Soluble Structure of CLIC and S100 Proteins Investigated by Atomic Force Microscopy


The ability to visualise proteins in their native environment and discern information regarding stoichiometry is of critical importance when studying protein interactions and function. We have used liquid cell atomic force microscopy (AFM) to visualise proteins in their native state in buffer and have determined their molecular volumes. The human proteins S100A8, S100A9, S100A12 and CLIC1 were used in this investigation. The effect of oxidation on the protein structure of CLIC1 was also investigated and we found that CLIC1 multimerisation could be discerned by AFM, which supports similar findings by other methods. We have found good correlation between the molecular volumes measured by AFM and the calculated volumes of the individual proteins. This method allows for the study of single soluble proteins under physiological conditions and could potentially be extended to study the structure of these proteins when located within a membrane environment.

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Valenzuela, S. , Berkahn, M. , Porkovich, A. , Huynh, T. , Goyette, J. , Martin, D. and Geczy, C. (2011) Soluble Structure of CLIC and S100 Proteins Investigated by Atomic Force Microscopy. Journal of Biomaterials and Nanobiotechnology, 2, 8-17. doi: 10.4236/jbnb.2011.21002.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] S. W. Schneider, J. Larmer, R. M. Henderson and H. Oberleithner, “Molecular Weights of Individual Proteins Correlate with Molecular Volumes Measured by Atomic Force Microscopy,” Pflugers Archiv-European Journal of Physiology, Vol. 435, No. 3, 1998, pp. 362-367. doi:10.1007/s004240050524
[2] D. J. Muller, H. Janovjak, T. Lehto, L. Kuerschner and K. Anderson, “Observing Structure, Function and Assembly of Single Proteins by AFM,” Progress in Biophysics and Molecular Biology, Vol. 79, No. 1-3, 2002, pp. 1-43. doi:10.1016/S0079-6107(02)00009-3
[3] N. P. Barrera, J. Betts, H. You, R. M. Henderson, I. L. Martin, S. M. Dunn, et al., “Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the alpha4beta3delta GABA(A) Receptor,” Molecular Pharmacology, Vol. 73, No. 3, 2008, pp. 960-967.
[4] N. P. Barrera, Y. Shaifta, I. McFadzean, J. P. Ward, R. M. Henderson and J. M. Edwardson, “AFM Imaging Reveals the Tetrameric Structure of the Trpc1 Channel,” Bioche- mical and Biophysical Research Communications, Vol. 358, No. 4, 2007, pp. 1086-1090. doi:10.1016/j.bbrc.2007.05.039
[5] S. Bahatyrova, R. N. Frese, C. A. Siebert, J. D. Olsen, K. O. Van Der Werf, R. Van Grondelle, et al., “The Native Architecture of a Photosynthetic Membrane,” Nature, Vol. 430, No. 7003, 2004, pp. 1058-1062. doi:10.1038/nature02823
[6] M. Stolz, D. Stoffler, U. Aebi and C. Goldsbury, “Monitoring Biomolecular Interactions by Time-Lapse Atomic Force Microscopy,” Journal of Structural Biology, Vol. 131, No. 3, 2000, pp. 171-180.
[7] I. A. Mastrangelo, M. Ahmed, T. Sato, W. Liu, C. Wang, P. Hough, et al., “High-Resolution Atomic Force Micro- scopy of Soluble Abeta42 Oligomers,” Journal of Mole- cular Biology, Vol. 358, No. 1, 2006, pp. 106-119. doi:10.1016/j.jmb.2006.01.042
[8] B. A. Cromer, C. J. Morton, P. G. Board and M. W. Parker, “From Glutathione Transferase to Pore in a CLIC,” European Biophysics Journal, Vol. 31, No. 5, 2002, pp. 356-364. doi:10.1007/s00249-002-0219-1
[9] S. J. Harrop, M. Z. DeMaere, W. D. Fairlie, T. Reztsova, S. M. Valenzuela, M. Mazzanti, et al., “Crystal Structure of a Soluble form of the Intracellular Chloride Ion Channel CLIC1 (NCC27) at 1.4-A Resolution,” Journal of Biological Chemistry, Vol. 276, No. 48, 2001, pp. 44993-5000. doi:10.1074/jbc.M107804200
[10] D. R. Littler, N. N. Assaad, S. J. Harrop, L. J. Brown, G. J. Pankhurst, P. Luciani, et al., “Crystal Structure of the Soluble Form of the Redox-Regulated Chloride Ion Channel Protein CLIC4,” FEBS Journal, Vol. 272, No. 19, 2005, pp. 4996-5007.
[11] K. S. Suh and S. H. Yuspa, “Intracellular Chloride Channels: Critical Mediators of Cell Viability and Potential Targets for Cancer Therapy,” Current Pharmaceutical Design, Vol. 11, No. 21, 2005, pp. 2753-2764.
[12] B. M. Tulk, S. Kapadia and J. C. Edwards, “CLIC1 Inserts from the Aqueous Phase into Phospholipid Membranes, where It Functions as an Anion Channel,” American Journal of Physiology-Cell Physiology, Vol. 282, No. 5, pp. C1103-C1112.
[13] K. Warton, R. Tonini, W. D. Fairlie, J. M. Matthews, S. M. Valenzuela, M. R. Qiu, et al., “Recombinant CLIC1 (NCC27) Assembles in Lipid Bilayers via a Ph-Dependent Two-State Process to Form Chloride Ion Channels with Identical Characteristics to Those Observed in Chinese Hamster Ovary Cells Expressing CLIC1,” Journal of Bio- logical Chemistry, Vol. 277, No. 29, 2002, pp. 26003- 26011. doi:10.1074/jbc.M203666200
[14] M. Berryman, J. Bruno, J. Price and J. C. Edwards, “CLIC-5A Functions as a Chloride Channel in Vitro and Associates with the Cortical Actin Cytoskeleton in Vitro and in Vivo,” Journal of Biological Chemistry, Vol. 279, No. 33, 2004, pp. 34794-34801.
[15] D. R. Littler, S. J. Harrop, W. D. Fairlie, L. J. Brown, G. J. Pankhurst, S. Pankhurst, et al., “The Intracellular Chloride Ion Channel Protein CLIC1 Undergoes a Redox-Con- trolled Structural Transition,” Journal of Biological Che- mistry, Vol. 279, No. 10, 2004, pp. 9298-9305. doi:10.1074/jbc.M308444200
[16] H. Singh and R. H. Ashley, “Redox Regulation of CLIC1 by Cysteine Residues Associated with the Putative Chan- nel Pore,” Biophysical Journal, Vol. 90, No. 5, 2006, pp. 1628-1638. doi:10.1529/biophysj.105.072678
[17] H. Singh and R. H. Ashley, “CLIC4 (p64H1) and Its Putative Transmembrane Domain Form Poorly Selective, Redox-Regulated Ion Channels,” Molecular Membrane Biology, Vol. 24, No. 1, 2007, pp. 41-52.
[18] H. Singh, M. A. Cousin and R. H. Ashley, “Functional Reconstitution of Mammalian Chloride Intracellular Channels’ CLIC1, CLIC4 and CLIC5 Reveals Differential Regu- lation by Cytoskeletal Actin,” FEBS Journal, Vol. 274, No. 24, 2007, pp. 6306-6316.
[19] R. Tonini, A. Ferroni, S. M. Valenzuela, K. Warton, T. J. Campbell, S. N. Breit, et al., “Functional Characterization of the NCC27 Nuclear Protein in Stable Transfected CHO-K1 Cells,” FASEB Journal, Vol. 14, No. 9, 2000, pp. 1171-1178.
[20] S. M. Valenzuela, D. K. Martin, S. B. Por, J. M. Robbins, K. Warton, M. R. Bootcov, et al., “Molecular Cloning and Expression of a Chloride Ion Channel of Cell Nuclei,” Journal of Biological Chemistry, Vol. 272, No. 19, 1997, pp. 12575-12582. doi:10.1074/jbc.272.19.12575
[21] T. Ravasi, K. Hsu, J. Goyette, K. Schroder, Z. Yang, F. Rahimi, et al., “Probing the S100 Protein Family through Genomic and Functional Analysis,” Genomics, Vol. 84, No. 1, 2004, pp. 10-22. doi:10.1016/j.ygeno.2004.02.002
[22] M. Pedrocchi, B. W. Schafer, H. Mueller, U. Eppenberger and C. W. Heizmann, “Expression of Ca(2+)-Binding Proteins of the S100 Family in Malignant Human Breast- Cancer Cell Lines and Biopsy Samples,” International Journal of Cancer, Vol. 57, No. 5, 1994, pp. 684-690.
[23] R. Donato, “Functional Roles of S100 Proteins, Calcium- Binding Proteins of the EF-hand Type,” Biochimica et Biophysica Acta, Vol. 1450, No. 3, 1999, pp. 191-231.
[24] R. Donato, “Intracellular and Extracellular Roles of S100 proteins,” Microscopy Research and Technique, Vol. 60, No. 6, 2003, pp. 540-551. doi:10.1002/jemt.10296
[25] R. J. Passey, K. Xu, D. A. Hume and C. L. Geczy, “S100A8: Emerging Functions and Regulation,” Journal of Leukocyte Biology, Vol. 66, No. 4, 1999, pp. 549-556.
[26] P. Lemarchand, M. Vaglio, J. Mauel and M. Markert, “Translocation of a Small Cytosolic Calcium-Binding Protein (MRP-8) to Plasma Membrane Correlates with Human Neutrophil Activation,” Journal of Biological Chemistry, Vol. 267, No. 27, 1992, pp. 19379-19382.
[27] C. Kerkhoff, C. Sorg, N. N. Tandon and W. Nacken, “Interaction of S100A8/S100A9-Arachidonic Acid Com- plexes with the Scavenger Receptor CD36 may Facilitate Fatty acid Uptake by Endothelial Cells,” Biochemistry, Vol. 40, No. 1, 2001, pp. 241-248.
[28] H. Kubista, R. Donato and A. Hermann, “S100 Calcium Binding Protein Affects Neuronal Electrical Discharge Activity by Modulation of Potassium Currents,” Neuro- science, Vol. 90, No. 2, 1999, pp. 493-508. doi:10.1016/S0306-4522(98)00422-9
[29] K. Hsu, R. J. Passey, Y. Endoh, F. Rahimi, P. Youssef, T. Yen, et al., “Regulation of S100A8 by Glucocorticoids,” Journal of Immunology, Vol. 174, No. 4, 2005, pp. 2318- 2326.
[30] T. Yen, C. A. Harrison, J. M. Devery, S. Leong, S. E. Iismaa, T. Yoshimura, et al., “Induction of the S100 Chemotactic Protein, CP-10, in Murine Microvascular Endothelial Cells by Proinflammatory Stimuli,” Blood, Vol. 90, No. 12, 1997, pp. 4812-4821.
[31] I. S. Thorey, J. Roth, J. Regenbogen, J. P. Halle, M. Bittner, T. Vogl, et al., “The Ca2+-Binding Proteins S100A8 and S100A9 are Encoded by Novel Injury- Regulated Genes,” Journal of Biological Chemistry, Vol. 276, No. 38, 2001, pp. 35818-35825. doi:10.1074/jbc.M104871200
[32] M. A. Grimbaldeston, C. L. Geczy, N. Tedla, J. J. Finlay-Jones and P. H. Hart, “S100A8 Induction in Keratinocytes by Ultraviolet A Irradiation is Dependent on Reactive Oxygen Intermediates,” Journal of Inves- tigative Dermatology, Vol. 121, No. 5, 2003, pp. 1168-1174. doi:10.1046/j.1523-1747.2003.12561.x
[33] F. Rahimi, K. Hsu, Y. Endoh and C. L. Geczy, “FGF-2, IL-1beta and TGF-beta Regulate Fibroblast Expression of S100A8,” FEBS Journal, Vol. 272, No. 11, 2005, pp. 2811-2827. doi:10.1111/j.1742-4658.2005.04703.x
[34] P. G. Sohnle, M. J. Hunter, B. Hahn and W. J. Chazin, “Zinc-Reversible Antimicrobial Activity of Recombinant Calprotectin (Migration Inhibitory Factor-Related Proteins 8 and 14),” Journal of Infectious Diseases, Vol. 182, No. 4, 2000, pp. 1272-1275.
[35] Y. Nakatani, M. Yamazaki, W. J. Chazin and S. Yui, “Regulation of S100A8/A9 (Calprotectin) Binding to Tumor Cells by Zinc Ion and Its Implication for Apop- tosis-Inducing Activity,” Mediators of Inflammation, Vol. 5, 2005, pp. 280-292. doi:10.1155/MI.2005.280
[36] C. van den Bos, J. Roth, H. G. Koch, M. Hartmann and C. Sorg, “Phosphorylation of MRP14, an S100 Protein Ex- pressed during Monocytic Differentiation, Modulates Ca(2+)-Dependent Translocation from Cytoplasm to Membranes and Cytoskeleton,” Journal of Immunology, Vol. 156, No. 3, 1996, pp. 1247-1254.
[37] K. Ishikawa, A. Nakagawa, I. Tanaka, M. Suzuki and J. Nishihira, “The Structure of Human MRP8, a Member of the S100 Calcium-Binding Protein Family, by MAD Phasing at 1.9 A Resolution,” Acta Crystallographica Section D-Biological Crystallography, Vol. 56, No. 5, 2000, pp. 559-566. doi:10.1107/S0907444900002833
[38] H. Itou, M. Yao, I. Fujita, N. Watanabe, M. Suzuki, J. Nishihira, et al., “The Crystal Structure of Human MRP14 (S100A9), a Ca(2+)-Dependent Regulator Protein in In- flammatory Process,” Journal of Molecular Biology, Vol. 316, No. 2, 2002, pp. 265-276.
[39] O. V. Moroz, A. A. Antson, G. G. Dodson, K. S. Wilson, I. Skibshoj, E. M. Lukanidin, et al., “Crystallization and Preliminary X-Ray Diffraction Analysis of Human Calcium- Binding Protein S100A12,” Acta Crystallographica Section D-Biological Crystallography, Vol. 56, No. 2, 2000, pp. 189-191. doi:10.1107/S0907444999014936
[40] O. V. Moroz, A. A. Antson, S. J. Grist, N. J. Maitland, G. G. Dodson, K. S. Wilson, et al., “Structure of the Human S100A12-Copper Complex: Implications for Host-Parasite Defence,” Acta Crystallographica Section D-Biological Crystallography, Vol. 59, No. 5, 2003, pp. 859-867.
[41] J. Goyette, W. X. Yan, E. Yamen, Y. M. Chung, S. Y. Lim, K. Hsu, et al., “Pleiotropic Roles of S100A12 in Coronary Atherosclerotic Plaque Formation and Rupture,” Journal of Immunology, Vol. 183, No. 1, 2009, pp. 593- 603. doi:10.4049/jimmunol.0900373
[42] N. Leukert, T. Vogl, K. Strupat, R. Reichelt, C. Sorg and J. Roth, “Calcium-Dependent Tetramer Formation of S100A8 and S100A9 is Essential for Biological Activity,” Journal of Molecular Biology, Vol. 359, No. 4, 2006, pp. 961-972. doi:10.1016/j.jmb.2006.04.009
[43] S. M. Valenzuela, M. Berkahn, D. K. Martin, T. Huynh, Z. Yang and C. L. Geczy, “Elucidating the Structure and Function of S100 Proteins in Membranes,” Proceedings of SPIE, Vol. 6036, No. 19, 2006, pp. 1-9.
[44] S. Berthier, M. H. Paclet, S. Lerouge, F. Roux, S. Vergnaud, A. W. Coleman, et al., “Changing the Confor- mation State of Cytochrome b(558) Initiates NADPH Oxidase Activation-MRP8/MRP14 Regulation,” Journal of Biological Chemistry, Vol. 278, No. 28, 2003, pp. 25499-25508.
[45] C. A. Harrison, M. J. Raftery, J. Walsh, P. Alewood, S. E. Iismaa, S. Thliveris, et al., “Oxidation Regulates the Inflammatory Properties of the Murine S100 Protein S100A8,” Journal of Biological Chemistry, Vol. 274, No. 13, 1999, pp. 8561-8569. doi:10.1074/jbc.274.13.8561
[46] M. Raftery, L. Collinson and C. Geczy, “Over Expression, Oxidative Refolding, and Zinc Binding of Recombinant Forms of the Murine S100 Protein MRP14 (S100A9),” Protein Expression & Purification, Vol. 15, No. 2, 1999, pp. 228-235. doi:10.1006/prep.1998.1015
[47] O. V. Moroz, A. A. Antson, G. N. Murshudov, N. J. Maitland, G. G. Dodson, K. S. Wilson, et al., “The Three- Dimensional Structure of Human S100A12,” Acta Crystallographica Section D-Biological Crystallography, Vol. 57, 2001, pp. 20-29.
[48] O. V. Moroz, A. A. Antson, E. J. Dodson, H. J. Burrell, S. J. Grist, R. M. Lloyd, et al., “The Structure of S100A12 in a Hexameric Form and Its Proposed Role in Receptor Signalling,” Acta Crystallographica Section D-Biological Crystallography, Vol. 58, No. 3, 2002, pp. 407-413. doi:10.1107/S0907444901021278

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