The Mechanism of Folding of Dihydrofolate Reductase

本研究的总体目标是了解蛋白质的氨基酸序列如何将其快速有效地折叠成其天然构象的机制。将采用生物物理和基因工程相结合的方法来探索来自常见细菌大肠杆菌和人类的同源二氢叶酸还原酶的折叠机制。圆二色性、核磁共振、荧光和x射线散射研究将提供对二级和三级结构的深入了解，以及先前观察到的两种蛋白质折叠过程中出现的瞬态、部分折叠形式的动力学和大小。超快混合将在100微秒内探测未展开形式的坍塌和非极性表面的发展。突变分析将探讨非极性侧链在几种疏水簇中的参与，这些疏水簇似乎在两类折叠中间体中起关键作用。蛋白质工程将测试片段折叠的潜力。最后，细菌伴侣GroEL在影响人二氢叶酸还原酶折叠中的作用将被研究。在了解折叠过程中出现的部分折叠物种的结构和动力学方面取得的进展，将有助于深入了解蛋白质的氨基酸序列自发地指导其独特三维结构形成的过程。蛋白质折叠问题的解决将对生物学、生物化学和生物技术工业产生重要影响。细胞外蛋白质的折叠和运输通常与它们自身的合成或与它们形成复合物的其他蛋白质的合成相协调。因此，折叠在细胞的正常功能中起着关键作用。由于蛋白质的生物活性与其三维结构直接相关，因此了解序列/结构关系是处理存储在DNA序列中的遗传信息的关键组成部分。

9604678 Matthews The overall goal of this research is to understand the mechanism by which the amino acid sequence of a protein directs its rapid and efficient folding to its native conformation. A combination of biophysical and genetic engineering methods will be used to probe the mechanism of folding of homologous dihydrofolate reductases from a common bacterium, E. coli, and from humans. Circular dichroism, NMR, fluorescence and x-ray scattering studies will provide insight into the secondary and tertiary structures, as well as the dynamics and sizes of transient, partially-folded forms that have previously been observed to appear during the folding of both proteins. Ultrafast mixing will probe the collapse of unfolded forms and the development of nonpolar surfaces within 100 microseconds. Mutational analysis will probe the involvement of nonpolar side chains in several hydrophobic clusters that appear to play key roles in two classes of folding intermediates. Protein engineering will test the potential of fragments to fold. Finally, the role of a bacterial chaperone, GroEL, in influencing the folding of human dihydrofolate reductase will be investigated. Progress in understanding the structures and dynamics of partially folded species that appear during folding should provide insight into the process by which the amino acid sequence of a protein spontaneously directs the formation of its unique three dimensional structure. A solution to the protein folding problem would have an important effect on biology, biochemistry and the biotechnology industry. The folding and transport of extracellular proteins is often coordinated with their own synthesis or with the synthesis of other proteins with which they form complexes. Thus, folding plays a key role in the normal functioning of cells. Because the biological activities of proteins are directly related to their three dimensional structures, an understanding of the sequence/structure relationship is a critical component of the processing of the genetic information stored in the DNA sequence.