Structural analysis of ClpL NTD. (A) 1H,15N-HSQC of ClpL NTD with resonance assignment labeled at each peak. Several residues exhibit dispersed peak position, indicative of folding. (B) 1H,1H-2D NOESY in D2O of ClpL NTD confirming NOEs between aromatic ring protons of the hydrophobic core formed by α2 and the β-sheet. NOEs of the second predicted core between α1 and α2 are absent, indicating that there is no interaction between α1 and α2. (C) Selected strips of the 13C-edited 3D NOESY-HSQC to illustrate that the predicted hydrophobic core between α2 and the β-sheet is present. Multiple cross-peaks (NOEs) are present between relevant residues (Y36, V38, L43, F48, Y51). (D) Selected strips of the 13C-edited 3D NOESY-HSQC illustrating the absence of NOEs expected to between residues of the second hydrophobic core between α1 and α2. For these residues (F19, F23, A34, Y36) only intra-residue or sequential NOEs are visible (except for Y36, which has NOEs to residues of the first hydrophobic core). Thus, despite transient formation of the α1-helix, there is no or only transient interactions between α1 and α2. (E) 15N spin relaxation data of ClpL NTD. Top: Longitudinal relaxation rates, middle: transverse relaxation rates, bottom: Heteronuclear NOEs. All three measurements show that the N-terminal region up to residue R35 is highly flexible, whereas the region of the hydrophobic core between α2 and the β-sheet higher heteronuclear NOE values and longer transverse relaxation rates indicating a higher degree of order. The altogether low heteronuclear NOE values, rarely above 0.6 indicate that the secondary structure elements (α1 vs. α2/β-sheet) are mobile with respect to each other in the ps-ns time scale. (F) Sequence alignment of ClpL NTDs. Similar and identical residues are highlighted in light and dark blue. The positions of secondary structural elements and of patches (A-E) composed of Y, F, N and Q residues are indicated.