PubMed · 8085149
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S A Benner, D L Gerloff, T F Jenny. 1994-09-16. Predicting protein crystal structures.. https://doi.org/10.1126/science.8085149
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Reconstitution of the tyrosyl radical in ribonucleotide reductase protein R2 requires oxidation of a diferrous site by oxygen. The reaction involves one externally supplied electron in addition to the three electrons provided by oxidation of the Tyr-122 side chain and formation of the mu-oxo-bridged diferric site. Reconstitution of R2 protein Y122F, lacking the internal pathway involving Tyr-122, earlier identified two radical intermediates at Trp-107 and Trp-111 in the vicinity of the di-iron site, suggesting a novel internal transfer pathway (Sahlin, M., Lassmann, G., Pötsch, S., Sjöberg, B. -M., and Gräslund, A. (1995) J. Biol. Chem. 270, 12361-12372). Here, we report the construction of the double mutant W107Y/Y122F and its three-dimensional structure and demonstrate that the tyrosine Tyr-107 can harbor a transient, neutral radical (Tyr-107(.)). The Tyr-107(.) signal exhibits the hyperfine structure of a quintet with coupling constants of 1.3 mT for one beta-methylene proton and 0.75 mT for each of the 3 and 5 hydrogens of the phenyl ring. Rapid freeze quench kinetics of EPR-visible intermediates reveal a preferred radical transfer pathway via Trp-111, Glu-204, and Fe-2, followed by a proton coupled electron transfer through the pi-interaction of the aromatic rings of Trp-(Tyr-)107 and Trp-111. The kinetic pattern observed in W107Y/Y122F is considerably changed as compared with Y122F: the Trp-111(.) EPR signal has vanished, and the Tyr-107(.) has the same formation rate as does Trp-111(.) in Y122F. According to the proposed consecutive reaction, Trp-111(.) becomes very short lived and is no longer detectable because of the faster formation of Tyr-107(.). We conclude that the phenyl rings of Trp-111 and Tyr-107 form a better stacking complex so that the proton-coupled electron transfer is facilitated compared with the single mutant. Comparison with the formation kinetics of the stable tyrosyl radical in wild type R2 suggests that these protein-linked radicals are substitutes for the missing Tyr-122. However, in contrast to Tyr-122(.) these radicals lack a direct connection to the radical transfer pathway utilized during catalysis.
The three-dimensional solution structure of the RNA self-complementary duplex [formula in text] was derived from two-dimensional NMR and the iterative relaxation matrix approach. Each GA mismatch forms two hydrogen bonds: A-NH6 to G-O6 and A-N1 to G-NH1 (imino). This GA structure differs from the sheared tandem GA structure in [formula in text] which also has two hydrogen bonds: A-N7 to G-NH2 and A-NH6 to G-N3 [SantaLucia, J., Jr & Turner, D. H. (1993) Biochemistry 32, 12612-12623], although the only difference between the two sequences is the order of the two GA mismatches. Inspection of three-dimensional structures indicates that substituting [formula in text] for [formula in text] makes GA mismatches unable to have stable sheared conformations. This may explain why the [formula in text] motif is rarely observed in nature, whereas [formula in text] common.
The crystal structure of desheptapeptide (B24-B30) insulin (DHPI), a virtually inactive analog of insulin, was determined at 1.6 A resolution. In the refined structure model, DHPI retains three alpha-helices (A1-A8, A12-A18, and B9-B19) as its structural framework, while great conformational changes occur in the N and C termini of B-chain. The beta-turn, which lies in B20-B30 in insulin and insulin analogs with high potency, no longer exists in DHPI. Relative motion is observed among the three alpha-helices, each as a rigid functional group. In contrast, a region covering B5-B6 and A6-A11 exhibits a relatively stable conformation. We interpret our results as identifying: (i) the importance of beta-turn in determining the receptor-binding potency of insulin and (ii) a leading role of PheB24 in maintaining the beta-turn structure.