Nucleic Acids Research, 2010, Vol. 38, No. 5 1731 Figure 7. KlcA136 secondary and tertiary structure. Colour code: alpha-helices, red; beta-strands, yellow and 310 helix, purple. (A) Secondary structure elements identiﬁed by STRIDE for the ensemble consensus shown as a topology fold schematic (55–57). (B) A cartoon representation of the closest-to-mean 3D structure in the ensemble. Note: loops are rendered smooth for clarity. and contains several hydrophobic amino acids at its N-terminus that are deeply buried. A stereo view of the ensemble of the 20 lowest energy structures of KlcA136 is shown in Supplementary Figure S2. Querying 3D-fold libraries and the PDB with the NMR-determined KlcA136 3D-structure demonstrated that its fold is novel, based upon its overall spatial arrangement and connectivity of secondary structure elements. For example, the strongest structural similarity found (z-score 5.1) using a DALI search of the PDB is to a discontinuous C-terminal portion of the 257-residue chain from G1/S-speciﬁc cyclin-D1 (PDB ID: 2W9Z), which is a purely a-helical protein. Although the z-score indicates structural similarity, the structurally aligned portions require a large 37-residue insertion between helices 2 and 3 (with respect to KlcA136) that encompass b-strands B–E, which are absent in the cyclin structure. Further conﬁrma- tion of the novelty of fold was obtained using SSM. These results are consistent with the lack of sequence identity/ signiﬁcance scores for the B. pertussis KlcA sequence against PDB sequences using a BLAST search, or remote similarity to any existing folds, when undertaking fold recognition. In the 1H-15N spectrum of KlcA136, two weak peaks were observed at 15N chemical shifts of 69 and 72 ppm. A F1 1H-coupled 1H-15N HSQC showed that these signals belong to NH2 moieties. The 15N chemical shifts are con- sistent with values reported for the guanidinium moiety of arginine from BioMagResBank (74). These NH2 groups would normally undergo chemical exchange with water and thus not be observed. The fact that they are present in the spectrum indicates reduced chemical exchange and suggests that these are involved in H-bonded salt bridges. Examination of the 3D structure of the protein, indicates that the Ng2 of R73 (one residue before strand D) and Oe2 of E86 (strand E) are separated by 2.97 A˚ , while this distance is 4.47 A˚ for R48 (loop) and E75 (strand D). The former pair meets the criterion (3.5 A˚ ) for a H-bonded salt bridge interaction (75). Based on this analysis the 15N peaks with chemical shifts of 69 and 72 ppm were tentatively assigned to NH2 moieties of R48 and R73. Our relaxation data, described below, indicate that the D and E strands are the most ﬂexible on the fast time scale, so a H-bonded salt bridge could stabilise these relatively short b-strands. The second inter- action could potentially maintain the relative orientation between the helix 2 and strands D and E, which connect to the rest of the protein via loops. The measured 15N longitudinal T1 and transverse T2 relaxation times and steady state 1H-15N NOEs were measured (Supplementary Figure S3). Due to spectral overlap, only 113 of the 137 non-proline residues of the protein could be used for the calculation of the relaxation parameters. The residues with higher amplitude of internal motions have simultaneously higher values of T1 and T2. In contrast, amide groups with the simultaneously lowest values of T1 and T2 values belong to the residues with the most restricted local mobility. Heteronuclear NOE values < 0.68 were used to identify residues of the protein that are dynamic on the ps–ns timescale. As the majority of heteronuclear NOEs are> 0.68, the backbone exhibits limited motions on this timescale (76). The only exceptions were residues 3–6 that exhibited negative NOE values indicating a ﬂexible N-terminus. The presence of internal motion and chemical exchange can lead to distortions of the T1/T2 ratio, and therefore the collection of residues used to determine the diﬀusion properties of the entire molecule excluded mobile