Exploring the Relationships between Protein Structures and Functions

• Main Authors: HUANG, CHIH-WEI, 黃志偉
• Other Authors: LEE, HWEI-JEN
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/wmf76d

博士 === 國防醫學院 === 醫學科學研究所 === 105 === Amino acid sequences determinate protein structures. Similar structures often produce similar functions, but exceptions abound. Some residues may play their crucial role in whether proteins exhibit specific functions. In this paper, the relationships between protein structures and functions is explored through investigations for two proteins with different characteristics. δ-Crystallin is the major structural protein in avian eye lenses and is homologous to the urea cycle enzyme argininosuccinate lyase. Two isoforms of proteins, the δ1- and δ2-crystallin, were expressed in avian lens, however, the ASL activity has only been detected in δ2-crystallin. δ-Crystallin is structurally assembled as double dimers. Lys-315 is the only residue arranged symmetrically at the diagonal subunit interfaces to interact with each other. This study found that both dimers and monomers existed in 2 ~ 4 M urea for wild-type protein while only monomers of the K315A mutant were observed under the same conditions, as judged by sedimentation velocity analysis. The assembly of monomeric K315A mutant was reversible in contrast to wild-type protein. Molecular dynamics simulations exhibited that the dissociation of primary dimers is prior to the diagonal dimers in wild-type protein. These results suggest the critical role of Lys-315 in stabilization of the diagonal dimer structure. Guanidinium hydrochloride (GdmCl) denatured wild-type or K315A mutant protein did not fold into functional protein. However, the urea dissociated monomers of K315A mutant protein in GdmCl were reversible folded through a multiple steps mechanism as measured by tryptophan and ANS fluorescence. Two partly unfolded intermediates were detected in the pathway. Refolding of the intermediates resulted in a conformation with greater amounts of hydrophobic regions exposed which was prone to the formation of protein aggregates. The formation of aggregates was not prevented by the addition of α-crystallin. These results highlight that the conformational status of the monomers is critical for determining whether reversible oligomerization or aggregate formation occurs. In the other section of this study, goose δ-crystallin with low activity was ameliorated by exchanging an N-terminal segment from human ASL. Stepwise replacement of the different residues in the exchanged segment was performed. It’s identified that the substitution of residues of Trp9, Tyr30 and Asp31 caused apparent reduction of the kcat value, while the KM value for variants of Gly6, Glu22, Asn25 and Ala26 were increased. These results highlight the important roles of these N-terminal residues in catalysis. The other protein we explored its structure and function is 4-Hydroxyphenylpyruvate dioxygenase (HPPD). HPPD is a non-haem iron (II)- dependent oxygenase that catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) to homogentisate (HG). In the active site, a strictly conserved 2-His-1-Glu facial triad coordinates the metal iron ready for catalysis. Substitution of these residues resulted in about a 10-fold decrease in the metal binding affinity, as measured by isothermal titration calorimetry, and a large reduction in enzyme catalytic efficiencies. The present study revealed the vital role of the ligand Glu349 in enzyme function. Replacing this residue with alanine resulted in loss of activity. The E349G variant retained 5% activity for the coupled reaction, suggesting that co-ordinating water may be able to support activation of the trans-bound dioxygen upon substrate binding. The reaction catalyzed by the H183A variant was not fully coupled. H183A variant catalytic activity resulted in protein cleavage between Ile267 and Ala268 and the production of an N-terminal fragment. The H266A variant was able to produce 4-hydroxyphenylacetate (HPA), demonstrating that decarboxylation had occurred but that there was no subsequent product formation. Structural modelling of the variant enzyme with bound dioxygen revealed the rearrangement of the coordination environment and the dynamic behavior of bound dioxygen in the H266A and H183A variants respectively. These models suggest that the residues regulate the geometry of the reactive oxygen intermediate during the oxidation reaction. The mutagenesis and structural simulation studies demonstrate the critical and unique role of each ligand in the function of HPPD, and which correlates with their respective co-ordination position. In conclusion, residues might play their critical functional roles in proteins depending on their positions in geometry of catalytic center or in assembly with different subunits. The results we have presented provide more information in the relationships between structures and functions protein and may trigger motivation on further investigation with big data analysis about them.