Pressure effects on the structure, kinetic, and thermodynamic properties of heat-induced aggregation of protein studied by FT-IR spectroscopy

Pressure can retrain the heat-induced aggregation and dissociate the heat-induced aggregates. We observed the aggregation-preventing pressure effect and the aggregatesdissociating pressure effect to characterize the heat-induced aggregation of equine serum albumin (ESA) by FT-IR spectroscopy. The results suggest the -helical structure collapses at the beginning of heat-induced aggregation through the swollen structure, and then the rearrangement of structure to the intermolecular -sheet takes place through partially unfolded structure. We determined the activation volume for the heat-induced aggregation (ΔV= +93 ml/mol) and the partial molar volume difference between native state and heat-induced aggregates (ΔV=+32 ml/mol). This positive partial molar volume difference suggests that the heat-induced aggregates have larger internal voids than the native structure. Moreover, the positive volume change implies that the formation of the intermolecular -sheet is unfavorable under high pressure. 1.Introduction Pressure can induce not only the formation of the protein aggregation, but also prevent protein from forming the heat-induced aggregates and dissociate the protein aggregates [1]. The use of the aggregation-preventing pressure effect allows us to investigate the kinetics of the formation of intermolecular -sheet structure more easily because the pressure reduces the reaction rate. Furthermore, the activation volume obtained from the kinetic analysis can also characterize the heatinduced aggregation. Using aggregates-dissociating pressure effect, we can assume that the formation of aggregation is quasi-reversible and the thermodynamic approach of the formation of aggregates is successful [2]. Recently, several results of pressure experiments on the protein aggregation implied that the aggregates have a large number of voids in the interior [3]. This consequence is in agreement with the thermodynamics principle that the pressure shifts the equilibrium to conformational states occupying smaller partial molar volumes. However, there have been no quantitative reports providing volume changes for protein aggregation. The structure of the protein aggregates is proposed to be predominantly dependent on the hydrogen bond network in the extended intermolecular -sheet architecture of the peptide backbones [4]. Therefore, studies from the secondary structural viewpoint are important to clarify the mechanism of Joint AIRAPT-22 & HPCJ-50 IOP Publishing Journal of Physics: Conference Series 215 (2010) 012156 doi:10.1088/1742-6596/215/1/012156 c © 2010 IOP Publishing Ltd 1 the protein aggregation. FT-IR spectroscopy is a powerful method for analyzing the secondary structure of proteins. The infrared amide I/I mode consists of the C=O stretching, C-N stretching, and C-C-N deformation modes of the peptide units, and appears in the region from 1620 to 1690 cm -1 [5]. This mode is diagnostically useful to examine the -sheet structure, which is one of the major structures of aggregates. Thus, we use FT-IR spectroscopy to elucidate the protein aggregation mechanism. 2. Secondary structure Figure 1-A shows typical FT-IR spectra in the amide I region of ESA solution in native state, pressure-, and heat-induced aggregated states. These aggregated states were checked by means of the optical microscopic observation in the diamond anvil cell under FT-IR spectroscopic measurement. The amide I mode consists of the C=O stretching, C-N stretching, and C-C-N deformation modes, and appears in the region from 1620 to 1690 cm -1 . This mode has been known to be highly sensitive to the secondary structure of polypeptides and proteins, so that it has served as an indicator of -helix and/or -sheet conformations. The dominant peak at around 1650cm -1 of the native state is characteristic for the helical secondary structure, which is the major structural component of ESA. The broadening of amide I and peak shift to around 1645-1640 cm -1 , which is discovered for pressure and heat induced aggregated sample, indicate an increase in population of disordered structure. The specific bands for intermolecular -sheet structure [6] are clearly observed at 1614 and 1685 cm -1 in the case of heataggregated state. Figures 1-B and 1-C show that the peak position of the dominant amide I band is plotted as functions of the pressure and temperature, respectively. The pressureand heat-induced aggregates separated from the ESA solution occur above 420 MPa and 45 C, respectively. In the pressure experiment (Figure 1-B), the peak position of the amide I band shifts from 1649 cm -1 at 0.1 MPa to 1642 cm -1 at 960 MPa, which means that the disordered structure becomes dominant component. The pressure-induced aggregation occurs at a pressure where the pressure induced peak shift exceeds more than half of total shift. Moreover, it is interesting that the pressure-induced aggregation process of ESA is quite reversible. In Figure 1-C, the peak position of amide I band slightly shifts to higher frequency with increasing temperature up to 45 C and sharply shifts to lower frequency with increasing temperature up to 70 C. The heat-induced aggregation occurs at 45 C. This suggests that a decrease of the -helical component induces cooperatively the formation of the heat-induced aggregates. Figure. 1. (A) FT-IR spectra of the amide I region of ESA in aqueous solution with 0.1 M NaCl (pD 4.4) at several conditions. Solid line; 0.1 MPa, 25 C (Native state), Broken line; 960 MPa, 25 C (Pressure-induced aggregated state), Dash dot line; 0.1 MPa, 90 C (heat-induced aggregated state). (B) The maximum position of the amide I band vs pressure for ESA. (C) The maximum position of the amide I band vs temperature for ESA. Solid and open circles indicate the pressure or temperature increase and decrease, respectively. The vertical broken line marks at beginning of the phase separation of ESA aggregates from solution induced by pressure and heat, respectively Joint AIRAPT-22 & HPCJ-50 IOP Publishing Journal of Physics: Conference Series 215 (2010) 012156 doi:10.1088/1742-6596/215/1/012156