atomic-level structure characterization of an ultrafast folding mini-protein denatured state原子水平结构特征的超快折叠mini-protein变性状态.pdfVIP

Atomic-Level Structure Characterization of an Ultrafast Folding Mini-Protein Denatured State Per Rogne1,2, Przemysław Ozdowy 1,2, Christian Richter3, Krishna Saxena3, Harald Schwalbe3, Lars T. Kuhn1,2* ¨ ¨ 1 European Neuroscience Institute Gottingen (ENI-G), Gottingen, Germany, 2 DFG Research Center Molecular Physiology of the Brain (CMPB)/EXC 171 ‘‘Microscopy at the ¨ Nanometer Range’’, Gottingen, Germany, 3 Institute for Organic Chemistry and Chemical Biology and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe- University Frankfurt, Frankfurt am Main, Germany Abstract Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate