| Summary: | 碩士 === 國立臺灣大學 === 生化學研究所 === 87 === SUMMARY
Abrin is one of a type II RIPs, and it is composed of an enzymatic A chain with N-glycosidase activity, and a lectin B chain which binds to galactosylated proteins and lipids on the surface of the cells. The two polypeptide chains are linked by a disulfide bond. This hetro-dimeric plant toxins inhibits protein synthesis through depurination of 28S rRNA at position 4324, mediated through the N-glycosidase activity of the A chain, known as one of ribosome inactivating proteins (RIPs) . The N-glycosidase activity causes the impairment of binding of elongation factor 2 to 28S ribosomal subunit, and thus inhibits protein synthesis at elongation process.
There are four isoforms of abrin; abrin-a, -b, -c and —d, among which abrin-a is the most abundant and the most toxic. Alignment of the known amino acid sequences of eleven RIPs shows that there are thirteen identical residues and five of them, Tyr74, Tyr113, Glu164, Arg167 and Trp198, are located at the active site of the abrin-a A chain.
In the present studies, site-directed mutagenesis was conducted for elucidation of the fuction of amphipathic-a Helix-A of the abrin-a A chain. The consensus hydrophobic scale for the amino acids proposed by Eisenberg et al. was used to distinguish the approximate boundaries between the hydrophobic and hydrophilic faces of the helices. Helical wheel diagrams were constructed fora-Helix-A in the wild type and a mutant. The changes of the amino acid residues , Phe 20 and Leu 24 residues of the wild type both to Asp residues decreased the hydrophobic moment from 0.44 to 0.18. In cell-free expression system, mutant F20D/L24D decreased its inhibitory activity 2-fold from that of wild type.
To compare the overall content of helical structure of the different proteins, far UV CD spectroscopy method was used. The evaluation of the helix content of the different proteins by standard methods shows the following approximate fractional helix contents: reAaTA: 36.3%; F20D/L24D reAaTA: 31.7%. The mutation leads to a lower percentage of helicity estimated about 4.6%.
The polarity of microenvironment of tryptophan residues in a protein can be evaluated by its accessibility to collide with fluorescence quenchers. The Stern-Volmer constant (KSV) for reAaTA and F20D/L24D reAaTA tryptophan quenching with
acrylamide was determined. In reAaTA, the tryptophan is in an environment 1.34-fold less accessible to acrylamide than F20D/L24D reAaTA tryptophan. The results of fluorescence quenching data suggested that Trp198 is more exposed to bulk phase in F20D/L24D reAaTA than in reAaTA. From the data presented above, we proposed that the mutant F20D/L24D AaTA has suffered a conformational change. We concluded that the mutation that disrupted the amphipathic property of the a- Helix-A of abrin a-A chian led to no dramatic effects on the inactivation of abrin a-A chain.
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