【作者】 王立旭；

【导师】 师同顺； 吴玉清；

【作者基本信息】 吉林大学 ， 无机化学， 2009， 博士

【摘要】 研究蛋白质去折叠路径及其在外扰下的构象变化,对揭示其折叠途径和机理至关重要。其中,如何区分蛋白质的折叠/去折叠途径是完全的协同过程、还是包含早期事件的非协同过程一直是蛋白质科学研究中重要的科学难题。红外吸收光谱是分析溶液中蛋白质二级结构含量及其变化的有力手段,而二维相关红外分析不但对相互重叠的蛋白质振动峰具有很强的分辨力,而且对于微弱的结构变化也具有独特的敏感性,它的提出更为利用红外吸收光谱揭示溶液中蛋白质的构象变化提供了广阔的前景。本论文中,我们分别选择热去折叠机制尚存争议的模型蛋白——核糖核酸酶A(RNase A)和压力去折叠机制仍有争议的多功能蛋白——泛素(ubiquitin)为研究对象,对其在温度、压力诱导下的预相变过程存在与否以及相应过程中的二级结构变化及顺序等进行了详尽的研究。主要研究内容及结论如下:(1)首次利用样本-样本二维相关分析和化学计量学中的主成分分析法(PCA)观测到变温过程中位于45 oC的RNase A的预相变过程,该信息由传统的单频红外谱峰分析无法揭示;同时还利用波数-波数二维相关分析对预相变过程所包含的构象微弱变化进行了详细分析,揭示出热诱导RNase A的预相变过程主要涉及α2和β1的结构变化。这些结果显示了二维相关光谱与PCA相结合方法在揭示预相变过程中二级结构微弱变化方面的独特优势。(2)我们还对比研究了还原剂存在条件下RNase A的热诱导去折叠过程,结果表明该过程也存在早期事件;还原剂的参与使得预相变过程发生的温度提前,同步相关光谱切谱的定量比较表明早期事件所包含的结构变化相对于主相变过程极其微弱。(3)本研究还利用二维相关红外光谱和主成分分析相结合揭示了压力扰动下ubiquitin去折叠过程中早期的微弱二级结构变化,结果表明压力诱导下的早期事件及相转变行为与压力诱导的水分子的结构变化及蛋白质水合密切相关。更多还原

【Abstract】 Over the past decades, much effort has been devoted to investigating the mechanism of protein folding and the main interactions that govern this process. Exploring the folding problem and the structure-function relationship will enable the modification and design of novel proteins or peptide mimetics, which can play crucial roles in bio- and nanotechnology. The protein structural transitions that occur in the pathway are at the heart of the specific conformational changes that form the individual intermediates involved in the rate-limited pathways that can distinguish one pathway from another.The analysis of protein unfolding pathways and the identification of specific conformational changes under perturbation are of fundamental importance for protein folding. Much effort has been devoted in recent years to elucidate whether the protein’s folding/unfolding is a fully cooperative process or whether it contains sequential events. To address such question, one needs the combined use of variable analytical tools. In this study, sample-sample two dimensional correlation spectroscopy (SS 2D), principal component analysis (PCA) in combination with variable-variable two-dimensional correlation spectroscopy (VV 2D) have provided answers to this question.Principal component analysis (PCA) is a well-established technique in statistics and chemometrics that gives a precise mathematical estimation of changes along the sample and variable vectors. This method is a well-known pattern recognition and multivariate data display method. Not only can it compare any object clusters, but it can also display any relationships among variables as well as among variables and objects. In sample-sample (SS) 2D correlation, the relationship between different samples observed under various states of perturbation is studied by examining the similarity or difference of their spectral trace patterns along the spectral variable. By use of SS 2D correlation methodology, one can follow the variable profiles directly. In variable-variable (VV) 2D correlation spectroscopy, it enables one to establish the correlation between different secondary structures of the protein through selective correlation peaks for a given perturbation. This provides detailed information on the process under investigation, including the sequence of events and the possibility of band assignments. Fourier transform infrared spectroscopy (FTIR) is a useful technique to investigate the secondary structure of proteins. 2D IR correlation spectroscopy is very powerful to unravel the highly complicated infrared bands of proteins since the conventional IR fail to tell the difference immediately for the minor structural variations.On the basis of the above, our studies will be focused on the following fields.1. Thermal kinetics study of ribonuclease A (RNase A) in solution by two-dimensional correlation infrared spectroscopyThermal unfolding of RNase A in Tris-DCl buffer solution is studied by Fourier transform infrared (FT-IR) spectroscopy. SS 2D correlation spectroscopy in combination with PCA are applied to these spectral data to reveal the thermal kinetics of RNase A. Using SS 2D spectroscopy subtle pretransition could be detected at 45 oC in addition to the main unfolding transition at 66 oC, which indicates that the thermal unfolding of RNase A does not proceed via a two-state mechanism, involving an intermediate. The second scores plot of PCA constructed from temperature-dependent IR spectra also certified a minor pretransition at 46 oC as well as a clear main transition at 66 oC. But the loadings plots of PCA suggest only the general structural variations because the second loadings plot has coupled the contribution both of pretransition and main transition conformational changes. Advantages of 2D correlation over PCA, including the identification of secondary structure and sequential order of conformational changes, were also discussed.Specific temperature regions identified by SS 2D were selected to construct VV 2D to monitor the conformational changes of RNase A during the observed transitions in detail. In the pretransition process, the observed structural variations are associated with local conformational changes ofα2 and the modification ofβ1; in the main unfolding, changes in irregular andα-helical structures are followed by those in theβ-sheet structure including the antiparallelβ-sheet components, resulting in the loss of secondary structure. Moreover, the gain ofβ-turn and a small amount of anti-parallelβ-strands reveal that the pretransition may involve changes in hydration and protein aggregations.Therefore, the present study demonstrates the great potential of SS 2D in revealing subtle phase transition of proteins in aqueous solutions. This pretransition cannot be detected by a single-frequency analysis of the original infrared spectra of RNase A. However, VV 2D correlation spectra reveal the variations in the structure of RNase A and their orders of variations that occur during these transitions.2. Thermally induced early events of RNase A under reducing conditions: Evidenced by principal component analysis and two-dimensional correlation infrared spectroscopyIn this chapter, thermally induced early unfolding events of ribonuclease A (RNase A) in the presence of 2-mercaptoethanol are evidenced successfully by PCA and two-dimensional correlation infrared (2D IR) spectroscopy. The analyses of secondary structural changes in different stages have clearly distinguished the early events from the main unfolding in the temperature course of RNase A.With fluorescence and infrared spectroscopy, we have demonstrated that the mechanism of the thermally induced reductive unfolding pathway and the related structural variations. Especially, the combined use of PCA and 2D IR correlation spectroscopy clarified that the early events of RNase A under reducing conditions are in a non-cooperative process, which in contrast to the unfolding under non-reducing conditions where a pretransitional temperature was revealed at 46 oC. 2D IR correlation analysis also revealed that in the early stage of the reductant thermal unfolding the subtle structural variations of the orderedβ-sheet in RNase A dominated; identical conformational changes occured at lower temperature for reductive RNase A as those under non-reducing conditions. It is reasonable that in stage I the change ofβ1 andα2 might be the main process. In stage II,β1 was further unfolded, changes ofα1 and another species ofβ2 andβ4 involved, that was, theβ-sheet was further unfolded as temperature increased.In general, the addition of the reductant to the solution of RNase A apparently induced a lower transition temperature of the pretransition. The quantitative analysis of the power spectra reveals that the early structural changes for reductive RNase A were definitely minor ones in relative to those in main unfolding. This study also revealed the usefulness of generalized 2D correlation spectroscopy and the quantitative utilization of its power spectrum to explore minor non-cooperative early events and the closely related subtle structural changes.3. High-pressure induced pretransition of ubiquitinPressure as an experimental variable provides unique information about the microscopic properties of the materials being studied, which is different from the traditional external perturbation variable (such as temperature). It is known that the measurements of temperature and/or pressure-induced process are extremely useful strategies. The non-covalent inter-molecular interaction of biological molecules is easy to be changed by pressure. Therefore, the coupling of high pressure, as an external perturbation, with 2D-IR and applying it to protein research has proven to be an insightful method for the investigation of proteins, although few concerning papers have been published so far. However, the requirement of a more sophisticated diamond anvil cell (DAC) for IR measurements makes it somewhat difficulty to employ pressure as a routine source of spectral intensity changes.In this chapter, the methodology will be applied to more meaningful and complicated protein, ubiquitin. By using SS 2D correlation spectroscopy and PCA,in addition to the main unfolding transition at around 689.9 MPa, subtle structural 7 transition could be detected at 392.2 MPa. This indicates that the pressure-induced unfolding of ubiquitin does not proceed via a two-state mechanism. Specific pressure regions identified by PCA were selected to construct VV 2D in order to monitor the conformational changes of ubiquitin during the observed transitions in detail. In the low pressure region, the structural variations are associated withα-helix, side chain and minor aggregation; in the main transition, disordered structure,α-helix and side chain proceeded beforeβ-sheet. The quantitative comparison of power spectra demonstrate that the intensity variations from early pressure region accounts for around 2.5 % that of the main pressure unfolding. Meanwhile, with the pressure increasing, the integration area of water peak and amide I has similar trends and it demonstrated that the early structural variations was associated with the water behavior and protein hydration. The present results suggest that the pressure-induced unfolding of ubiquitin was not a complete two state process or only because of water behavior. Further exploration of the pressure stability of ubiquitin is currently being investigated in our laboratory.更多还原