title: Transient enzyme-substrate recognition monitored by real-time NMR.
abstract: Slow protein folding processes during which kinetic folding intermediates occur for an extended time can lead to aggregation and dysfunction in living cells. Therefore, protein folding helpers have evolved, which prevent proteins from aggregation and/or speed up folding processes. In this study, we present the structural characterization of a long-living transient folding intermediate of RNase T1 (S54G/P55N) by time-resolved NMR spectroscopy. NMR resonances of this kinetic folding intermediate could be assigned mainly by a real-time 3D BEST-HNCA. These assignments were the basis to investigate the interaction sites between the protein folding helper enzyme SlyD(1-165) (SlyD*) from Escherichia coli (E. coli) and this kinetic intermediate at a residue resolution. Thus, we investigated the Michaelis-Menten complex of this enzyme reaction, because the NMR data acquisition was performed during the actual catalysis. The interaction surface of the transient folding intermediate is restricted to a region around the peptidyl-prolyl bond (Y38-P39), whose isomerization is catalyzed by SlyD*. The interaction surface regarding SlyD* extends from specific amino acids of the FKBP domain forming the peptidyl-prolyl cis/trans-isomerase active site to almost the entire IF domain. This illustrates an effective interplay between the two functional domains of SlyD* to facilitate protein folding catalysis.
authors.affiliation: Institut für Physik, Biophysik, Martin-Luther-Universität Halle-Wittenberg , D-06120 Halle (Saale), Germany.
authors.forename: Caroline
authors.initials: C
authors.lastname: Haupt
authors.forename: Rica
authors.initials: R
authors.lastname: Patzschke
authors.forename: Ulrich
authors.initials: U
authors.lastname: Weininger
authors.forename: Stefan
authors.initials: S
authors.lastname: Gröger
authors.forename: Michael
authors.initials: M
authors.lastname: Kovermann
authors.forename: Jochen
authors.initials: J
authors.lastname: Balbach
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; C085606:SlyD protein, E coli; D006163:Ribonuclease T1; D019696:Peptidylprolyl Isomerase