Nucleation and Growth of Membrane Protein Crystals In Meso—A

Communication pubs.acs.org/crystal

Nucleation and Growth of Membrane Protein Crystals In MesoA Fluorescence Microscopy Study Andrey Bogorodskiy,† Fedor Frolov,† Alexey Mishin,‡ Ekaterina Round,§,#,¶,□ Vitaly Polovinkin,§,#,¶,□ Vadim Cherezov,‡,● Valentin Gordeliy,†,§,#,¶,□ Georg Büldt,†,⊥ Thomas Gensch,∥ and Valentin Borshchevskiy*,†,§ †

Laboratory for Advanced Studies of Membrane Proteins and ‡Laboratory for Structural Biology of GPCRs Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow region, Russia § ICS-6: Structural Biochemistry, ∥ICS-4: Cellular Biophysics, and ⊥ICS-5: Molecular Biophysics, Institute of Complex Systems (ICS), Research Centre Jülich GmbH, 52425 Jülich, Germany # Université Grenoble Alpes, ¶CNRS, and □CEA, Institut de Biologie Structurale, 38044 Grenoble, France ● Bridge Institute, Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States S Supporting Information *

ABSTRACT: Since the introduction of in meso crystallization of membrane proteins in lipidic cubic phase (LCP) by Landau and Rosenbusch in 1996, numerous studies attempted to elucidate the mechanism of in meso crystal nucleation and growth. Here we present a fluorescence microscopy study of the crystallization process of the light-driven proton pump bacteriorhodopsin. The crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. These observations suggest an Ostwald ripening mechanism of the in meso crystal growth. Analysis of the microcrystal spatial distribution suggests that microcrystal nucleation occurs predominately at the LCP domain boundaries.

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crystallization of several difficult targets.12−19 Over the last 15 years, the development of the in meso method include variations such as a detergent-free approach,20 sponge phase crystallization, 21,22 combination in meso method with vapor diffusion,23 overcoming bR crystal twinning,24,25 and usage of amphipoles26 and non-MO host lipids.27−29 Automation and miniaturization of the crystallization setup30 made this method accessible to a variety of different targets. Finally, the use of LCP-grown microcrystals for serial femtosecond X-ray crystallography at X-ray free electron lasers enabled highresolution structures from micron-sized crystals of challenging membrane proteins and complexes.31−35 Despite increasing popularity of the in meso method during recent years, its mechanism continues to be poorly understood. Here we present an in meso crystallization study of bacteriorhodopsin (bR) that uses confocal laser scanning microscopy (CLSM) to observe the formation of crystals during the nucleation phase followed by continual monitoring of the entire growth phase over a span of several weeks. bR Distribution During In Meso Crystallization. At an early stage of the work we followed the crystallization process by CLSM using the weak native fluorescence of bR retinal36,37

ith the introduction of membrane protein crystallization in lipidic cubic phase (LCP) in 1996 (also known as in meso or in cubo crystallization), Landau and Rosenbusch established a new era of membrane protein structural research.1−3 Although in the beginning this method was regarded as an exotic way to produce diffraction quality crystals of microbial rhodopsins, in recent years, especially with the successful crystallization of G-protein coupled receptors, this method became associated with a number of major breakthroughs in the field.4 Soon after the launch of the in meso method, Qiu and Caffrey published a very detailed phase diagram for monoolein (MO),5 the most common host lipid used in LCP crystallization, establishing the basis for further studies of the in meso method. Later, a hypothetical mechanism for in meso crystallization was proposed, suggesting that the lipidic mesophase adopts a multilamellar structure in the vicinity of the growing crystal.6−8 This idea stemmed from the observation that all crystals produced by this method feature a multilamellar packing of protein molecules. Electron microscopy and X-ray scattering supported this mechanism.9 A neutron scattering study showed that Cubic-Pn3m was the dominating lipidic phase during the course of the crystallization process.10 It was recently proposed by Gordeliy and Moiseeva11 that it is the physical property of the lipid bilayer, not its particular chemical composition that drives the crystallization process, with these principles applied to the successful © 2015 American Chemical Society

Received: July 24, 2015 Revised: October 29, 2015 Published: November 2, 2015 5656

DOI: 10.1021/acs.cgd.5b01061 Cryst. Growth Des. 2015, 15, 5656−5660

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Communication

(centered around 740 nm) excited by 561 nm irradiation. Crystallization trials were carried out in meso starting with bR in purple membranes (PM) or in a detergent-solubilized state as described in detail in Supporting Information. The first CLSM fluorescence image of in meso PM crystallization samples were taken ∼20 min after initiating the crystallization trial. Fluorescent aggregates of 0.5−4 μm in size with emission characteristics of bR were observed (Figure 1A).

Figure 1. (A) CLSM fluorescence image of a bR crystallization sample taken within 20 min after addition of the precipitant. (B) Image after 8 h. (C) Image after 16 h. (D) Image after 24 h; the crystal (marked by arrow) is fully formed.

Figure 1A−D depicts crystal nucleation and growth within the first 24 h. Crystal growth started from one of the aggregates, within 8 h showing the first signs of a hexagonal shape, typical for bR in-meso-crystals. The crystal reached a size of ∼5 μm by the end of day one. Two short clips of bR crystal growth are available in Supporting Information. Since the fluorescence intensity from a growing crystal on day one after nucleation was significantly stronger than the intensity from small aggregates, crystal growth was tracked using two separate laser scans with different excitation powers, one for the crystal and the second for its surroundings (Figure 2). After 2 days the crystal reached a size of 7 μm (Figure 2A), and continued growing until it plateaued at its final size of 30 μm across and 5 μm thick by day 20. A second nearby crystal appeared 1 week after initiation of crystallization (Figure 2B− D). Interestingly, the growing crystal was surrounded by a depletion zone of maximal 100−150 μm radius with a reduced amount of aggregates. This area became evident on day seven and enlarged in size as the crystal continued to grow (see Figure 2B,C). The insets to Figure 2 demonstrate radially averaged normalized fluorescence intensity with increasing distance from the crystal. The strong decrease in fluorescence intensity near the crystal indicates that the crystal is growing by incorporation of bR molecules from the surrounding aggregates. To rule out the possibility that the aggregates observed during in meso crystallization were produced by aggregation of PM patches naturally present in the starting solution, we

Figure 2. (A−D) Time series of in meso bR crystal growth. To enhance contrast, the area outside the crystal was scanned with a higher laser power and merged with the fluorescence image of the crystal. Red dots are bR aggregates, which form ring-like structures. The growing crystal absorbs bR from aggregates in its vicinity. Insets show the dependency of normalized fluorescence intensity on the distance from the crystal. 5657

DOI: 10.1021/acs.cgd.5b01061 Cryst. Growth Des. 2015, 15, 5656−5660

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To further elucidate the nature of the aggregates we used a sample with ∼1% Cy3-labeled and 99% unlabeled solubilized bR for setting up new crystallization trials. Similar to previous crystallization trials with unlabeled bR and PM, we found aggregates, crystals, and depletion zones formed by unlabeled protein (Figure 5). Cy3 fluorescence was, however, very low inside the crystals and aggregates compared to bR native fluorescence (Figure 5A,B). Moreover, Cy3-labeled bR did not form a depletion zone around the growing crystal (Figure 5C). We rationalize the apparent absence of labeled bR in the crystals by a tight crystal packing of protein molecules, which could not accommodate the bulky dye (although the possibility of severe Cy3-fluorescence quenching in crystals could not be totally discarded). The idea of labeled bR exclusion due to tight crystal packing is also supported by the absence of Cy3fluorescent depletion zone around the crystal; by analogy to unlabeled bR, a depletion zone should have formed if labeled bR was incorporated into the crystal. The absence of labeled bR in the aggregates suggests that they are likely three-dimensional microcrystals rather than aggregates of two-dimensional crystals. Formation of microcrystals which are later absorbed by the growing crystal paired with the appearance of a depletion zone shows evidence of Ostwald ripening40,41 during in meso crystal growth of bR. This phenomenon describes dissolution of small crystals and redeposition of molecules onto larger crystals, which is driven by the minimization of overall surface energy. Ostwald ripening was observed for the first time in gelatin plates with formation of crystallization rings of salts in the end of 19th century.40 Later, this phenomenon was observed in many inorganic systems42 as well as several viruses (tobacco mosaic virus,43 tomato bushy stunt virus44) and water-soluble proteins (αamylase, thermostable aspartyl-tRNA synthetase,44 and lysozyme, where it was described in great detail45). Here, we demonstrated for the first time that Ostwald ripening also takes place during in meso crystallization of membrane proteins. Distribution of Microcrystals. Another interesting observation was that the aggregates were nonuniformly distributed in the sample, forming characteristic “honeycomb” patterns, particularly apparent during the first day after crystallization setup (Figure 2A,B). The size of regions surrounded by microcrystals (10−20 μm) coincides with the typical domain size of the lipidic cubic phase reported previously (10−15 μm).9 Therefore, we believe that the domain boarders might serve as areas of inhomogeneities which facilitate crystal nucleation in the sample. In conclusion, here we report the observations of crystal nucleation occurring at the LCP domain boundaries and Ostwald ripening of crystals in meso. The work sheds additional

performed an additional crystallization trial starting from solubilized bR instead of PM, in which no bR aggregates were present. Similarly, small bR aggregates were evident in the crystallization sample captured on day seven (Figure 3).

Figure 3. In meso crystallization sample made from solubilized bR. CLSM fluorescence image was taken on day seven after setting up crystallization. Figure shows small crystals, aggregates, and a depletion zone surrounding the growing crystal.

Hexagonal bR crystals also formed in this sample, surrounded by depletion zones, as observed previously for crystallization trials starting from PM. The observation suggests that small bR aggregates do form during the crystallization process (both starting from PM and solubilized bR) rather than being introduced in the sample during setup. Elucidating the Nature of Aggregates. To investigate the nature of the bR aggregates we applied second-harmonic generation (SHG) imaging of the crystallization sample. The unique symmetry requirements for SHG signal result in destructive interference and no coherent signal from randomly oriented assemblies of proteins (e.g., proteins in solution or in amorphous aggregates) but produce bulk-allowed SHG from crystals of homochiral molecules, such as proteins.38 This phenomenon is frequently used for membrane protein microcrystal detection in the method known as second-order nonlinear optical imaging of chiral crystal (SONICC).39 The same area of a crystallization sample made from solubilized bR was imaged on day seven after setup using CLSM (Figure 4B) and SHG (irradiation 1000 nm) (Figure 4A). The superposition of A and B in Figure 4C shows high colocalization of fluorescence and SHG signals suggesting that bR forms ordered aggregates. However, it remained unclear whether these aggregates were three-dimensional microcrystals or aggregates of two-dimensional crystals (PM)the generation of SHG signal is possible in both cases.

Figure 4. (A) SHG signal generated in in meso crystallization sample of solubilized bR (excitation at 1000 nm). (B) CLSM image of the same region of sample (excitation at 561 nm). (C) Superposition of (A) and (B). Co-localized SHG and fluorescence signals (yellow) indicate that not only crystal but also most aggregates formed by bR molecules have crystalline order. 5658

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Figure 5. (A) CLSM image of in meso crystallization sample containing 1% Cy3-labeled (fluorescent signal shown in yellow) and 99% unlabeled bR (fluorescent signal shown in red). The inset shows fluorescence spectra of Cy3-labeled and unlabeled bR. No Cy3-fluorescence signal was detected from the crystal. (B) Zoomed-out CLSM image of another region of the same sample. The bR crystal located in the middle of the figure was not scanned (black rectangle) because of its high fluorescent signal oversaturating detector. (C) Distribution of normalized fluorescence intensity of Cy3labeled and native bR at the distance R from the crystal shown in (B). Unlabeled bR molecules form a depletion zone around the crystal whereas Cy3-labeled bR molecules do not. The moderate negative incline of normalized fluorescence intensity of Cy3-labeled protein (yellow graph in C) is due to greater concentration of unlabeled bR (in comparison with the crystal vicinity) which displaced Cy3-labeled bR molecules. (5) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223−234. (6) Nollert, P.; Qiu, H.; Caffrey, M.; et al. FEBS Lett. 2001, 504 (3), 179−186. (7) Qutub, Y.; Reviakine, I.; Maxwell, C.; et al. J. Mol. Biol. 2004, 343, 1243−1254. (8) Caffrey, M. Cryst. Growth Des. 2008, 8 (12), 4244−4254. (9) Cherezov, V.; Caffrey, M. Faraday Discuss. 2007, 136, 195−212. (10) Efremov, R.; Shiryaeva, G.; Bueldt, G.; et al. J. Cryst. Growth 2005, 275 (1−2), e1453−e1459. (11) Gordeliy, V. I.; Moiseeva, E. S. In Molecules: Nucleation, Aggregation and Crystallization; Sedzik, J., Riccio, P., Eds.; World Scientific Publishing Co. Pte. Ltd.: Singapore, 2009; pp 259−281. (12) Nogly, P.; Gushchin, I.; Remeeva, A.; et al. Nat. Commun. 2014, 5, 4169. (13) Shevchenko, V.; Gushchin, I.; Polovinkin, V.; et al. PLoS One 2014, 9 (12), e112873. (14) Borshchevskiy, V.; Round, E.; Erofeev, I.; et al. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2014, 70 (10), 2675−2685. (15) Gushchin, I.; Chervakov, P.; Kuzmichev, P.; et al. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (31), 12631−12636. (16) Ishchenko, A.; Round, E.; Borshchevskiy, V.; et al. J. Photochem. Photobiol., B 2013, 123, 55−58. (17) Gushchin, I.; Reshetnyak, A.; Borshchevskiy, V.; et al. J. Mol. Biol. 2011, 412 (4), 591−600. (18) Borshchevskiy, V. I.; Round, E. S.; Popov, A. N.; et al. J. Mol. Biol. 2011, 409 (5), 813−825. (19) Gushchin, I.; Shevchenko, V.; Polovinkin, V.; et al. Nat. Struct. Mol. Biol. 2015, 22 (5), 390−395. (20) Nollert, P.; Royant, A.; Pebay-Peyroula, E.; et al. FEBS Lett. 1999, 457, 205−208. (21) Cherezov, V.; Clogston, J.; Papiz, M. Z.; et al. J. Mol. Biol. 2006, 357 (5), 1605−1618. (22) Wadsten, P.; Wöhri, A. B.; Snijder, A.; et al. J. Mol. Biol. 2006, 364 (1), 44−53. (23) Kubicek, J.; Schlesinger, R.; Baeken, C.; et al. PLoS One 2012, 7, e35458. (24) Borshchevskiy, V.; Efremov, R.; Moiseeva, E.; et al. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (1), 26−32. (25) Gordeliy, V.; Borshchevskiy, V. In Modern Aspects of Bulk Crystal and Thin Film Preparation; Kolesnikov, N., Ed.; InTech, 2012; pp 477−496. (26) Polovinkin, V.; Gushchin, I.; Sintsov, M.; et al. J. Membr. Biol. 2014, 247 (9−10), 997−1004. (27) Borshchevskiy, V.; Moiseeva, E.; Kuklin, A.; et al. J. Cryst. Growth 2010, 312 (22), 3326−3330. (28) Li, D.; Lee, J.; Caffrey, M. Cryst. Growth Des. 2011, 11 (2), 530− 537. (29) Salvati Manni, L.; Zabara, A.; Osornio, Y. M.; et al. Angew. Chem., Int. Ed. 2015, 54 (3), 1027−1031. (30) Cherezov, V. Curr. Opin. Struct. Biol. 2011, 21 (4), 559−566.

light on the mechanism of in meso crystallization, which could facilitate rational design of future in meso crystallization experiments with new membrane protein targets.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b01061. Crystallization from PM. Crystallization from solubilized bR. Crystallization of labeled bR. Confocal fluorescence microscopy. (PDF) Video clip showing crystal growth from PM over time 0h−44h (AVI) Video clip showing crystal growth from PM over time 0h−90h (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +7 964 632-8650. Author Contributions

The authors are grateful for the help and consultations of Alexei Grichine and Michael Sintsov. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by Russian Science Foundation research project 14-14-00995 (A.B., F.F., M.S., A.M., V.P., V.G., V.B.). The work was done in the framework of CEA (IBS) − HGF(FZJ) STC 5.1 specific agreement and was supported by FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX49-01) within the Grenoble Partnership for Structural Biology (PSB) (E.R., V.P., V.G.).

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DOI: 10.1021/acs.cgd.5b01061 Cryst. Growth Des. 2015, 15, 5656−5660