Introduction
The ability of small, highly charged cations to induce protein structural changes following binding is a fundamental process behind many biological signaling pathways. Whilst the signaling cascades controlled by cation binding are very diverse, individual cation-binding motifs are often conserved between different proteins. One such cation binding motif is the "Repeat in Toxin" (RTX) motif present on many virulence factors that is secreted by pathogenic microbes. RTX sequences are glycine- and aspartaterich domains that constitute a specific type of Ca2+ binding site that is essential for the function of such secreted toxins, including the pore-forming cytolytic toxin, adenylate cyclase toxin (CyaA). Indeed, CyaA, from Bordetella pertussis, the causative agent of whooping cough, has been shown to bind calcium in solution. The calcium dependence of these secreted toxins has been proposed to facilitate their secretion into the extracellular medium, as newly synthesized peptides would need to pass through the narrow channels of the secretion machinery. With the low calcium concentration inside the bacterial cytosol, it has been suggested that RTX motifs would form an unfolded conformation, favorable for secretion. However, once in the extracellular space, the higher calcium concentration would cause the RTX domain to bind calcium ions, and form a structured, active protein. In order to better characterize the mechanism of calcium-dependent RTX function, a series of biophysical methods were employed to study changes in both conformation and size of the RTX repeat domain (RD) of CyaA toxin, following calcium binding.Materials and methods
Materials and proteins were prepared as described (1, 2, 3) Intrinsic Viscosity and Molecular Mass Measurements with SEC-TD - Size exclusion chromatography (SEC) experiments were performed using a Superdex 200 column (GE Healthcare) controlled by a GPCmax module and connected on-line to a triple detector array (TDA) model 302 (Malvern, UK). TDA contained (i) a static light scattering cell with two photodiode detectors, at 7° for low angle (LALS) and at 90° for right angle laser light scattering (RALS), (ii) a differential refractive index detector, (iii) a photometer, and (iv) a differential viscometer. Protein concentration was determined using both the photometer and the refractive index detector. The RALS and LALS data, in combination with the concentration, provided the molecular mass M. Intrinsic viscosity (η) was calculated using the differential viscometer. Both molecular mass and intrinsic viscosity were calculated with the OmniSEC software. Zeta Potential - zeta potential and electrophoretic mobility experiments were performed using a Zetasizer Nano ZS instrument (Malvern) which monitors the forward light scattering at an angle of 17°. Buffers and samples were filtrated on 0.2 μm filters prior to acquisition. RTX polypeptide concentrations ranged between 70 to 130 μM and were diluted in 20 mM Hepes, 20 mM NaCl, pH 7.4. A dedicated cuvette DTS1070 and a high concentration microcuvette ZEN1010 were used for the acquisition of the electrophoretic mobility values using the fast field reversal mode. The quality of the instrumentation was checked using the standard DTS1235. For each sample, five to ten independent measurements (one run each) were acquired in fast field reversal mode. The quality criteria to keep a measurement were based on: (i) the zeta quality factor (signal-to-noise ratio of the frequency shift); (ii) the mean count rate that should not change throughout the duration of the data acquisition; (iii) the quality of the phase plot (radian amplitude and frequency) and the Fourier-transform of the phase plot. The acceptable electrophoretic mobility measurements were averaged and the standard deviation was computed. These data were used to generate the electrophoretic mobility distribution of each state (Apo and Holo) of RCL and RCS. Other methods used in this study, but not described here, include Circular Dichroism (CD) and Analytical Ultracentrifugation (AUC), and Nuclear Magnetic Resonance (NMR) Spectroscopy.Results
The changes in the protein conformation and biophysical properties of RD, following the binding of calcium were studied. In particular, the utilization of Size Exclusion Chromatography, coupled to a Triple Detector Array, yielded key information regarding the behavior of the peptide. These data are described below. Binding of Calcium to RD Induces Formation of Ordered Structures Data from Far-UV CD and NMR suggested that RD in the absence of calcium (Apo-RD) existed in a disordered state, with little structure, while RD bound to calcium (Holo-RD) formed distinct secondary structures (1). In order to further explore these differences, Apo- and Holo-RD was subjected to size exclusion chromatography (SEC), combined with a triple detector array (TDA). Holo-RD eluted at a retention volume (RV) of 13.8 mL, close to the expected RV for a globular protein of the appropriate size (Fig. 1A, B). However, Apo-RD eluted at a RV of 10.4 mL, which corresponds to a natively folded protein of approximately 600 kDa. However, data from right angle light scattering intensities (Fig. 1B) indicated that the molecular mass was unchanged, demonstrating the change in RV was not due to protein oligomerization. Moreover, the differential viscometer peak sizes were very different between the two states (Fig 1B), yielding intrinsic viscosity values (η) of 5.5 mL.g-1 for Holo-RD, and 35.1 mL.g-1 for Apo-RD. This unusually high value for Apo-RD explains the low RV, as RV is related to hydrodynamic volume. Supporting studies using AUC confirmed that Holo- and Apo-RD have the same molecular mass, of 73 kDa, but different sedimentation coefficients (1). These data yielded a hydrodynamic radius (RH) of 3.2 nm for Holo-RD and 6.5 nm for Apo-RD (1). Together, these data demonstrate that following binding of calcium, RD changes from a disordered peptide into a more tightly formed peptide with ordered secondary structure. >> Download the full Application Note hereMalvern provides the materials and biophysical characterization technology and expertise that enables scientists and engineers to investigate, understand and control the properties of dispersed systems. These systems range from proteins and polymers in solution, particle and nanoparticle suspensions and emulsions, through to sprays and aerosols, industrial bulk powders and high concentration slurries. Used at all stages of research, development and manufacturing, Malvern’s instruments provide critical information that helps accelerate research and product development, enhance and maintain product quality and optimize process efficiency. Our products reflect Malvern’s drive to exploit the latest technological innovations. They are used by both industry and academia, in sectors ranging from pharmaceuticals and biopharmaceuticals to bulk chemicals, cement, plastics and polymers, energy and the environment. Malvern systems are used to measure particle size, particle shape, zeta potential, protein charge, molecular weight, mass, size and conformation, rheological properties and for chemical identification, advancing the understanding of dispersed systems across many different industries and applications. www.malvern.com Material relationships http://www.malvern.com/en/
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