| Co-Investigator(Kenkyū-buntansha) |
MIZUNO Hiroshige University of Kyusyu Kyoritsu, 情報処理センター, 助教授 (90209768)
HIKICHI Kunio Department of Polymer Science, Faculty of Science, Hokkaido University, 高分子学科, 教授 (30000805)
ROBART.H. KR バージニア大学, 生物学科, 教授
KRETSINGER Robert H. Department of Biology, University of Virginia
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| Research Abstract |
During the past 5 years many protein sequences have been noted to contain unique tandem repeats having either a Tyr or Phe and 20-60% Pro with abundantly Gly, Gln, and Ser. Matsushima, Creutz and Kretsinger have developed a group of related helical models for seven proteins (molluscan rhodopsin, synaptophytsin, synexin, gliadin, RNA polymerase II, hordein, and gluten) based on the polyproline conformation combined with one or two beta-turns. In the present project we puropose to confirm the existence of polyproline, beta-turn helices using both theoretical and experimental methods.
First method is the measurements of proton nuclear magnetic resonance (NMR) of synthetic repeat pentapeptide, (Tyr-Pro.Pro.Gln.Gly)n, of which the tandem repeat is contained at the carboxy-terminal domain of molluscan rhodopsin. Ac-(Tyr-Pro.Pro.Gln.Gly)n-NH_2 (N=2, 4, 6, and 8) was synthesized by solid-phase methods and was purified by HPLC. The two-dimensional NMR studies were carried out on DMS0-d_6 solutio
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ns of Ac-(Tyr.Pro.Pro.Gln.Gly)n-NH2 (n=2 and 4). Some inter-residue NOEs were observed, containing two NOEs between the Tyr alphaCH and the Pro deltaCH and the Pro deltaCH. Two observed NOEs indicate that bith the Tyr-Pro and the Pro-Pro bonds favor the trans-form in DMSO. The results of amide protons temperature coefficients, ^3J_Nalpha coupling constants, NOEs observed between inter-residues in Ac-(Tyr.Pro.Pro.Gln.Gly)_2-NH2 also indicated that Pro.Gln or Gly.Tyr may be involved in a type II beta-turn.
Second method is small-angle x-ray scattering of one high molecular weight (HMW) subunit of wheat glutenin. The radius of gyration of whole particles, Ro, in aq. 50% (v/v) 1-propanol and 0/1M acetic acid 16.6 * 0.1nm and 22.8nm, respectively, and the corresponding radius of gyration of cross-section, Rc, was 2.82 * 0.02nm and 2.23 * 0.01nm, which indicate that the glutenin HMW subunit exists as very anisotropy particles in both solutions.
Third method is molecular modeling by computer graphics and energy minimization for some tandem repeats. We tried to model the tandem repeats consisting of Pro.Gln.Gln.Pro.Phe.Pro.Gln within gamma-gliadin and Leu・Pro・Pro・Pro・Val・His within zein or glutelin-2 and developed a group of helical models closely related with polyproline beta-turn helices.
These studies support that these tandem repeats take polyproline beta-turn helices, which are a fundamental basic supersecondary structure. Less
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