AFM study of Escherichia coli RNA polymerase σ70 subunit aggregation

BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 54 – 62

Research Article

nanomedjournal.com

AFM study of Escherichia coli RNA polymerase σ70 subunit aggregation Evgeniy V. Dubrovin, PhDa,⁎, Olga N. Koroleva, PhDb , Yulia A. Khodak, MScc , Natalia V. Kuzminaa , Igor V. Yaminsky, DSca , Valeriy L. Drutsa, PhDc a

Department of Physics of Polymers and Crystals, Faculty of Physics, Moscow State University, Moscow, Russia b Faculty of Chemistry, Moscow State University, Moscow, Russia c Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Received 14 January 2011; accepted 21 May 2011

Abstract The self-assembly of Escherichia coli RNA polymerase σ70 subunit was investigated using several experimental approaches. A novel rodlike shape was reported for σ70 subunit aggregates. Atomic force microscopy reveals that these aggregates, or σ70 polymers, have a straight rodlike shape 5.4 nm in diameter and up to 300 nm in length. Atomic force microscopy data, Congo red binding assay, and sodium dodecyl sulfate gel electrophoresis confirm the amyloid nature of observed aggregates. The process of formation of rodlike structures proceeds spontaneously under nearly physiological conditions. E. coli RNA polymerase σ70 subunit may be an interesting object for investigation of amyloidosis as well as for biotechnological applications that exploit self-assembled bionanostructures. Polymerization of σ70 subunit may be a competitive process with its three-dimensional crystallization and association with core RNA polymerase. From the Clinical Editor: In this basic science study, the self-assembly of Escherichia coli RNA polymerase σ70 subunit was investigated using atomic force microscopy and other complementary approaches. © 2012 Elsevier Inc. All rights reserved. Key words: E. coli RNA polymerase σ70 subunit; Self-assembly; Atomic force microscopy; Amyloid fibrils; Congo red

For specific initiation of transcription by bacterial RNA polymerase the temporary association of core enzyme with sigma factor is required. The primary sigma factor in E. coli is σ70, which provides the transcription of housekeeping genes.1,2 Over the past few years a great deal of information on the structure and function of σ70 has been accumulated.3,4 Several functional domains responsible for binding with core enzyme, promoter DNA, and regulatory factors, have been revealed in its structure.3,5,6 However, there are no comprehensive data on physicochemical properties of the protein. In particular, there are few reports that σ70 subunit can form aggregates, especially at high (above 5 μM) protein concentration7,8 and upon storage of the protein solution for prolonged periods.9 In the earliest studies of Lowe et al7 it was found that heating of σ70 subunit above 49°C facilitates the formation of aggregates, which according to electron microscopy (EM) data are “sigma polymers” with diameter of 8 nm and length This work is supported by the federal target program “Scientific and educational research personnel to innovative Russia” and “Grants of the President of Russian Federation” (МК-5121.2010.2). ⁎Corresponding author: Department of Physics of Polymers and Crystals, Moscow State University, Leninskie Gory, 1/2, 119991, Moscow, Russia. E-mail address: [email protected] (E.V. Dubrovin).

of 100 nm. Since that time the morphology of these aggregates has not been practically studied. Recently, thermal-induced aggregation was also observed for another related primary sigma subunit, σA of Staphylococcus aureus.10 In recent years, atomic force microscopy (AFM) has been widely used to study the morphology of protein aggregates.11-17 Using this approach it was demonstrated that many globular proteins under certain conditions are able to form amyloid-like fibrils in vitro.18,19 The interest in studying these structures stems from several observations. First of all, in vivo aggregation (fibril formation) of most of them is associated with development of a number of prevalent human diseases, such as Alzheimer's, Parkinson's, and diabetes.20,21 Second, their high stability and ability to self-assemble makes amyloid fibrils suitable for nanotechnological applications.14,22 Although much attention has been directed to the investigation of amyloidogenesis in various eukaryotic and model systems, there are little data on the aggregation properties of bacterial proteins.23,24 Several recent articles report the presence of amyloid-type structures, formed by overexpressed proteins inside bacterial inclusion bodies.25,26 However, there is no evidence as to whether intracellular proteins in bacteria are capable of forming amyloid-like aggregates.

1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.05.014 Please cite this article as: E.V. Dubrovin, O.N. Koroleva, Y.A. Khodak, N.V. Kuzmina, I.V. Yaminsky, V.L. Drutsa, AFM study of Escherichia coli RNA polymerase σ70 subunit aggregation. Nanomedicine: NBM 2012;8:54-62, doi:10.1016/j.nano.2011.05.014

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Despite a few reports where the formation of some aggregates by RNA polymerase σ70 subunit was mentioned,7,9 the aggregation of this protein has not been systematically studied. Moreover, to the best of our knowledge, there are no AFM data on the morphology of RNA polymerase σ70 subunit and its aggregates. Because this protein plays a key role in transcription initiation, it is difficult to overestimate the importance of this study. Self-association of σ70 subunit may dramatically influence the regulation of transcription processes in the cell (e.g., by inactivation of σ subunit), as was demonstrated for temperature-sensitive mutant σ subunit upon its extensive aggregation.7 The aim of our work was to investigate the aggregation properties of Escherichia coli RNA polymerase σ70 subunit using AFM and to characterize its aggregates.

Methods Tris and EDTA were purchased from Merck (Darmstadt, Germany), Congo red from Sigma-Aldrich (St. Louis, Missouri). All other reagents were of analytical grade. Escherichia coli ER1821 was used as host strain for protein expression and purification. Construction of the plasmid pC4-d for overexpression of σ70 subunit fusion protein, containing intein and chitin binding tag, and isolation of pure σ70 subunit with single extra Gly residue using IMPACT system were described elsewhere.27 Sample preparation for AFM Before AFM imaging, aliquots of σ70 subunit stock solutions (concentration 0.5–1 μg/μL) in 50–100 mM NaCl buffer were diluted 3–100 times with deionized water or NaCl- and MgSO4containing buffer, so that final concentration of σ70 in solution constituted 2.5–160 μg/mL and ionic strength was in the range of 1–20 mM NaCl and 0–5 mM MgSO4. pH value of deposited solutions varied in the range 7.4–7.8. A freshly cleaved mica slice was placed on top of a 10-μL droplet of σ70-containing solution and left for adsorption for 10 minutes. After that, the surface was dried in air flow and rinsed by placing of the mica slice on the top of a 100-μL droplet of deionized water for 40 minutes. In case of highly oriented pyrolytic graphite (HOPG) as a substrate, a 25-μL droplet of σ70 subunit-containing solution was deposited on the surface of freshly cleaved HOPG and left for adsorption for 10 minutes, then dried in air flow and rinsed in a 100-μL droplet of deionized water for 40 minutes. After rinsing, all samples were dried in air flow. AFM imaging and image processing All experiments were carried out using Nanoscope IIIa multimode atomic force microscope (Veeco Instruments, Santa Barbara, California) in tapping mode in air. Commercial silicon NSC11 (spring constant 48 N/m, resonance frequency 330 kHz) cantilevers (MikroMasch, Tallinn, Estonia) were used. The scan rate was typically 2 Hz, and the number of lines was 512. To minimize a mechanical impact of the

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cantilever on the sample we have used large setpoint values (90–95%). By the example of investigation of tobacco mosaic virus particles adsorbed on a mica surface, we have recently demonstrated the possibility to obtain correct height values in tapping-mode AFM even on soft objects.28 Image processing was performed using the FemtoScan software.29 Values for the diameter of rodlike aggregates were estimated from their height at different points (N N 100) in different AFM images. This value did not depend on the cantilever used. Congo red binding assay The Congo red binding assay was performed as described by Klunk et al.30 A stock solution of Congo red was prepared by dissolving the dye in a buffer containing 0.15 M NaCl and 5 mM potassium phosphate, pH 7.4, to a final concentration of 100 μM. Congo red solution was passed through a 0.2-μm filter immediately before use and then was mixed with protein solutions to a final concentration of 10 μM Congo red and 0.1– 1 μM σ70 subunit. Solutions of 10 μM Congo red lacking protein were also prepared. The samples were vortexed for 15 s and incubated at 20°C for 15 minutes. The absorption spectrum of each sample was measured after 5 minutes' equilibration and recorded from 400 nm to 650 nm on a Varian Cary 50 UV-Vis spectrophotometer (Varian Inc., Palo Alto, California) using the cuvette with 1-cm pathlength. The spectrum of Congo red alone was compared with that of Congo red solutions containing protein. A red-shift absorption band toward 540 nm was taken to be indicative of the formation of amyloid structures. Electrophoresis in SDS and native polyacrylamide gels Electrophoretic analysis of protein preparations in 10% sodium dodecyl sulfate (SDS)-Tris-glycine polyacrylamide gel was performed by the Laemmli method.31 Samples of the proteins (2–5 μg), either freshly prepared or incubated for different periods under variable conditions, were mixed with SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, loaded onto the SDS gels, and electrophoresed for 1.5 hours. Electrophoresis of the protein samples in native 5% polyacrylamide gel was performed in Tris-glycine buffer (pH 8.6) at electric field intensity of 20 V/cm for 3–4 hours, as described by Ilag et al.32 After electrophoresis, the gels were stained with PageBlue protein staining solution (Fermentas, Vilnus, Lithuania).

Results E. coli RNA polymerase σ70 subunit was isolated by affinity chromatography using IMPACT system as described.30 This approach allows us to obtain highly pure preparations of σ70 protein, containing a single extra C-terminal Gly residue. The functional activity of preparations was checked in a variety of in vitro transcription systems.33,34

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Figure 1. (A) Tapping-mode height AFM image of E. coli RNA polymerase σ70 subunit deposited from 160 μg/mL solution in the buffer (10 mM NaCl) and (B) its height distribution histogram. Image size is 3.15 μm.

We have studied the aggregation of E. coli RNA polymerase σ70 subunit using AFM, Congo red binding assay, and PAGE. Atomic force microscopy A typical AFM image of E. coli RNA polymerase σ70 subunit, deposited on the mica surface, is presented in Figure 1, A. Under the conditions indicated, σ70 is an asymmetrical particle with a height on the AFM images varying in the range of 1–4 nm. This can be concluded from the analysis of height distribution of the molecules, presented in Figure 1, B: the broadened peak (full width height maximum constitutes 2.3 nm) demonstrates that different molecules adsorb to mica via their different sites, therefore providing different height values.35 According to a 20-Å EM reconstruction of intact activatordependent transcription initiation complex, σ70 subunit within RNA polymerase holoenzyme has an asymmetrical shape with a length-to-width ratio of about 4.36 However, the three-dimensional structure of the full-length σ70 in a free state is still unknown, and our data demonstrate that it might be rather similar to its state in complex with core enzyme. We have found that rodlike aggregates accompany monomeric molecules of σ70 subunit in most AFM images (Figures 13). These straight aggregates are 5.4 ± 0.2 nm in diameter and vary considerably in their length. The mass content of rodlike aggregates in the total pool of σ70 particles can be estimated from AFM images assuming that σ70 polymers have the same density as free-σ70 monomers. This estimation serves as an indicator, allowing us to understand changes in the content of σ70 polymers under different conditions. For this purpose, the total volume of all adsorbed σ70 subunit as well as the volume of rodlike polymers should be calculated. The content of polymers adsorbed on mica from σ70 protein solution, containing b2 mM NaCl, is within the range of 0–1% depending on the particular specimen. Such small amounts of

protein could not usually be reliably detected using electrophoresis techniques. The ability to image single binding events of molecules and their assemblies via AFM allows one to detect even extremely small concentrations of polymers present in the suspension. Deposition of σ70 on mica surface from 20 mM NaCl-5 mM MgSO4 buffer solution results in considerable (5- to 10-fold) increase in the surface density of adsorbed protein molecules (Figure 2, A). For example, estimation of the total volume of adsorbed σ70 subunit per 1 μm2 gives 6100 and 56,500 nm3 for protein deposited from 1 mM NaCl and from 20 mM NaCl-5 mM MgSO4 buffer solutions, respectively. The values presented should be considered as upper estimates of volumes due to the well-known broadening effect of the AFM tip. The most likely scenario of σ70 adsorption implies that this protein is negatively charged, and the increase of electrolyte concentration leads to the decrease of the electrical double-layer repulsion between mica and protein surfaces and therefore to the increase of protein physisorption. This effect was described for negatively charged purple membrane adsorption on mica and explained using Derjaguin-Landau-Verwey-Overbeek theory.37 The negative charge of the N-terminal region 1.1 of primary σ proteins was reported by Gruber and Gross.2 Rodlike σ70 aggregates are clearly visible in Figure 2, A. For clarity, they are enlarged in the insets beneath the AFM image. The content of adsorbed σ70 polymers, calculated from AFM images, increases up to 8% at electrolyte concentration 20 mM NaCl-5 mM MgSO4. Because the storage of σ70 subunit was reported to be one of the factors responsible for aggregates formation,9 we have deposited σ70 solution stored for 3 months (aged) onto mica using the same procedure as was utilized for fresh protein. AFM images of the aged preparation reveal a lot of large aggregates of various sizes and irregular shapes along with rodlike polymer structures (Figure 2, B; insets demonstrate the enlarged σ70 polymers). These aggregates have not been observed in the case

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Figure 2. Tapping-mode height AFM image of E. coli RNA polymerase σ70 subunit deposited from (A) 160 μg/mL and (B) 55 μg/mL solution in the buffer (20 mM NaCl-5 mM MgSO4). (A) Fresh sample. (B) Sample stored for 3 months at −20°C in 50% glycerol. Insets demonstrate zoomed sigma polymers images from corresponding AFM data. Image size is 3.15 μm. Inset sizes are (A) 250 × 215 nm, (B) 390 × 265 nm.

of fresh σ70 subunit (compare Figure 2, A and B). Numerical estimate demonstrates that there is no appreciable change in the content of σ70 polymers in the aged samples, although it may be higher due to possible presence of the “rods” masked by large aggregates that could not be directly observed by AFM and therefore taken into account. We have studied samples obtained by the deposition of σ70 protein in varied conditions, such as initial concentration of the protein and ionic strength of a buffer. Figure 3 illustrates the formation of polymers in the solutions of 2.5 and 5 μg/mL σ70 with different ionic strengths. Their content did not change significantly upon the dilution of σ70 solution within the concentration range 2.5–5 μg/mL and was estimated as 7.2% and 7.9% for 5 and 2.5 μg/mL, respectively. In this series of experiments the increase of ionic strength of the deposition buffer has also improved the protein adsorption, as it was observed for samples with more concentrated protein solution, for which AFM images are presented in Figure 2. σ70 polymers were also revealed at significantly higher concentrations of NaCl and MgCl2 (up to 100 and 50 mM correspondingly; see Figure 3, E and F), indicating the ability of σ70 to polymerize at a wide range of electrolyte concentrations. The length distributions of σ70 polymers show the main maximum at approximately 125 nm (Figure 4). However, the dispersion is rather large, and maximal length values grow with the increase of ionic strength of the deposition buffer (Figure 4, B). We can also conclude that the tail up to 13 nm

in height distribution of particles in Figure 1, B may correspond to early aggregates of σ polymers, adsorbed on mica by one of their termini. To exclude the possibility that the observed polymers could be artifacts upon deposition on mica due to its particular surface properties (e.g., negative surface charge in aqueous environment), we have also deposited a solution of σ70 subunit on the surface of HOPG, which is neutral and hydrophobic. In this experiment, similar straight rodlike aggregates of about 5 nm in diameter and of 50−200 nm length were observed on AFM images (see Supplementary Materials, Figure S1, available online at http://www.nanomedjournal.com). The content of σ70 polymers adsorbed on HOPG constituted about 5% (sigma protein was deposited from 20 mM NaCl-5 mM MgSO4 buffer). To clarify whether the formation of observed linear aggregates is the result of epitaxial polymerization or binding of already assembled aggregates from solution, another technique that is not surface-based should be utilized. For this reason, we have analyzed σ70 subunit solution using Congo red binding and SDS-PAGE. Analysis of aggregates using Congo red binding The dimensions of σ70 polymers observed by AFM are quite typical for amyloid-like protofibrils, described in the literature for dozens of other amyloidogenic proteins.12,38 To get more evidence of amyloid-like character of the aggregates formed by

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Figure 3. Tapping-mode height AFM images of E. coli RNA polymerase σ70 subunit deposited on mica from 5 μg/mL (A, B), 2.5 μg/mL (C, D), and 20 μg/mL (E, F) solution in the buffer, containing 2 mM NaCl (A, C); 20 mM NaCl-5 mM MgSO4 (B, D); 20 mM NaCl-50 mM MgSO4 (E); 100 mM NaCl-5 mM MgSO4 (F). Image sizes are 2.5 μm (A-D) and 1.7 μm (E, F).

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Figure 4. Length distributions of σ polymers deposited from (A) 2 mM NaCl and (B) 20 mM NaCl-5 mM MgSO4 solutions.

σ70, we have used the Congo red binding assay. Specific interaction of this dye with β-sheet structures, present in fibrils, causes a red shift of the optical absorption spectrum of Congo red from 490 nm to 530-540 nm.30,39 For these experiments we have used samples of freshly isolated σ70 subunit and preparations, stored for prolonged periods, in particular for 3 months, at –20°C in the presence of glycerol. The absorption peak of Congo red undergoes a red shift when aliquots of protein solutions are added to the dye (Figure 5, A). Subtraction of spectrum of Congo red alone from experimentally measured spectrum of protein solution in the presence of Congo red yields the difference spectrum with a maximum at 530–540 nm (Figure 5, B). The difference spectra correspond to the aggregate-bound Congo red. Such a result is expected only if ordered amyloid-like aggregates are present in the solution.30 Fibril content was higher in case of the long-term stored (aged) samples (Figure 5, A, B). Addition of small quantities (about 2–5% wt/wt) of aged σ70 to new preparations (seeding) also facilitates fibril formation (see, for example, Figure 5, C). The difference spectrum absorption of solution containing σ70, incubated for 5 days at 10°C with seeds, is noticeably increased compared with that without seeding. Such an effect, indicating that the rate-limiting step of the overall process is apparently a nucleation stage, has been described for many other amyloidogenic proteins.40,41 Therefore, σ70 subunit is prone to generate amyloid-like structures, and this process depends on duration of protein incubation and availability of primer particles. Analysis of aggregates by PAGE There are several reports demonstrating that oligomers of amyloidogenic proteins are stable enough to be analyzed by SDS-PAGE.42,43 Thus, the samples of σ70 preparations (1–10 μM), both freshly prepared and aged by incubation for various periods (1–10 days at 10–20°C or 2–10 months at –20°C in 50% glycerol solution), were subjected to electrophoresis in 10% denaturing SDS-polyacrylamide gel (pH 8.3). Figure 6, A shows, that fresh preparations exhibit mainly a single band of σ70 monomer in the SDS electrophoregrams, whereas in the case of aged preparations there are smeared bands,

located at the stacking-separating gel interface or at the top of the stacking gel, with much higher than expected molecular mass, which can be interpreted as aggregates. Bands with apparent molecular masses (140, 210, 280 kDa, etc.) corresponding to soluble oligomers, were also observed at early stage of aging. The preparations of σ70, kept for longer periods (especially for several months) at –20°C, contain more aggregates, than freshly isolated preparations, although the sensitivity of the electrophoretic method does not allow detecting small quantities of initial aggregates in the newly obtained preparations. The main trend is that the aggregates slowly accumulate over time. This was also demonstrated by nondenaturing gel electrophoresis (Figure 6, B). Being negatively charged at neutral pH, σ70 subunit is able to migrate to the anode under conditions of native gel electrophoresis, performed in Tris-glycine buffer, pH 8.6.32 One can observe that the proportion of high-molecular-mass species on the top of the native gel even exceeds that observed in corresponding samples, analyzed by SDS-PAGE, indicating that not all of the aggregates observed under different conditions are of the same morphology, and rodlike structures are only part of the aggregates that could potentially form σ70 subunit. Moreover, the predominance of irregular high-molecular-weight protein aggregates over rodlike sigma polymers was confirmed by us for stored samples in AFM experiments (Figure 2, B).

Discussion More than 20 proteins are presently known to form amyloid fibrils, and there is a belief that perhaps all proteins are potentially able to form amyloid fibrils under appropriate conditions.44 Amyloid fibrils from different proteins display many common properties including a core cross-β-sheet structure and similar morphologies, such as a twisted, ropelike structure consisting of two to six protofilaments, 2–5 nm in diameter.39,44 According to AFM data, straight rodlike shape of amyloid fibrils formed in vitro was observed for amyloid β-peptide,13-15 Ig light chain,11 insulin,16 α-synuclein,17 and some others. The morphology of RNA polymerase σ70 subunit

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Figure 6. Electrophoretic characterization of σ70 subunit aggregates. Aliquots af σ70 subunit preparations (about 1–2 μg per lane), either freshly isolated (lanes 1) or stored for 3 months (lanes 2) and 10 months (lanes 3) at –20°C in 50% glycerol, were subjected to electrophoresis on (A) 10% SDSTris-glycine or (B) 5% native Tris-glycine (pH 8.6) polyacrylamide gels. Lanes M contain protein markers with indicated molecular masses (in kilodaltons). The arrow points to position of σ70 monomer. The position of aggregates, which are visible in aged preparations (lanes 2 and 3), is shown by vertical brackets.

Figure 5. (A) Congo red (CR) spectra in the absence and presence of 0.7 μM of either freshly isolated σ70 subunit (CR + σ70 new) or stored for 3 months (CR + σ70 aged). The spectrum of the 5 mM phosphate buffer, 150 mM NaCl, pH 7.4, was subtracted from all the spectra. (B) Difference spectra, obtained by subtracting the spectra of the protein alone and of CR alone from the spectra of the protein in the presence of CR, presented in A. (C) Difference spectra, obtained for CR solution, containing 0.4 μM of freshly isolated σ70 subunit, incubated for 5 days at 10°C either in the presence of 0.02 μM of aged preparation (CR+ σ70 + seeds) or without seeding (CR+ σ70).

fibrils is very typical for that of amyloid fibrils: straight fibers of 5.4 nm in diameter and of up to 300 nm length. The diameter distribution of σ70 rodlike polymers is monodisperse, indicating that there is only one particular type of mature fibrils. Finally, in our work we have found that the presence of σ70 protein in Congo red solution results in a typical red shift in the spectrum of the dye, which is an additional proof of the amyloid nature of aggregates. According to published data, amyloid fibrils formation is mainly the result of partial or complete unfolding of proteins.45 However, there is evidence that some proteins are capable of forming this type of aggregates as a result of temporary fluctuations in their tertiary structure without disruption of native conformation.46 These fluctuations lead to exposure of some internal (hidden inside the globule) regions, facilitating intermolecular interactions. Previously in the work of Lowe et al.7 it was shown by EM that σ70 subunit is capable of forming high temperature-induced polymer aggregates, having a linear flexible structure of 8.5 nm width and several hundred nanometers length. Notably, specimen preparation there implied nonphysiological negative staining in uranyl formate that may influence the structure and properties of protein aggregates.

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There were no data on the amyloid morphology of these aggregates. In our work we have discovered that aggregates formed by σ70 under not extreme conditions are close to physiological ones. We can speculate that σ70 subunit is prone to form amyloid-like aggregates when in the native conformation. Mild conditions of σ70 subunit isolation and experiments on seeding confirm our suggestion. Differences in the type of σ70 subunit used, specimen preparation technique, and conditions of investigations, as well as the absence of vertical profile of aggregates observed by EM, do not allow us to compare σ70 polymers obtained by us with those described by Lowe et al.7 However, the comparison of AFM measured height of monomeric σ70 (see Figure 1, B) with that of its aggregates (5.4 nm) allows us to make the similar suggestion that the repeat unit of σ70 polymer probably consists of two or more aggregated σ70 molecules. We have shown that σ70 amyloid-like aggregates exist in rather small quantities (0–8%) under not extreme and nearly physiological conditions (at pH 7.4–7.8 and ionic strength 1–20 mM NaCl and 0–50 mM MgSO4). Such a low content of rodlike aggregates, especially in 1–2 mM NaCl buffer, could hardly be detected and correctly interpreted using conventional biochemical techniques alone, as a result of their rather low sensitivity. However, AFM can detect even a single protein aggregate within a scanned surface area. Remarkably, the content of σ70 rodlike aggregates adsorbed on neutral HOPG surface (5%) was similar to that for mica surface (7%–8%), indicating that aggregates formation could not be attributed to specific charge properties of mica surface. In accordance with earlier work,9 our electrophoresis and Congo red binding assay results indicate a significant increase of the amount of σ70 subunit aggregates upon protein storage for several months at –20°C in 50% glycerol, although AFM data demonstrate that most of these aggregates are of irregular rather than rodlike shape. At this point there are x-ray data only for the fragment (114– 442) of the subunit.4 The crystallization of the full-length protein is apparently unsuccessful as a result of the presence of rather extended highly disordered N- and C-domains. However, one can speculate that aggregation with formation of one-dimensional ordered structures is one of the competing processes in attempts to obtain the three-dimensional crystals of full-length σ70 subunit. σ70 amyloid-like polymer is a promising object for investigation, because its formation may be connected with the regulation of transcription in the cell. It is well known that there are several minor sigma factors in bacterial cells (at least six in E. coli, such as sigma 32, 38, and 54) that are active under specific (usually stress) conditions (osmotic shock, heat shock, etc.) that are inhibitory for σ70. One can speculate that such conditions can cause some changes in protein structure, facilitating the ability of σ70 to form aggregates inside living cells. In this case the aggregation may be one of the stages of a regulatory process. In particular, the aggregation may be a way of sequestering the active σ70 subunit from cytoplasm, with subsequent degradation of aggregates under conditions when σ70 concentration exceeds the required level at a specific phase of bacterial growth. On the other hand, the amyloids can serve as a temporary storage depot of σ70

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subunit in the cell and maintain the appropriate concentration of active protein in cytoplasm, as was proposed by Maji et al.47 The identification of amyloid structures by bacterial proteins in vivo is a complex problem, in that the approaches available (for example, Congo red staining of inclusion bodies or laser confocal microscopy) are associated with invasive manipulations (leading to destruction of the cells), that can affect the aggregation process; therefore, the results cannot be considered as rigorous proofs of in vivo amyloid formation. The in vivo aggregation was successfully demonstrated (using Congo red specific binding) for bacterial curli proteins that were secreted from the cell.26,48,49 The phenomenon observed in the present work may be utilized to create convenient model systems to study the mechanism of amyloidosis. Besides, the controlled growth of σ70 polymers may be interesting for creating bionanostructures in biotechnological applications. For this reason the means must be found to increase fibrillization. Most likely, the use of sigma subunit mutant variants will permit a significant increase in the degree of its polymerization. Identification of the factors influencing (in particular, stimulating) the aggregation process (temperature variations or some low-molecular-weight ligands) also paves the way for targeted regulation of gene expression at the transcriptional level in specially designed strains (including biotechnological ones). Undoubtedly, further investigation of different aspects of σ70 polymerization and fibril formation is an interesting and challenging task.

Appendix A. Supplementary data Supplementary materials related to this article can be found online at doi:10.1016/j.nano.2011.05.014.

References 1. von Hippel PH, Bear DG, Morgan WD, McSwiggen JA. Protein-nucleic acid interactions in transcription: a molecular analysis. Ann Rev Biochem 1984;53:389-446. 2. Gruber TM, Gross CA. Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 2003;57:441-66. 3. Burgess RR, Anthony L. How sigma docks to RNA polymerase and what sigma does. Curr Opin Microbiol 2001;4:126-31. 4. Malhotra A, Severinova E, Darst SA. Crystal structure of a σ70 subunit fragment from E. coli RNA polymerase. Cell 1996;87:127-36. 5. Helmann JD, Chamberlin MJ. Structure and function of bacterial sigma factors. Ann Rev Biochem 1988;57:839-72. 6. Severinova E, Severinov K, Fenyö D, Marr M, Brody EN, Roberts JW, et al. Domain organization of the Escherichia coli RNA polymerase sigma 70 subunit. J Mol Biol 1996;263:637-47. 7. Lowe PA, Aebil U, Gross C, Burgess RR. In vitro thermal inactivation of a temperature-sensitive σ subunit mutant (rpoD8OO) of Escherichia coli RNA polymerase proceeds by aggregation. J Biol Chem 1981;256:2010-5. 8. Ferguson AL, Hughes AD, Tufail U, Baumann CG, Scott DJ, Hoggett JG. Interaction of σ70 with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance. FEBS Lett 2000;481:281-4. 9. Callaci S, Heyduk E, Heyduk T. Conformational changes of Escherichia coli RNA polymerase s70 factor induced by binding to the core enzyme. J Biol Chem 1998;273:32995-3001.

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