ChemComm

Published on 25 June 2014. Downloaded by Tulane University on 19/10/2014 18:43:16.

COMMUNICATION

View Article Online View Journal | View Issue

Cite this: Chem. Commun., 2014, 50, 8955

epi-Fluorescence imaging at the air–water interface of fibrillization of bovine serum albumin and human insulin†

Received 26th May 2014, Accepted 25th June 2014

Kristen Sessions, Stuart Sacks, Shanghao Li and Roger M. Leblanc*

DOI: 10.1039/c4cc04030a www.rsc.org/chemcomm

Protein fibrillization is associated with many devastating neurodegenerative diseases. This process has been studied using spectroscopic and microscopic methods. In this study, epi-fluorescence at the air–water interface was developed as an innovative technique for observing fibrillization of bovine serum albumin and human insulin.

Fibrillization is a degenerative process in amyloid peptides, and is associated with a wide variety of human diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, type II diabetes, and prion disease.1–3 Protein fibrillization involves formation of intermolecular hydrogen bonding of extended polypeptide strands (also called ‘‘cross-b’’ architecture) as a consequence of protein misfolding. Studies have shown that this process is favoured by conditions that denature or partially unfold the protein such as high temperatures, low pH, hydrophobic interface or surface, and agitation.4,5 Several microscopic methods have already been established to characterize structural and morphological changes of noncrystalline protein fibrils, including electron microscopy (EM) and atomic force microscopy (AFM).6–8 A high vacuum environment is needed to run specimens in methods of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Furthermore, either staining or extremely low temperature is necessary for TEM in order to enhance the contrast of protein samples. These requirements limit the conditions for observing protein morphology changes over time. Although atomic force microscopy (AFM) does not need special sample treatment and can monitor protein fibrillization in ambient air or even a liquid environment,9,10 this technique is limited by its slow scanning speed and small scanning area size. Since proteins fibrillate in the body at the biological interfaces, a technique such as epi-fluorescence microscopy would be interesting to more closely mimic biological conditions. Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, USA. E-mail: [email protected]; Fax: +1-305-284-4571; Tel: +1-305-284-2194 † Electronic supplementary information (ESI) available: About the experimental details of materials, incubation, and measurements of CD, ThT fluorescence, AFM, isotherm and epi-fluorescence microscopy. See DOI: 10.1039/c4cc04030a

This journal is © The Royal Society of Chemistry 2014

The fluorescent dye Thioflavin T (ThT) has become one of the most widely used methods with high sensitivity and convenience to identify amyloid fibrils.11,12 As the percentage of b-sheet increases throughout fibrillization, ThT is able to bind more effectively with the fibrils. It is widely accepted that the fluorescence enhancement upon binding to fibrils results from the selective immobilization of a subset of the ThT conformers with high quantum yields of fluorescence.13,14 While the increase of fluorescence intensity correlates with the fibril formation, the linear dye ThT needs to bind to the surfaces of cross-b structures. Thus the binding may alter the intrinsic structures of fibrils. As the ThT binding is a non-imaging technique, it does not illustrate the morphology changes. The purpose of this study was to develop an epi-fluorescence microscopic technique, at the air–water interface, for qualitatively observing protein fibrillization. It is cost-effective and able to give results quickly. It involves spreading a protein solution as a Langmuir monolayer, then depositing 5-(octadecanoylamino)-fluorescein on top of it, allowing for image detection.15,16 The experimental details are provided in the ESI.† If proven effective, epi-fluorescence microscopy can be a useful technique to detect or image protein fibrillization in future studies. Characteristic transition by fibrillization from a-helix to b-sheet was monitored using CD spectra, which measure the components of the secondary structures. BSA showed a decrease in a-helix from 80 to 41% and an increase in b-sheet from 6 to 17% over the 48 hour incubation at 65 1C (Fig. 1A). Similarly, the percentage of a-helix in HI decreased from 31 to 1% while b-sheet increased from 17 to 74% in 10 hour incubation at 65 1C (Fig. 1B). The typical kinetics of protein fibrillization monitored by a fibril-specific dye of ThT usually commences with a lag time to form nucleus, followed by a relatively short elongation phase.17,18 In the present study, BSA incubated at 65 1C does not have a lag phase and shows a continuing increase in the fluorescence of ThT (Fig. 2A). As suggested by the CD spectra in Fig. 1A, the fibrils of BSA still retain significant amounts of a-helix, suggesting that the BSA fibrils do not possess the same structural rigidity as classic protein fibrils. In fact, AFM images during the fibrillization of BSA confirm that BSA in the incubation develops amorphous fibrils

Chem. Commun., 2014, 50, 8955--8957 | 8955

View Article Online

Communication

Published on 25 June 2014. Downloaded by Tulane University on 19/10/2014 18:43:16.

Fig. 1 CD spectra of (A) 0.1 mg mL 1 of BSA in phosphate buffer at pH 7.4 with 50 mM NaCl; (B) 0.1 mg mL 1 of HI at pH 2 with 100 mM NaCl. Both proteins were incubated at 65 1C with moderate agitation.

ChemComm

Fig. 4 Surface pressure-area isotherms of (A) 0.1 mg mL 1 BSA in phosphate buffer with 50 mM NaCl at pH 7.4; and (B) 0.33 mg mL 1 human insulin with 100 mM NaCl at pH 2. Both proteins were incubated at 65 1C with agitation.

pressure-area isotherms and epi-fluorescence images at the corresponding times deposited at the air–water interface (Fig. 4 and 5). Langmuir monolayer technique has been widely applied to study properties and structures of molecules deposited at the air–water interface, such as proteins, lipids, polymers, and

Fig. 2 Fluorescence intensity of ThT at 487 nm for (A) BSA incubated at 65 1C for 48 h; (B) HI incubated at 65 1C for 14 h.

(Fig. 3A), which have different morphologies from the classic protein fibrils with rope-like structures. The term of ‘‘aggregates’’ may be properer than ‘‘fibrils’’ for BSA. All these observations are consistent with previous studies.19–21 Human insulin (HI) did have a lag phase and no increase in intensity of fluorescence was observed for the first two hours. Once fibrillization is complete, the intensity of fluorescence will reach a plateau. This was observed for HI after nine hours (Fig. 2B). The AFM images of HI during the fibrillization show the morphology of fibril growth (Fig. 3B). The characterizations of the CD spectra, the enhancement of ThT fluorescence, and AFM confirm that fibrils of BSA and HI formed throughout incubation. The changes in morphology observed from solution (Fig. 1–3) appear to correlate with changes of surface

Fig. 3 AFM images of the BSA (A) and HI (B) fibrillization incubated at 65 1C for various hours. The scale bar in each image shows 1 mm.

8956 | Chem. Commun., 2014, 50, 8955--8957

Fig. 5 epi-Fluorescence microscopic images (image size: 895 mm  713 mm) at the air–water interface of (A) BSA incubated at 65 1C for 0, 12, 24, and 48 h; (B) HI incubated at 65 1C for 0, 2, 6, and 10 h.

This journal is © The Royal Society of Chemistry 2014

View Article Online

Published on 25 June 2014. Downloaded by Tulane University on 19/10/2014 18:43:16.

ChemComm

nanomaterials.22–25 Fig. 4 shows the surface pressure-area isotherms at the air–water interface over the incubation timeperiod, which was used to characterize the Langmuir monolayer. BSA at time 0 had a lift-off point at 3000 Å2 per molecule and a limiting molecular area of about 2500 Å2 per molecule. Fibrillization of BSA slightly decreased the lift-off point and limiting molecular area, but not significantly even after 48 hours of incubation at 65 1C (Fig. 4A). HI at time 0 had a lift-off point at 750 Å2 per molecule and a limiting molecular area of 635 Å2 per molecule (Fig. 4B), which are comparable to previously published values at the air–water interface.26,27 The surface pressure-area isotherms of HI at a lag phase (2 h) and an elongation phase (6 h) appeared similarly. However, after 10 hours of incubation, no surface pressure was observed for HI at the air–water interface. This is probably because the mature fibrils of HI with rich cross-b architecture were not surface active and sank into the subphase. This could be also one limitation of the method, as one may not be able to get the images of mature fibrils at the air– water interface. The distinct behaviours of surface pressure between HI at 10 hours and BSA at 48 hours probably result from the structural difference between the HI and BSA fibrils as shown by CD spectra in Fig. 1 and AFM images in Fig. 3. Once the presence of fibrils in solution was confirmed, epi-fluorescence images were taken at the air–water interface to monitor the process of fibrillization. The dye ODFL used in experiment has a planar aromatic fluorescein headgroup and a C18 carbon chain. ODFL does not have specific binding sites with protein fibrils. Therefore, it should have no or very limited impact on the kinetics or outcomes of protein fibrillization. For both BSA and HI, a change in morphology was observed throughout incubation and corresponded to the absence or presence of fibrils in solution. It is worth noticing that the morphology obtained by epi-fluorescence was not the actual fibrils, but the domains of ODFL confined by protein fibrils. When protein fibrils present at the air–water interface, the fluidity of the ODFL monolayer was disturbed or confined by the fibrils. The morphology of the BSA Langmuir monolayer at time 0 with ODFL was mostly circular, domain-like structures with some areas of brighter green, which indicated a higher concentration of ODFL (Fig. 5A: 0 h). As fibrillization of BSA continued, the circular domains became less regular (Fig. 5A: 12 h). The morphology became more web-like and less fluid at 24 h (Fig. 5A: 24 h) probably due to the formation of BSA aggregates (Fig. 3A). After 48 hours, no circular structures remained and the entire Langmuir monolayer showed the stagnant web structure (Fig. 5A: 48 h). The morphology of the HI monolayer with ODFL also changed throughout fibril formation. Initially, HI showed shapes of straight lines (Fig. 5B: 0 h). Compared with epi-fluorescence image of BSA at time 0, the shape difference might be due to nature of the two proteins, such as the size and conformation. By the end of the lag phase (Fig. 5B: 6 h), some thickening of lines could be observed and the images became more web-like, similar to the transition observed for BSA. At the end of fibrillization (Fig. 5B: 10 h), however, the images were solid dark green. This also corresponded to the flat surface-pressure area isotherm at 10 hours, and indicated that the fibrils were not surface active to remain at the air–water interface.

This journal is © The Royal Society of Chemistry 2014

Communication

In summary, the epi-fluorescence microscopic images showed a change in morphology that corresponded to the different phases of fibril formation observed by the measurements of the CD spectra, ThT fluorescence, and AFM. This confirms that epi-fluorescence microscopy is a viable method for qualitatively observing fibril formation. Although epi-fluorescence microscopy has much lower resolution compared with other microscopic methods, such as SEM, TEM, and AFM, it has its own advantages due to its low cost, fast scanning, and ease to use. It does not require high vacuum environments and is able to mimic more closely cellular conditions. Also, unlike the ThT fluorescence, the ODFL used in these images does not bind to the fibrils being formed. The epi-fluorescence microscopy provides snap-shot images that represent a rapid view of fibril formation with low cost and fast speed of operation.

Notes and references 1 D. H. Small and C. A. McLean, J. Neurochem., 1999, 73, 443. 2 E. H. Koo, P. T. Lansbury and J. W. Kelly, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 9989. 3 M. Stefani, Biochim. Biophys. Acta, Mol. Basis Dis., 2004, 1739, 5. 4 V. Sluzky, J. A. Tamada, A. M. Klibanov and R. Langer, Proc. Natl. Acad. Sci. U. S. A., 1991, 88, 9377. 5 S. Li and R. M. Leblanc, J. Phys. Chem. B, 2014, 118, 1181. 6 B. Chen, K. R. Thurber, F. Shewmaker, R. B. Wickner and R. Tycko, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 14339. 7 S. Campioni, G. Carret, S. Jordens, L. Nicoud, R. Mezzenga and R. Riek, J. Am. Chem. Soc., 2014, 136, 2866. 8 R. Jansen, W. Dzwolak and R. Winter, Biophys. J., 2005, 88, 1344. 9 S. Bolisetty, J. Adamcik and R. Mezzenga, Soft Matter, 2011, 7, 493. 10 M. Zhu, S. Han, F. Zhou, S. A. Carter and A. L. Fink, J. Biol. Chem., 2004, 279, 24452. 11 Q. Ma, G. Wei and X. Yang, Nanoscale, 2013, 5, 10397. 12 T. Ban, D. Hamada, K. Hasegawa, H. Naiki and Y. Goto, J. Biol. Chem., 2003, 278, 16462. 13 V. I. Stsiapura, A. A. Maskevich, V. A. Kuzmitsky, K. K. Turoverov and I. M. Kuznetsova, J. Phys. Chem. A, 2007, 111, 4829. 14 E. S. Voropai, M. P. Samtsov, K. N. Kaplevskii, A. A. Maskevich, V. I. Stepuro, O. I. Povarova, I. M. Kuznetsova, K. K. Turoverov, A. L. Fink and V. N. Uverskii, J. Appl. Spectrosc., 2003, 70, 868. 15 J. Orbulescu, S. V. Mello, Q. Huo, G. Sui, P. Kele and R. M. Leblanc, Langmuir, 2001, 17, 1525. 16 S. Li, M. Micic, J. Orbulescu, J. D. Whyte and R. M. Leblanc, J. R. Soc., Interface, 2012, 9, 3118. 17 J. D. Harper and P. T. Lansbury, Annu. Rev. Biochem., 1997, 66, 385. 18 J. T. Ben, F. L. Chiu, J. V. David and J. Letitia, Biochem. J., 2013, 456, 67. 19 N. K. Holm, S. K. Jespersen, L. V. Thomassen, T. Y. Wolff, P. Sehgal, L. A. Thomsen, G. Christiansen, C. B. Andersen, A. D. Knudsen and D. E. Otzen, Biochim. Biophys. Acta, Proteins Proteomics, 2007, 1774, 1128. ´rez, P. Taboada and V. Mosquera, Biophys. J., 2009, 96, 2353. 20 J. Jua `, M. Leone, A. Ponzoni, G. Sberveglieri 21 V. Vetri, M. D’Amico, V. Fodera and V. Militello, Arch. Biochem. Biophys., 2011, 508, 13. 22 S. Li, A. J. Stein, A. Kruger and R. M. Leblanc, J. Phys. Chem. C, 2013, 117, 16150. 23 A. P. Costa, X. Xu and D. J. Burgess, Langmuir, 2012, 28, 10050. 24 S. Li, J. Guo, R. A. Patel, A. L. Dadlani and R. M. Leblanc, Langmuir, 2013, 29, 5742. 25 I. I. Perepichka, K. Borozenko, A. Badia and C. G. Bazuin, J. Am. Chem. Soc., 2011, 133, 19702. 26 S. Johnson, W. Liu, G. Thakur, A. Dadlani, R. Patel, J. Orbulescu, J. D. Whyte, M. Micic and R. M. Leblanc, J. Phys. Chem. B, 2012, 116, 10205. ´rez, N. M. Blanco-Vila and N. Vila-Romeu, Thin Solid 27 M. Nieto-Sua Films, 2013, 548, 509.

Chem. Commun., 2014, 50, 8955--8957 | 8957

epi-Fluorescence imaging at the air-water interface of fibrillization of bovine serum albumin and human insulin.

Protein fibrillization is associated with many devastating neurodegenerative diseases. This process has been studied using spectroscopic and microscop...
2MB Sizes 0 Downloads 2 Views