Bellavia Giuseppe, Paccou Laurent, Guinet Yannick, Hédoux Alain
UMET, UFR de Physique, BAT P5 UMR CNRS 8207, Université Lille 1 , 59655 Villeneuve d'Ascq, France.
J Phys Chem B. 2014 Jul 31;118(30):8928-34. doi: 10.1021/jp500673b. Epub 2014 Jul 16.
We present Raman investigations on lysozyme/trehalose/glycerol solutions at low water content, from room temperature up to the occurrence of the protein thermal denaturation. We studied the Amide I band and the low-frequency spectrum as a function of the glycerol content. The former allows us to monitor the protein unfolding; the latter probes the protein and solvent dynamics in anharmonic and quasi-harmonic regimes. It was shown that adding a small amount of glycerol to trehalose stiffens the dry matrix in which proteins are embedded, thus improving their stability. The analysis of the Amide I band reveals that glycerol enhances the stabilization effect of trehalose on proteins for low water content, but still liquid, systems. Data show that the protein unfolding temperature has a maximum value around 5% Glyc/TRE g/g. The overlapping low-frequency contributions, corresponding to fast anharmonic and quasi-harmonic motions, respectively, related to the mean square displacement ⟨u(2)⟩ and the vibrational density of states (VDOS) usually determined by neutron scattering experiments, have been carefully analyzed to understand the effect of glycerol. The intensity of the quasi-elastic scattering (QES) peak reveals a dynamical-like transition at high temperatures, close to the denaturation temperature. This one, as well as the low-frequency vibrational modes, reflects the same enhanced trend of the Amide I band with respect to the glycerol concentration, but at lower temperatures. A linear correlation is found among the transition temperatures of both the dynamical-like transition and the low-frequency modes, as well as the temperature dependent change of the Amide I frequency. This confirms the solvent dynamics as a necessary precursor to promote protein unfolding. Glycerol anti-plasticizes the matrix with respect to the trehalose by enhancing the stability of the protein in a more rigid trehalose/water/glycerol matrix. As expected from the analysis of the Amide I band, the maximum effect of glycerol on trehalose is determined for 5% Glyc/TRE content.
我们展示了对低含水量的溶菌酶/海藻糖/甘油溶液进行的拉曼研究，研究温度范围从室温直至蛋白质发生热变性。我们研究了酰胺I带以及低频光谱随甘油含量的变化。前者使我们能够监测蛋白质的展开；后者则探测了蛋白质和溶剂在非谐和准谐区域的动力学。结果表明，向海藻糖中添加少量甘油会使蛋白质所嵌入的干燥基质变硬，从而提高其稳定性。对酰胺I带的分析表明，对于低含水量但仍为液态的体系，甘油增强了海藻糖对蛋白质的稳定作用。数据显示，蛋白质展开温度在约5%（甘油/海藻糖，克/克）时具有最大值。分别对应于快速非谐运动和准谐运动的重叠低频贡献，与通常由中子散射实验确定的均方位移〈u(2)〉和振动状态密度（VDOS）相关，已被仔细分析以了解甘油的作用。准弹性散射（QES）峰的强度揭示了在高温下接近变性温度时类似动力学的转变。这一转变以及低频振动模式，相对于甘油浓度而言，显示出与酰胺I带相同的增强趋势，但温度较低。在类似动力学转变和低频模式的转变温度以及酰胺I频率随温度的变化之间发现了线性相关性。这证实了溶剂动力学是促进蛋白质展开的必要前提。相对于海藻糖，甘油通过在更刚性的海藻糖/水/甘油基质中增强蛋白质的稳定性来使基质抗塑化。正如从酰胺I带的分析所预期的那样，甘油对海藻糖的最大作用在甘油/海藻糖含量为5%时确定。
