Thermal unfolding of calreticulin studied by DCS and FTIR

DECCA M.B.; DURAND, S.; HALLAK, M. H.; MONTICH, G

Tucuman

Congreso; SAB 2012: XLI Reunion Anual Sociedad Argentina de Biofisica; 2012

Calreticulin (CRT) is a multifunctional Ca2+-binding protein involved in a variety of cellular processes. In spite of been a multidomain protein with non-globular shape, and high conformational flexibility, it is widely accepted that CRT unfolds in a two-state transition. Here we revisited the thermal unfolding of CRT by differential scanning calorimetry (DSC) and FTIR spectroscopy. We observed that the heat capacity profile of CRT is consistent with a highly dynamic protein, with a complex thermotropic behavior. Clearly, the unfolding process is not a simple two state transition, and at least three stages can be identified. (i) In the low temperature region (10-30°C) the heat capacity increases in function of temperature from the very beginning of heating with a large slope, characteristic of local disruptions in labile protein structures. No cooperative transitions in secondary structure were observed within this low temperature region. The amount of â-sheet determined by FTIR spectroscopy structure is in agreement with the number of residues involved in â chains in the partially solved structure of CRT domais, but the amount of á helix is higher than expected, suggesting that unsolved regions could adopt fluctuating helix conformations. (ii) Between 30 and 45 °C the heat capacity profile shows a low cooperative low enthalpy transition which is coupled with a decrease of â structure. (iii) The main unfolding transition is centered at 49 °C with a complete loss of secondary structure as revealed by FTIR. CRT unfolding is reversible up to 55°C; and after that a subtle aggregation process is detected. The thermotropic behavior above described corresponds to a protein with the high affinity calcium binding site fully occupied. Heat capacity profiles of the apo CRT show a broader main unfolding transition which is shifted to lower temperature and overlaps with the pre-transition. Within the low temperature region the heat capacity profile is essentially the same as that of the holo protein. +-binding protein involved in a variety of cellular processes. In spite of been a multidomain protein with non-globular shape, and high conformational flexibility, it is widely accepted that CRT unfolds in a two-state transition. Here we revisited the thermal unfolding of CRT by differential scanning calorimetry (DSC) and FTIR spectroscopy. We observed that the heat capacity profile of CRT is consistent with a highly dynamic protein, with a complex thermotropic behavior. Clearly, the unfolding process is not a simple two state transition, and at least three stages can be identified. (i) In the low temperature region (10-30°C) the heat capacity increases in function of temperature from the very beginning of heating with a large slope, characteristic of local disruptions in labile protein structures. No cooperative transitions in secondary structure were observed within this low temperature region. The amount of â-sheet determined by FTIR spectroscopy structure is in agreement with the number of residues involved in â chains in the partially solved structure of CRT domais, but the amount of á helix is higher than expected, suggesting that unsolved regions could adopt fluctuating helix conformations. (ii) Between 30 and 45 °C the heat capacity profile shows a low cooperative low enthalpy transition which is coupled with a decrease of â structure. (iii) The main unfolding transition is centered at 49 °C with a complete loss of secondary structure as revealed by FTIR. CRT unfolding is reversible up to 55°C; and after that a subtle aggregation process is detected. The thermotropic behavior above described corresponds to a protein with the high affinity calcium binding site fully occupied. Heat capacity profiles of the apo CRT show a broader main unfolding transition which is shifted to lower temperature and overlaps with the pre-transition. Within the low temperature region the heat capacity profile is essentially the same as that of the holo protein. This work was supported by grants from the Agencia Nacional de Promocion Cientifica y Tecnologica,  CONICETand  SECyT-Universidad Nacional de Cordoba