NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein

Abstract

The growing understanding of partially unfolded proteins increasingly points to their biological relevance in allosteric regulation, complex formation, and protein design. However, the structural characterization of disordered proteins remains challenging. NMR methods can access both the dynamics and structures of such proteins, yet suffering from a high degeneracy of NMR signals. Here, we overcame this bottleneck utilizing a salt-inducible split intein to produce segmentally isotope-labeled samples with the native sequence, including the ligation junction. With this technique, we investigated the NMR structure and conformational dynamics of TonB from Helicobacter pylori in the presence of a proline-rich low complexity region. Spin relaxation experiments suggest that the several nano-second time scale dynamics of the C-terminal domain (CTD) is almost independent of the faster pico-to-nanosecond dynamics of the low complexity central region (LCCR). Our results demonstrate the utility of segmental isotopic labeling for proteins with heterogenous dynamics such as TonB and could advance NMR studies of other partially unfolded proteins.

Heterogeneous dynamics in partially disordered proteins

“…In more realistic Amber ff03ws simulations, the disordered region with more extended conformations has only a marginal effect on the rotational dynamics of C-terminal domain, in line with the interpretation from experimental spin relaxation data. 16 Overcondensation of disordered proteins in Amber ff99SB-ILDN and CHARMM36m simulations is reported also previously. 19,20 Even though the spin relaxation times from the Amber ff03ws simulation are close to the experimental values in Fig.…”

“…16 We have recently determined the NMR structure of the folded C-terminal domain in HpTonB from a construct containing the last 107 residues (HpTonB 179-285 ) and measured the backbone N-H spin relaxation times of almost all residues in the periplasmic region of TonB protein from Helicobacter pylori (HpTonB 36-285 ) using the segmental isotopic labeling with salt-inducible split intein. 16,29 The main conclusion from these experiments is that the structure and dynamics of the folded C-terminal domain is largely independent of the proline rich disordered region. However, more detailed interpretation of the conformational ensemble and dynamics of HpTonB 36-285 containing disordered region cannot be made from the experimental data alone.…”

“…This is in line with the small changes in experimental spin relaxation times upon increasing the length of linker observed in our previous work. 16 These results suggest that the linker is very flexible and that the rotational dynamics of the folded C-terminal domain is almost independent of the attached flexible disordered linker. This conclusion is supported by the similar results for overall timescales estimated from experimental T 1 /T 2 ratios and from MD simulation analysis in Table 2.…”

“…, are taken from our previous work 16 D planes were found to have multiple timescales. 42 Because large number of parameters are needed to describe such dynamics, the multiple timescale dynamics is very difficult to detect even with the excessive amount of experimental data measured at different conditions.…”

“…4,[11][12][13][14] We have recently resolved the problem of spectral overlap for partially disordered TonB protein with 101 folded and 149 disordered residues using segmental isotopic labeling with salt-inducible split intein. 16 Moreover, we have used molecular dynamics (MD) simulations to interpret the rotational dynamics of asymmetrically shaped folded proteins from NMR spin relaxation times. 15 However, the accuracy of force field parameters limits the applications of MD simulations to interpret the NMR data.…”