CP12 is a small chloroplast protein involved in the Benson-Calvin cycle. Since it was demonstrated that the CP12 protein shared different conformational properties between reduced and oxidized states we took advantage of the segregational properties of the Traveling Wave Ion Mobility (TWIM) guide to study subtle conformational changes related to redox changes.


Electrospray ionization mass (ESI-MS) spectra of the CP12 protein were recorded in the positive ion mode using an ESI source fitted on a quadrupole time-of-flight (QToF) hybrid mass spectrometer equipped with a TWIM cell (Synapt HDMS G1, Waters Corp., Manchester) under non-denaturing conditions. Non-covalent experiments were performed using the same instrument without the use of the TWIM device.


Whatever the CP12 form studied, our results showed that CP12 protein was represented by two conformers in equilibrium that displayed very slight differences. These observations led us to propose that CP12 protein structure is rather undergoing transient subtle structural changes than having two different conformational populations in solution. In addition, using non-denaturing experiments, NAD and CP12 stoichiometry were determined with respect to the GAPDH tetramer and the redox state of CP12.


In this study we showed that the use of the segregational property of the ion mobility (TWIM, Synapt G1 HDMS, Waters, Manchester, UK) allowed differentiation of subtle conformational changes between redox states of the CP12 protein. Standard non-denaturing experiments revealed different binding stoichiometry according to the redox state of the CP12 protein. Copyright © 2012 John Wiley & Sons, Ltd.