [32]. However,However, it it should should be mentioned thatthat thethe rolerole of of disulfide disulfide bond bond in in amyloid amyloid fibril fibril formation formation is notis notalways always interpreted interpreted using using the paradigm the paradigm of native of state native stabilization. state stabilization. Thus, it has Thus, been demonstratedit has been demonstratedfor the highly for amyloidogenic the highly amyloidogenic two-chain H-fragment two-chain of H-fragment insulin that of the insulin presence that of the a disulfidepresence bondof a disulfideplays a decisive bond plays role fora decisive its amyloidogenicity: role for its amyl associationoidogenicity: of H-fragment association monomers of H-fragment to fibrils monomers decreased toabruptly fibrils decreased in the presence abruptly of in an the agent presence reducing of an disulfide agent reducing bonds [33 disulfide]. In a similar bonds manner,[33]. In a disulfide similar bond rupture induced by sodium tetrathionate inhibited amyloidogenesis of hen egg white lysozyme (HEWL) at pH 2.0 [34]. Hence, the role played by disulfide bond in amyloidogenesis is rather protein-specific; it can both stimulate and inhibit the amyloidogenesis. In the case of amyloid formation by albebetin protein, disulfide bond has a stimulating effect.

3.3. Design of Mutant Variants of ABB and Study of Their Amyloidogenicity As mentioned above, the process of amyloid formation of ABB is sensitive to pH changes in the region of histidine residues titration. Such residues of ABB are located at positions 30 and 65. An analysis of the surface distribution of charged and polar residues in the predicted spatial ABB structure (Figure7a) shows, residues in these positions are surrounded by glutamic acids, which have a negative charge in examined pH region. Titration of histidine residues leads to the positive charge appearance and a significant change in the surface charge distribution, which in turn contributes to amyloid formation. Since there are two identical repeats in ABB sequence, we decided to check whether they are identical in the process of amyloid formation. For this, two mutant variants were designed that carry H30F and H65F substitutions. The second issue that we wanted to clarify with the help of mutational analysis is the effect of cysteine residues on the process of amyloid formation. As can be seen in Figure7c, cysteine residues Biomolecules 2020, 10, 241 11 of 16 that did not form a disulfide bond are located on opposite sides of the protein. This leads to the fact that either intermolecular disulfide bonds are formed, or the protein structure is strongly deformed. This leads to heterogeneity in the albebetin population with oxidized cysteines. At the same time, for this entire population, a significantly reduced ability to amyloid formation is observed. To eliminate the heterogeneity of the population, we made mutations replacing existing cysteines and obtained a double mutant C8S/C43S. The expected practical use of amyloid fibrils is associated with the creation of fibrils decorated with functionally active domains. The intermediate stage of isolation of ABB is a fusion protein with thioredoxin. In the course of present work it was noted that under certain conditions this construct also forms amyloid-like aggregates. It was decided to study this process in some more details. Great importance was the determination of the role of each of the parts of the fusion protein in amyloid formation. This did not require the creation of additional genetic constructs.

3.3.1. Studying the Kinetics of Amyloid Formation by the Mutant Variants ABB(H30F) and ABB(H65F) Are the same ABB repeats the same? When studying amyloid formation by the mutant variant ABB(H30F), we demonstrated that the rates of increase intensity of ThT fluorescence and aggregate accumulation are slower than those for the original ABB (Figure8a). This fact is consistent with the results of electrophoretic analysis of protein specimens under non-denaturing conditions, which demonstrate that the aggregated form of both the original type and the mutant ABB proteins emerges during the first hour of incubation; however, accumulation of the aggregated form of ABB(H30F) protein is a slower process (Figure8b,c). Identically to the case of ABB, the monomeric form is almost completely converted into the aggregated form after 32 h incubation. It is important to mention that the monomeric form of ABB(H30F) protein was retained even after the specimen had been incubated for more than 50 h. No significant differences were observed for the kinetics of amyloid formation by ABB(H65F) and the morphology of its amyloid structures (data not shown); they appeared to be largely similar to those of the original ABB (see Figure1, Figure2, and Figure4). Substitution H30F reliably slows down the rate of amyloid formation compared to original ABB and ABB(H65F). Hence, the experimental data infer that the H65F and H30F substitutions are not equivalent for fibrillogenesis despite the fact that theyBiomolecules are located 2020, 10 in, 241 identical positions - two identical α-β-β protein repeats. The amyloid structures12 of of16 all these proteins observed by AFM have similar morphologies and sizes (data not shown). Thus,Thus, fromfrom thethe pointpoint ofof viewview ofof amyloidamyloid formation,formation, thethe albebetinalbebetin repeatsrepeats areare notnot identical,identical, andand thisthis shouldshould bebe taken taken into into account account in in the the further further design design of of structures structures based based on on it. it. The The same same can can apply apply to manyto many other other proteins proteins containing containing amino amino acid acid repeats. repeats.

Figure 8. The kinetics of amyloid formation by the native ABB and its mutant variant ABB(H30F). Figure 8. The kinetics of amyloid formation by the native ABB and its mutant variant ABB(H30F). (a) Time dependences of the intensity of ThT ﬂuorescence. Analysis of the contents of monomeric and (a) Time dependences of the intensity of ThT fluorescence. Analysis of the contents of monomeric aggregated forms at diﬀerent stages of incubation using gel electrophoresis in 9% PAGE gel under and aggregated forms at different stages of incubation using gel electrophoresis in 9% PAGE gel non-denaturing conditions for original ABB (b) and mutant variant ABB(H30F) (c). under non-denaturing conditions for original ABB (b) and mutant variant ABB(H30F) (c).

Figure 9. (a) The CD spectra of the mutant variant ABB(C8S/C43S) and original ABB in the presence of the reducing agent tris(2-carboxyethyl)phosphin hydrochloride (TCEP). (b) The kinetics of Biomolecules 2020, 10, 241 12 of 16

Thus, from the point of view of amyloid formation, the albebetin repeats are not identical, and this should be taken into account in the further design of structures based on it. The same can apply to many other proteins containing amino acid repeats.

Figure 8. The kinetics of amyloid formation by the native ABB and its mutant variant ABB(H30F). (a) Time dependences of the intensity of ThT fluorescence. Analysis of the contents of monomeric and aggregated forms at different stages of incubation using gel electrophoresis in 9% PAGE gel under non-denaturing conditions for original ABB (b) and mutant variant ABB(H30F) (c).

3.3.2. Studying the Kinetics of Amyloid Formation by the Mutant Variant ABB(C8S/C43S) According to the CD spectroscopy data, the structure of the mutant protein ABB(C8S/C43S) was similar to that of the original ABB treated with an agent reducing disulfide bonds (Figure 9a). As expected, the introduction of a C8S/C43S double mutation can enhance the amyloidogenic properties of ABB protein. In addition, these mutations can save us from the need to use agents breaking the S-S bond and modifying cysteine residues. This in turn would allow us to eliminate the uncertainties associated with improper tying of intra- or intermolecular S-S bonds. BiomoleculesHowever,2020, 10 this, 241 fact caused some difficulties when studying the kinetics of amyloid formation.12 of 16 In the experiments with original ABB, the instant of initiation of amyloid aggregation coincided with the instant when the agent reducing disulfide bonds was added. For the mutant variant 3.3.2.ABB(C8S/C43S), Studying the the Kinetics amyloid of Amyloidstructures Formation started forming by the Mutantat the specimen Variant ABB(C8S preparation/C43S) stage (Figure 9b), whileAccording the increasing to the CD temperature spectroscopy and data, changing the structure pH only of accelerated the mutant this protein process. ABB(C8S According/C43S) to wasthe AFM similar data, to thatthe ofmorphology the original ofABB the resulting treated with fibril ans was agent close reducing to the disulﬁdeone observed bonds for (Figure the fibrils9a). Asformed expected, by original the introduction ABB (data of not a C8S shown)./C43S double mutation can enhance the amyloidogenic properties of ABBThus, protein. eliminating In addition, the ABB these ability mutations to form can disulf saveide us bonds, from thewe needdeprive to useourselves agents of breaking an amyloid the S-Sformation bond and control modifying tool. cysteineThe use residues.of such Thisa mutation in turn wouldis justified allow only us to when eliminate the theintended uncertainties use of associatedamyloid fibrils with precludes improper tyingthe use of of intra- a reducing or intermolecular agent. S-S bonds.

Figure 9. (a) The CD spectra of the mutant variant ABB(C8S/C43S) and original ABB in the presence of Figure 9. (a) The CD spectra of the mutant variant ABB(C8S/C43S) and original ABB in the presence the reducing agent tris(2-carboxyethyl)phosphin hydrochloride (TCEP). (b) The kinetics of amyloid of the reducing agent tris(2-carboxyethyl)phosphin hydrochloride (TCEP). (b) The kinetics of formation by the mutant variant ABB(C8S/C43S) and original ABB in the presence and absence of TCEP according to rising ﬂuorescence of ThT. The thickness of the solid lines corresponds to the instrumental errors; the hatching shows the dispersion during repeated repetition of the experiment.

However, this fact caused some difficulties when studying the kinetics of amyloid formation. In the experiments with original ABB, the instant of initiation of amyloid aggregation coincided with the instant when the agent reducing disulfide bonds was added. For the mutant variant ABB(C8S/C43S), the amyloid structures started forming at the specimen preparation stage (Figure9b), while the increasing temperature and changing pH only accelerated this process. According to the AFM data, the morphology of the resulting fibrils was close to the one observed for the fibrils formed by original ABB (data not shown). Thus, eliminating the ABB ability to form disulfide bonds, we deprive ourselves of an amyloid formation control tool. The use of such a mutation is justified only when the intended use of amyloid fibrils precludes the use of a reducing agent.

3.3.3. Studying the Kinetics of Amyloid Formation by the ABB-TRX Hybrid Protein As mentioned above, it was noted that under certain conditions, the TRX-ABB fusion protein also forms amyloid-like aggregates. This process has been investigated in more detail. It was demonstrated in this study that both ABB-TRX in the presence of TCEP and ABB(C8S/C43S)-TRX can form amyloid fibrils (Figure 10a). We determined that under the examined conditions, TRX with a linker, separately from ABB, does not form amyloid fibrils. This indicates that it is ABB that is the amyloidogenic part in the composition of the fusion protein. We see that the ABB has not lost its ability to form amyloid fibrils. This suggests that the N-end of ABB (by which it is connected to TRX) in the amyloid fibril is located on the surface and is accessible for the attachment of other proteins. In addition, the linker used is long enough to allow the free placement of TRX molecules around the fibril. The next question is what state TRX is in the fibril. To determine TRX state we measured tryptophan fluorescence during amyloid formation. Note that there are no tryptophan residues in ABB, which means that in this way we observe only TRX. As we see, in the process of amyloid formation, Biomolecules 2020, 10, 241 13 of 16

amyloid formation by the mutant variant ABB(C8S/C43S) and original ABB in the presence and absence of TCEP according to rising fluorescence of ThT. The thickness of the solid lines corresponds to the instrumental errors; the hatching shows the dispersion during repeated repetition of the experiment.

3.3.3. Studying the Kinetics of Amyloid Formation by the ABB-TRX Hybrid Protein As mentioned above, it was noted that under certain conditions, the TRX-ABB fusion protein also forms amyloid-like aggregates. This process has been investigated in more detail. It was demonstrated in this study that both ABB-TRX in the presence of TCEP and ABB(C8S/C43S)-TRX can form amyloid fibrils (Figure 10a). We determined that under the examined conditions, TRX with a linker, separately from ABB, does not form amyloid fibrils. This indicates that it is ABB that is the amyloidogenic part in the composition of the fusion protein. We see that the ABB has not lost its ability to form amyloid fibrils. This suggests that the N-end of ABB (by which it is connected to TRX) in the amyloid fibril is located on the surface and is accessible for the attachment of other proteins. In addition, the linker used is long enough to allow the free placement of TRX molecules around the fibril. The next question is what state TRX is in the fibril. To determine TRX state we measured

Biomoleculestryptophan2020 fluorescence, 10, 241 during amyloid formation. Note that there are no tryptophan residues13 of 16in ABB, which means that in this way we observe only TRX. As we see, in the process of amyloid formation, the position of the peak of tryptophan fluorescence (I320/I380 intensity ratio) practically thedoes position not change of the (Figure peak 10b). of tryptophan In addition, fluorescence we analyzed (I320 the/ I380stability intensity of the ratio)thioredoxin practically structure. does notFor changethis, denaturation (Figure 10 b).by urea In addition, was investigated we analyzed and co thempared stability free of TRX, the thioredoxin TRX as part structure. of a fusion For protein this, denaturationand TRX in mature by urea amyloid was investigated fibrils. Structural and compared changes free were TRX, recorded TRX as by part the ofchange a fusion in the protein position and TRXof the in peak mature of amyloidtryptophan fibrils. fluorescence. Structural The changes experiment were recorded showed by a thecoincidence change in of the denaturation position of thecurves peak (Figure of tryptophan 10c). This fluorescence. leads to two The conclusions. experiment First: showed thioredoxin a coincidence maintains of denaturation its stable curvesspatial (Figurestructure. 10 c).Second: This leadsthe interaction to two conclusions. of TRX with First: ABB thioredoxin and its amyloid maintains fibril its is stable negligible. spatial Otherwise, structure. Second:the stability the interaction of the TRX of TRX structure with ABB would and itsbe amyloidsignificantly fibril ischanged. negligible. This Otherwise, fact opens the stabilityup broad of theprospects TRX structure for further would usage be significantly of ABB and changed. its variants This factas a opens platform up broad for rational prospects design for further of amyloid usage offibrils ABB carrying and its variants functional as adomains. platform for rational design of amyloid fibrils carrying functional domains.

Figure 10. The kinetics of amyloid formation by the hybrid protein ABB-TRX observed by ThT Figure 10. The kinetics of amyloid formation by the hybrid protein ABB-TRX observed by ThT ﬂuorescence (a) and the ratio of the intensities of tryptophan ﬂuorescence at 320 and 380 nm (b). TRX fluorescence (a) and the ratio of the intensities of tryptophan fluorescence at 320 and 380 nm (b). structure unfolding by urea for free TRX, TRX-ABB fusion and TRX-ABB fusion in the amyloid state (c). TRX structure unfolding by urea for free TRX, TRX-ABB fusion and TRX-ABB fusion in the amyloid 4. Conclusionsstate (c).

4. ConclusionsThus, we characterized ABB as an amyloidogenic protein and as a promising object for the amyloid structures engineering. The main stages of its amyloid formation were shown. In addition, ways to controlThus, this we process characterized were shown. ABB as an amyloidogenic protein and as a promising object for the amyloidThe structures results of detailedengineering. study The focused main stages on the of kinetics its amyloid of amyloid formation formation were shown. by ABB In usingaddition, the spectralways to andcontrol microscopic this process methods were shown. infer that this process involves four main stages. (1)The Non-fibrillar results of detailed aggregates study formation focused andon the accumulation kinetics of takeamyloid place formation at the first by stage ABB of using the ABB the amyloidspectral and fibril microscopic formation as methods evidenced infer by that changes this inprocess the CD involves spectra four of the main specimen stages. and changes in the morphology detected by electron microscopy. No binding of ThT amyloid specific dye is detected here. (2) The second stage is characterized by formation of the short fibrils with cross-beta structure as evidenced by the data on increasing intensity of ThT fluorescence. (3) Further changes in the amyloid morphology indicate that there is a third stage. According to the EM and AFM data, growth and accumulation of fibrils forming a dense network take place at this stage. (4) According to the fluorescence microscopy data, the fourth stage of amyloid formation by ABB can be differentiated, which involves the formation of large fibril bundles observed at long incubation times. The formation of the suprafibrillar structure is attributed to the fact that the backbone of an amyloid fibril typically contains only a small portion of the protein (~8–10 AAR) [1], while the remaining portion is somehow involved in the interaction between fibrils or in the formation of large bundles by them. Undoubtedly, the protein engineering of amyloid structures and their use in practice requires knowledge of the properties of a current protein and methods for controlling the process of its amyloid formation. Such knowledge for ABB was the goal of our work. ABB, is one of many proteins for which the possibility of amyloid formation is shown, and at the same time one of the few that can be put into practice. For ABB, in our opinion, the most obvious application is to use it as a structural basis for functional amyloid fibrils. We have shown the possibility of this using TRX as an example. However, this requires verification and confirmation with other proteins or functional domains. A remarkable property of the ABB-based fusion protein is the possibility of its preparation and purification in monomeric water-soluble form and the subsequent controlled start of amyloid formation. To control Biomolecules 2020, 10, 241 14 of 16 the launch, as we have shown in this work, one can use a change in pH or disulfide bonds breaking. The rate of fibril formation can be accurately controlled by temperature and ionic strength. In conclusion, we want to note the joyful fact that the albebetin, one of the first artificial proteins in the world, engineered under the leadership of O.B. Ptitsin, continues to evolve and find its new uses.

Author Contributions: The manuscript was written in collaboration of all authors, R.C. and V.B.(Vitalii Balobanov) are authors with equal contribution. R.C., V.B. (Valentina Bychkova), and D.D. planned and designed experiments; V.B. (Valentina Bychkova) and R.C. performed the analysis of protein structure; R.C. and V.B. (Vitalii Balobanov) participated equally by performing the mutant genes construction and protein purification; V.B. (Vitalii Balobanov) and A.E. performed the preparation of amyloid fibrils, measurement of kinetics of amyloid formation and of CD spectra; V.B. (Vitalii Balobanov), A.E., and R.C. performed the data analysis; V.B. (Valentina Bychkova), D.D., and M.K. contributed reagents/materials/analysis tools; all authors participated in discussion of the results; R.C., V.B. (Vitalii Balobanov), D.D. wrote the manuscript; V.B. (Valentina Bychkova), D.D., and M.K. supervised the project. All authors have read and agreed to the published version of the manuscript. Funding: The initial part of this study was supported by the Russian Science Foundation (grant № 14-24-00157), and its final part by the Program for Molecular and Cellular Biology of the Russian Academy of Sciences. Acknowledgments: The authors are grateful to Machulin A.V. and Eliseeva I.A. for help by AFM and fluorescence microscopy measurement. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Lutter, L.; Serpell, C.J.; Tuite, M.F.; Xue, W.F. The molecular lifecycle of amyloid—Mechanism of assembly, mesoscopic organisation, polymorphism, suprastructures, and biological consequences. BBA Proteins Proteom. 2019, 11, 140257. [CrossRef][PubMed] 2. Eanes, E.D.; Glenner, J. X-ray diffraction studies on amyloid filaments. Histochem. Cytochem. 1968, 16, 673–677. [CrossRef][PubMed] 3. Nizhnikov, A.A.; Antonets, K.S.; Inge-Vechtomov, S.G. Amyloids: From Pathogenesis to Function. Biochemistry (Moscow) 2015, 80–89, 1127–1144. [CrossRef] 4. Maji, S.K.; Perrin, M.H.; Sawaya, M.R.; Jessberger, S.; Vadodaria, K.; Rissman, R.A.; Singru, P.S.; Nilsson, K.P.; Simon, R.; Schubert, D.; et al. Functional amyloids as natural storage of peptide hormones in pituitary secretory granules. Science 2009, 325, 328–332. [CrossRef] 5. Guyonnet, B.; Egge, N.; Cornwall, G.A. Functional amyloids in the mouse sperm acrosome. Mol. Cell. Biol. 2014, 34, 2624–2634. [CrossRef][PubMed] 6. Li, J.; McQuade, T.; Siemer, A.B.; Napetschnig, J.; Moriwaki, K.; Hsiao, Y.S.; Damko, E.; Moquin, D.; Walz, T.; McDermott, A.; et al. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell 2012, 150, 339–350. [CrossRef] 7. Romero, D.; Aguilar, C.; Losick, R.; Kolter, R. Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. Proc. Natl. Acad. Sci. USA 2010, 107, 2230–2234. [CrossRef] 8. Dueholm, M.S.; Petersen, S.V.; Sonderkaer, M.; Larsen, P.; Christiansen, G.; Hein, K.L.; Enghild, J.J.; Nielsen, J.L.; Nielsen, K.L.; Nielsen, P.H.; et al. Functional amyloid in Pseudomonas. Mol. Microbiol. 2010, 77, 1009–1020. [CrossRef] 9. Alteri, C.J.; Xicohtencatl Cortes, J.; Hess, S.; Caballero Olin, G.; Giron, J.A.; Friedman, R.L. Mycobacterium tuberculosis produces pili during human infection. Proc. Natl. Acad. Sci. USA 2007, 104, 5145–5150. [CrossRef] 10. Fowler, D.M.; Koulov, A.V.; Alory Jost, C.; Marks, M.S.; Balch, W.E.; Kelly, J.W. Functional amyloid formation within mammalian tissue. PLoS Biol. 2006, 4, e6. [CrossRef] 11. Dolgikh, D.A.; Kolomiets, A.P.; Bolotina, I.A.; Ptitsyn, O.B. Molten-globule state accumulates in carbonic-anhydrase folding. FEBS Lett. 1984, 165, 88–92. [CrossRef] 12. Krobath, H.; Estacio, S.G.; Faisca, P.F.N.; Shakhnovich, E.I. Identification of conserved aggregation-prone intermediate state in the folding pathways of Sps-SH3 amyloidogenic variants. J. Mol. Biol. 2012, 422, 705–722. [CrossRef][PubMed] Biomolecules 2020, 10, 241 15 of 16

13. Fedorov, A.N.; Dolgikh, D.A.; Chemeris, V.V.; Chernov, B.K.; Finkelshtein, A.V.; Shulga, A.A.; Alakhov, Y.B.; Kirpichnikov, M.P.; Ptitsyn, O.B. De novo design, synthesis and study of albebetin, a polypeptide with a predetermined 3-dimensional structure—Probing the structure at the nanogram level. J. Mol. Biol. 1992, 225, 927–931. [CrossRef] 14. Dolgikh, D.A.; Gabrielyan, A.E.; Navolotskaya, Y.V.; Chemeris, V.V.; Kirpichnikov, M.P. Artificial protein with a set spatial structure and biological activity. Biophysics 1993, 38, 59–65. 15. Aphasizheva, I.Y.; Dolgikh, D.A.; Abdullaev, Z.K.; Uversky, V.N.; Kirpichnikov, M.P.; Ptitsyn, O.B. Can grafting of an octapeptide improve the structure of de novo protein? FEBS 1998, 425, 101–104. [CrossRef] 16. Dolgikh, D.A.; Uversky, V.N.; Gabrielyan, A.E.; Chemeris, V.V.; Fedorov, A.N.; Navolotskaya, Y.V.; Zav’yalov, V.P.; Kirpichnikov, M.P. The de novo protein with grafted biological function: Transferring of interferon blast-transforming activity to albebetin. Protein Eng. 1996, 9, 195–201. [CrossRef] 17. Aphasizheva, I.Y.; Dolgikh, D.A.; Kirpichnikov, M.P. Study of immunogenic properties of albeferon, biologically active de novo protein. Mol. Biol. 1999, 33–34, 602–606. 18. Chertkova, R.V.; Abdullaev, Z.K.; Dolgikh, D.A.; Zav’yalov, V.P.; Kirpichnikov, M.P. Artificial proteins with antiviral properties of alpha-interferons. Russ. J. Bioorg. Chem. 2003, 29, 231–241. [CrossRef] 19. Chertkova, R.V.; Kostanyan, I.A.; Astapova, M.V.; Surina, E.A.; Dolgikh, D.A.; Kirpichnikov, M.P. An artificial protein with the biological activity of the differentiation factor for the HL-60 cell line of human promyelocyte leukemia. Russ. J. Bioorg. Chem. 2003, 29, 30–37. 20. Bocharova, O.V.; Moshkovskii, S.A.; Chertkova, R.V.; Kolesanova, E.F.; Dolgikh, D.A.; Kirpichnikov, M.P. The immune response to artificial proteins containing biologically active fragments of human IFN-alpha 2 and insulin. Mol. Biol. 2002, 36, 65–70. [CrossRef] 21. Bocharova, O.V.; Moshkovskii, S.A.; Chertkova, R.V.; Abdullaev, Z.K.; Kolesanova, E.F.; Dolgikh, D.A.; Kirpichnikov, M.P. Attachment of biologically active fragments of interferon-alpha 2 and insulin to the artificial protein, albebetin, affects immunogenicity of the resulting construct. Vopr. Meditsinskoi Khimii 2002, 48, 94–102. 22. Morozova-Roche, L.A.; Zamotin, V.; Malisauskas, M.; Ohman, A.; Chertkova, R.; Lavrikova, M.A.; Kostanyan, I.A.; Dolgikh, D.A.; Kirpichnikov, M.P. Fibrillation of carrier protein albebetin and its biologically active constructs. multiple oligomeric intermediates and pathways. Biochemistry 2004, 43, 9609–9619. [CrossRef][PubMed] 23. Lavrikova, M.A.; Zamotin, V.V.; Malisauskas, M.; Chertkova, R.V.; Kostanyan, I.A.; Dolgikh, D.A.; Kirpichnikov, M.P.; Morozova-Roche, L.A. Amyloidogenic properties of the artificial protein albebetin and its biologically active derivatives. The role of electrostatic interactions in fibril formation. Biochemistry (Moscow) 2006, 3, 386–395. [CrossRef] 24. Zamotin, V.; Gharibyana, A.; Gibanova, N.V.; Lavrikova, M.A.; Dolgikh, D.A.; Kirpichnikov, M.P.; Kostanyan, I.A.; Morozova-Roche, L.A. Cytotoxicity of albebetin oligomers depends on cross-beta-sheet formation. FEBS Lett. 2006, 580, 2451–2457. [CrossRef] 25. Schagger, H.; Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 1987, 166, 368–379. [CrossRef] 26. Provencher, S.W.; Glocker, J. Estimation of globular protein secondary structure from circular dichroism. Biochemistry 1981, 20, 33–37. [CrossRef] 27. Brender, J.R.; Krishnamoorthy, J.; Sciacca, M.F.; Vivekanandan, S.; D’Urso, L.; Chen, J.; La Rosa, C.; Ramamoorthy, A. Probing the sources of the apparent irreproducibility of amyloid formation: Drastic changes in kinetics and a switch in mechanism due to micellelike oligomer formation at critical concentrations of IAPP. J. Phys. Chem. B 2015, 119, 2886–2896. [CrossRef] 28. Dovidchenko, N.V.; Finkelstein, A.V.; Galzitskaya, O.V. How to determine the size of folding nuclei of protofibrils from the concentration dependence of the rate and lag-time of aggregation. I. Modeling the amyloid photofibril formation. J. Phys. Chem. B 2014, 118, 1189–1197. [CrossRef] 29. Galzitskaya, O.V.; Garbuzynskiy, S.O.; Lobanov, M.Y. Is it possible to predict amyloidogenic regions from sequence alone? J. Bioinform. Comput. Biol. 2006, 4, 373–388. [CrossRef] 30. Raman, S.; Vernon, R.; Thompson, J.; Tyka, M.; Sadreyev, R.; Pei, J.; Kim, D.; Kellogg, E.; DiMaio, F.; Lange, O.; et al. Structure prediction for CASP8 with all-atom refinement using Rosetta. Proteins 2009, 77 (Suppl. 9), 89–99. [CrossRef] Biomolecules 2020, 10, 241 16 of 16

31. Li, Y.; Gong, H.; Sun, Y.; Yan, J.; Cheng, B.; Zhang, X.; Huang, J.; Yu, M.; Guo, Y.; Zheng, L.; et al. Dissecting the role of disulfide bonds on the amyloid formation of insulin. Biochem. Biophys. Res. Commun. 2012, 423, 373–378. [CrossRef][PubMed] 32. Vinther, T.N.; Norrman, M.; Ribe, U.; Huus, K.; Schlein, M.; Steensgaard, D.B.; Pedersen, T.A.; Pettersson, I.; Ludvigsen, S.; Kjeldsen, T.; et al. Insulin analog with additional disulfide bond has increased stability and preserved activity. Protein Sci. 2013, 22, 296–305. [CrossRef][PubMed] 33. Piejko, M.; Dec, R.; Babenko, V.; Hoang, A.; Szewczyk, M.; Mak, P.; Dzwolak, W. Highly amyloidogenic two-chain peptide fragments are released upon partial digestion of insulin with pepsin. J. Biol. Chem. 2015, 290, 5947–5958. [CrossRef] 34. Sarkar, N.; Kumar, M.; Dubey, V.K. Effect of sodium tetrathionate on amyloid fibril: Insight into the role of disulfide bond in amyloid progression. Biochimie 2011, 93–95, 962–968. [CrossRef][PubMed]

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