Monitoring calcium-induced conformational changes of Calmodulin with Taylor Dispersion Analysis

Summary

Taylor Dispersion Analysis (TDA) with UV-detection as implemented in the Viscosizer instrument, can be used to determine the diffusion coefficient and to calculate the hydrodynamic radius of molecules in solution[1]. This microcapillary flow-based technique uses ultra-low sample consumption, is highly sensitive >100 Daltons, and is compatible with complex solvents or formulations, including those that contain aggregates and/or certain surfactants and excipients which can significantly interfere with light scattering measurements. The instrument baselines on the dispersant itself and uses active referencing, enabling measurement even at very low concentrations. This application note evaluates the sizing analysis of Calmodulin samples by TDA and assesses how this technology can be used to complement light scattering analysis in the protein characterization workflow.

The majority of calcium-binding proteins utilize structural domains known as EF-hands. The EF-hand motif contains two α-helices which are positioned at right angles to each other, separated by an interhelical loop, which forms a single calcium binding site. The ability of these proteins to transport calcium and bind a variety of ligands means they have an important role in many essential processes such as cell signaling, cell motility, growth, proliferation and apoptosis[2]. Their regulatory role in such processes is entirely dependent on the presence or absence of bound calcium ions on the EF-hand regions[3].

Calmodulin (CaM - an abbreviation for 'calcium-modulated protein') is a calcium-binding messenger protein with a number of isoforms, which is abundant in all eukaryotic cells. CaM has a molecular weight of approximately 17kDa, and contains two homologous globular domains with a flexible linker that connects them. Each of these domains contains two EF-hands, which will each bind one Ca²⁺ ion. Binding of calcium at all four binding sites causes the interhelical angles in the EF-hand motifs to shift, resulting in a "closed" to "open" conformational change. This change exposes hydrophobic interfaces within the protein that are able to bind and subsequently activate a large number of target protein ligands[3].

Figure 1: Images courtesy RCSB: 1QX5[4] and 1CLL[5]

Method

Calmodulin (Sigma Aldrich, Poole, UK) was prepared to a final concentration of 100µg/mL in 10mM HEPES buffer (1M HEPES stock solution (Sigma Aldrich, Poole, UK); Fresenius sterile water (Fresenius Kabi, UK) and adjusted to pH 7 with 0.25M hydrochloric acid and 0.5M sodium hydroxide). Calcium chloride (Sigma Aldrich, Poole, UK) from a 100mM stock CaCl₂ solution was added to a 1mL aliquot of the Calmodulin sample, to a final concentration of 0.1mM. Samples for analysis were sterile-syringe filtered (0.02µm (Fisher Scientific, 50-822-149)) and stored at 4ºC until analyzed using the Viscosizer.

All measurements were carried out at 25ºC using default settings, with variable settings adjusted where required. The Viscosizer was fitted with the 214 nm filter unless otherwise stated.

Results & Discussion

Calmodulin hydrodynamic radius changes according to the presence/absence of calcium chloride

Calmodulin remains in the EF-hand "closed" conformation in the absence of calcium ions, but changes to the EF-hand "open" conformation when all four binding sites are associated with a calcium ion. The estimated hydrodynamic radius (R_H) of the "closed" ca. 17kDa globular protein is 2.0nm, with the R_H of the protein's "open" linear structure estimated to be 3.23nm[6].

Figure 2: The above image shows 100µg/mL Calmodulin in the absence of CaCl₂ measured using the Viscosizer, showing UV absorbance (214nm) of the sample at the two windows of the microcapillary. Overlaid traces from two separate measurements show good agreement and repeatability. From Taylor Dispersion Analysis with two window detection, the calculated size of Calmodulin from these measurements is 1.7nm R_H.

Figure 3: The above image shows 100µg/mL Calmodulin in the presence of 0.1mM CaCl₂ measured using the Viscosizer, showing UV absorbance (214nm) of the sample at the two windows of the microcapillary. Overlaid traces from two separate measurements show average agreement, with a calculated size of 3.4nm R_H. There is a deviation from the fit noticeable in both peaks, resulting from the presence of two isoforms of the protein. A one-component model fit was used (as above, Figure 2) to ensure a robust comparison between data sets.

The size of Calmodulin was measured at 1.7nm R_H (Figure 2) in the "closed" conformation (absence of CaCl₂) of the protein (see Figure 1), which is in line with the estimated theoretical value of 2.0nm R_H [4]. The presence of 0.1mM calcium chloride caused a significant change in the measured hydrodynamic radius, from 1.7nm to 3.4nm (Figure 3), which agrees with the theoretical size for the "open" conformation of Calmodulin. The same single-component model was used to characterize the data from both data sets, to ensure a robust comparison. 

This application note demonstrates that sizing analysis using Taylor Dispersion Analysis (TDA) can be useful as an orthogonal technology to dynamic light scattering (DLS), and explores how the system may be used to monitor the conformational state of proteins in relation to interactions, formulation or stress.

References

1. Taylor Dispersion Analysis Compared to Dynamic Light Scattering for the Size Analysis of Therapeutic Peptides and Proteins and Their Aggregates. Andrea Hawe & Wendy L. Hulse & Wim Jiskoot & Robert T. Forbes Pharm Res 2011 28:2302-2310.
2. Calcium-Binding Allergens: From Plants to Man. Valenta, R, Hayek B, Seiberler, S, Bugajska-Schretter, A, Niederberger, V, Twardosz, A, Natter, S, Vangelista, L, Pastore, A, Spitzauer, S, Kraft, D. Int Arch Allergy Immunol 1998;117:160-166.
3. Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer. Park, HY, Kim, SA, Korlach, J, Rhoades, E, Kwok, LW, Zipfel, WR, Waxham, MN, Webb, WW, Pollack, L. Proceedings of the National Academy of Sciences 2008; 105: 542-547.
4. Crystal structures of apocalmodulin and an apocalmodulin/SK potassium channel gating domain complex. Schumacher, M.A., Crum, M., Miller, M.C. Structure 2004 12: 849-860 - Source RCSB PDB.
5. Calmodulin structure refined at 1.7 A resolution. Chattopadhyaya, R., Meador, W.E., Means, A.R., Quiocho, F.A. J.Mol.Biol. 1992 228: 1177-1192 - Source RCSB PDB.
6. Relationship Between Molecular Weight and Hydrodynamic Size. P Claes, M Dunford, A Kenney and P Vardy "An on-line dynamic light scattering instrument for macromolecular characterisation" in "Laser light scattering in biochemistry" S E Harding, D B Sattelle and V A Bloomfield (eds) pp 66-76, RSC (1992).