| Proteins are an indispensable class of molecules involved in the whole life process. The execution of protein biological functions is closely related to the particularly native structure which requires correct folding. Proteins’correct folding into unique native structures enables them to fully perform their particular biological functions. However, some conditions can cause protein misfolding which adversely affects proper functioning and can result in a loss of functions. Thus, protein folding into the native state is one of the central problems in molecular and cell biology. Dimerized Protein folding process is one of the problems which have attracted the attention of scientists all along. By using Forster resonance energy transfer (FRET) method, we monitored the distance of protein monomers changing with respect to time, and compared folding rate constants of the protein to further obtain information about cooperability of protein dimerization and folding. We also studied the protein unfolding behavior adsorbed onto nanoparticle surface. Due to its properties such as charged, hydrophobic and big curvature, nanoparticle surfaces can cause protein unfolding, which is a protential pitfall in medicinal applications. Hence, understanding the protein unfolding process on nano surfaces can help scientists to optimize the nanoparticle design. From the two aspects above, we chose β-lactoglobulin as the model protein to study the dimerization kinetics. In addition, we used GB1and Human Serum Albumin to study the interation between nanoparticles and the unfolding process induced by absorption, and tried to find the physical mechanism of these processes.The main contents of my thesis are as follow:We introduced the background information of protein, protein folding and protein aggregation in Chapter1, especially the importance of protein dimerization in biological system. The self-association of proteins to form dimers and higher-order oligomers is a very common phenomenon. Recent structural and biophysical studies show that protein dimerization or oligomerization is a key factor in the regulation of proteins. Specific protein dimerization is integral to biological function, structure and control. It is essential to help people understanding cell even life further. In addition, we introduced the experimental methods used in these studies:stopped-flow technique and Forster resonance energy transfer (FRET) technique. Stopped-flow is an early used technique studying protein folding kinetics, which helped people understanding the protein folding/unfolding pathways. FRET technique provides a facilitated way to study the details of life activities at the molecular level. FRET is related to the distance between the fluorophores which can provide structural information of different domains of biomacromolecules, so that scientists are able to monitor conformational changes during the motion or regulation of molecules using optical methods. Moreover, by using recently developed single-molecule FRET techniques, we can understand more deeply about the relationship between structures and functions of biomolecules.In Chapter2, we studied the dimerization kinetics of β-lactoglobulin (p-1g) using stopped-flow techniques combined with FRET method. The stability and dimeric state of β-lactoglobulin can be dramatically affected by labeling thiophilic agent to Cys121, but the underlining mechanism of such effect is still unclear. In this study, we labeled a FRET pair of fluorescence donor (1,5-IAEDANS) and acceptor (5-IAF) dyes to Cys121of β-lg monomers to investigate the effect of bulky thiophilic modification on the structure and stability of β-1g. It is found that the modification dramatically destroys the native structure of β-1g and results in an obvious increase of its α-helical content, coincident with the accumulation of non-native α-helical intermediates during its folding process. Importantly, the dimeric state of β-lg can still be reached whereas its dimerization rate decreases dramatically, allowing us to characterize the dimerization process using a FRET method based on a stopped-flow apparatus. Our results reveal that the dimerization process occurs before the completely folding of individual monomers, providing direct evidence on the cooperativity of folding andbinding processes.In Chapter3we studied the binding interaction of protein and nanoparticles. Owing to their many outstanding features, such as small size, large surface area, and cell penetration ability, nanoparticles have been increasingly used in medicine and biomaterials as drug carriers and diagnostic or therapeutic agents. However, our understandings of the interactions of biological entities, especially proteins, with nanoparticles are far behind the explosive development of nanotechnology. In typical protein-nanoparticle interactions, two processes, i.e. surface binding and con-formational changes of proteins, are intermingled with each other and have not been quantitatively described yet. Here, by using a stopped-flow fast mixing technique, we were able to shed light onto the kinetics of the adsorption induced protein unfolding on nanoparticle surfaces in detail. We observed a biphasic denaturation behavior of protein GB1on latex nanoparticle surfaces. Such kinetics can be adequately described by a fast equilibrium adsorption followed by a slow reversible unfolding of GB1. Based on this model, we quantitatively measured all rate constants that are involved in this process, from which the free energy profile is constructed. This allows us to evaluate the effects of environmental factors, such as pH and ionic strength, on both the adsorption and the conformational change of GB1on latex nanoparticle surface. These studies provide a general physical picture of the adsorption induced unfolding of proteins on nanoparticle surfaces and a quantitative description of the energetics of each transition. We anticipate that it will greatly advance our current understanding on protein-nanoparticle interactions and will be helpful for rational control of such interactions in biomedical applications.Based on the results from Chapter3, we further researched the size effects on the protein binding and unfolding behavior on nanoparticle surfaces in Chapter4. Taking Human Serum Albumin (HSA) as model protein, we found that bigger nanoparticle size caused more secondary structure disturbance. This can also be confirmed by the kinetic experiments, in which the protein unfolding rate is faster when adsorbed onto bigger nanoparticles. HSA is a massive globular protein and is a predominantly alpha-helix protein, it is possible that the interaction interface of protein and the small size nanoparticle surface is not strong enough to hold the opened helix. Thus, though the adsorption rate is fast, damages to the secondary structure were insignificant.In Chapter5, we observed and studied the conformational changes of DNA Holliday junction with or without Mg2+in buffers by using single-molecule FRET technique. Using the new designed junction sequence, we found that the conformational propensity altered comparing to former reported results. The newly designed junction is biased to a high energy transfer efficiency conformation, unlike the near-equal populations in each conformer of the original junction. It is worthy noting that the high transfer efficiency of the new junction is slightly lower than the original one, indicating that the distance between the fluorophore-labeled junction arms was further than the original one. This could be due to the enhanced flexibility of the new junction, which would provide more alternatives for conformation. Understanding Holliday junction conformational transition dynamics may help us reinforce a new junction sequence designs to advance our enzyme digestion kinetics. |
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