Using a Viscotek Triple Detector Array (TDA302) system coupled to a GPCmax chromatographic system, the team characterised the structural and hydrodynamic properties of a fragment of the adenylate cyclase (CyaA) toxin, a major virulence factor of Bordetella pertussis, the causative bacteria of whooping cough. The research team demonstrated that calcium binding induces important hydrodynamic changes in the protein, gaining important insights into its biological function. Results were published in the Journal of Biological Chemistry (Jan 2009; 284: 1781 – 1789).
Conventional calibration of size exclusion chromatography (SEC) is based on known hydrodynamic volume of standard proteins. Such a calibration procedure suffers from the drawback of possible interactions between the protein of interest and the SEC matrix. Hence, conventional calibration can provide neither the molecular mass nor reliable hydrodynamic information.
The Viscotek TDA302 uses a series of detectors to analyse the eluting sample, including: a UV-Visible spectrophotometer, a differential refractometer (RI), a 7° Low Angle Light Scattering (LALS), a 90° Right Angle Light Scattering (RALS) detector and a differential pressure (DP) Wheatstone bridge viscometer. OmniSEC software analyses all the collected data, presenting it in an information-rich format. The UV-detector and the RI are used to measure protein concentration, which is required to determine both the absolute molecular mass (MM) and the intrinsic viscosity (IV). MM is calculated directly from the light scattering data and IV from the viscometer. The IV results give insight into protein hydration and shape. In this application, IV is the only hydrodynamic parameter significantly affected by calcium binding. The information that the system supplies is therefore essential towards understanding protein binding and folding behaviours which can be key towards understanding how these molecules interact with the human body.
About the Institut Pasteur
The Institut Pasteur is one of the world's leading biomedical research centres. Founded by Louis Pasteur in 1887, it is a private, non-profit foundation dedicated to the prevention and treatment of diseases through biological research, education and public-health activities. Close to 2,600 people work on its campus in Paris, where a large part of its research activities are devoted to infectious diseases. The Paris institute is the heart of an International network bringing together 30 institutes across all five continents. Over a period of 120 years, 10 Institut Pasteur researchers have received the Nobel Prize, the latest of these in 2008, when it was awarded to Professors Françoise Barré-Sinoussi and Luc Montagnier for the discovery of the AIDS virus.
www.pasteur.fr
