奈米液珠和胜太-奈米金複合物奈米系統的熱力學及動力學研究

Abstract

在目前的生物醫學研究範疇中，生物分子與奈米介面的物理特性已儼然成為 當今最重要的課題，尤其於組織工程與藥物釋放具有極大的應用價值。當奈米級 表面與蛋白質進行物理性的交互作用，所產生的影響可能具有改變蛋白質結構形 態，並導致蛋白質功能的未知變化。然而相關的主要結構性與物理性變化的研究 至今仍處早期摸索階段。 生物分子與奈米介面的交互作用有可能是專一性或非專一性的結合，而在本 實驗室先前的研究發現，免疫球蛋白具有專一性辨識奈米金粒子的特性。(Huang, Chen et al. 2006; Chen, Hung et al. 2008; Chen, Hung et al. 2010; Hung, Pan et al. 2010)我們也發現奈米金粒子對於溶菌酶的物理性作用為非專一性反 應，並影響溶菌酶的功能變化與外部構形，經由圓二色光譜(CD)分析得到，在溶 菌酶與奈米金粒子作用後的確改變了蛋白質自身的二級結構，而構形的改變程度 則取決於奈米金粒子的粒徑大小，尤其當奈米金粒徑小於2~3 奈米，其構形變化 有明顯的提升。經NMR 液相光譜分析亦顯示溶菌酶蛋白質結構會被奈米金粒所影 響，在TOXY 以及COSY 光譜分析結果中，溶菌酶與2nm 奈米金結合後在指紋區 有輕微的頻率性改變，意味著溶菌酶的蛋白質構形變換是由於與2nm 奈米金反應 後的結果。為了進一步研究奈米金粒子對溶菌酶蛋白質結構的影響，我們以 2nm、5nm 奈米金與溶菌酶共結晶的觀點作為探討，藉由結晶構形分子模型的分 析可得到主要的結構變化。然而目前的實驗結果，仍難以對奈米金誘導蛋白質產 生物理性結構變化現象作出一個完整的邏輯性規則。因此為了探討並歸類出奈米 金與蛋白質構形之間的作用機制，我們利用胜肽分子與奈米金作結合，並以CD 光譜分析後發現蛋白質螺旋結構有顯著的改變。接續的實驗模型將以探討胜肽- 奈米金複合物的摺疊結構及熱力學為主軸。 本計劃主要目的 1.分析胜肽分子與不同粒徑奈米金粒子接合後的分子構形變化。 2.研究以物理性熱力學參數改變胜肽分子與奈米金粒子的結合反應。 3.探討胜肽分子與奈米金粒子的動力學機制。

The physical property of bio-nano interface has become an important issue in the biomedical research, especially in tissue engineering and drug delivery. Nano-surface interacts with protein and somehow alters the structure and function of protein. However, fundamental study of structure and dynamics is still at the early stage. The interaction at bio-nano interface can be specific or non-specific. We have shown that specific recognition of nanoparticles can be found using immunoglobulin. We have shown that non-specific interaction of GNPs to lysozyme marginally changed the conformation and function of lysozyme. CD spectrum of GNP-lysozyme conjugate indicates that the secondary structure was changed. The degree was size-dependent. In particular, the effect is mostly enhanced when smaller GNPs of 2-nm and 3-nm in diameter are used. NMR solution structure also indicates conformational change of lysozyme. 1H-NMR TOXY and COSY spectra for lysozyme in the presence and in the absence of 2-nm GNP indicated that slight frequency change was found in the fingerprint region, which indicates conformation change of lysozyme in the presence of 2-nm GNP. To investigate the structural change of lysozyme in the presence of GNP, lysozyme os co-crystallized with 2-nm and 5-nm GNPs. Molecular modeling for the crystal structure indicates that minor change in the structure. However, it is difficult to summarize a general rule for the structural change induced by the conjugation of GNPs. To investigate the general rule underlying the non-specific interaction between GNP and protein, structural investigation to peptide-GNP conjugates is performed. CD study indicated significant enhancement of helical structure. The current model thus will be applied as model system in the investigation of structure and dynamics of bio-nano interaction. The specific aims of this proposal will be: 1. To investigate the structural change of model peptide when conjugated to GNPs with various diameters. 2. To study the thermodynamic parameters of peptide folding in the presence of conjugated GNPs. 3. To study the folding dynamics of conjugated GNPs. Taiwanese group has demonstrated sufficient ability in the biophysical research with publication in the related field.