RESUMO
The phototriggered unbinding of the intrinsically disordered S-peptide from the RNase S complex is studied with the help of transient IR spectroscopy, covering a wide range of time scales from 100 ps to 10 ms. To that end, an azobenzene moiety has been linked to the S-peptide in a way that its helicity is disrupted by light, thereby initiating its complete unbinding. The full sequence of events is observed, starting from unfolding of the helical structure of the S-peptide on a 20 ns time scale while still being in the binding pocket of the S-protein, S-peptide unbinding after 300 µs, and the structural response of the S-protein after 3 ms. With regard to the S-peptide dynamics, the binding mechanism can be classified as an induced fit, while the structural response of the S-protein is better described as conformational selection.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Peptídeos/metabolismo , Ribonucleases/metabolismo , Sequência de Aminoácidos , Compostos Azo/química , Compostos Azo/efeitos da radiação , Proteínas Intrinsicamente Desordenadas/química , Cinética , Luz , Peptídeos/química , Ligação Proteica/efeitos da radiação , Conformação Proteica em alfa-Hélice , Desdobramento de Proteína/efeitos da radiação , Ribonucleases/químicaRESUMO
Age-onset cataracts are believed to be expedited by the accumulation of UV-damaged human γD-crystallins in the eye lens. Here we show with molecular dynamics simulations that the stability of γD-crystallin is greatly reduced by the conversion of tryptophan to kynurenine due to UV-radiation, consistent with previous experimental evidences. Furthermore, our atomic-detailed results reveal that kynurenine attracts more waters and other polar sidechains due to its additional amino and carbonyl groups on the damaged tryptophan sidechain, thus breaching the integrity of nearby dry center regions formed by the two Greek key motifs in each domain. The damaged tryptophan residues cause large fluctuations in the Tyr-Trp-Tyr sandwich-like hydrophobic clusters, which in turn break crucial hydrogen-bonds bridging two ß-strands in the Greek key motifs at the "tyrosine corner". Our findings may provide new insights for understanding of the molecular mechanism of the initial stages of UV-induced cataractogenesis.