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. Author manuscript; available in PMC: 2015 Nov 5.
Published in final edited form as: Biochemistry. 2015 Apr 27;54(17):2727–2738. doi: 10.1021/acs.biochem.5b00036

Figure 9.

Figure 9

RNA is a key component of Trx quinary structure. (a) Overlay of in-cell 1H–15N CRINEPT–HMQC–TROSY spectra of 15 μM REDPRO-labeled purified wild-type Trx with (red) and without (blue) 30 mg/mL total E. coli RNA. The indole NH of W29 exhibits a downfield shift in the RNA-bound and in-cell NMR spectra (Figures 1 and 2). The indole NH of W32, along with backbone amide peaks of E31, C33, C36, K37, I39, and A40 (boxed), is broadened due to the protein–RNA interaction that is similar to the quinary interaction observed in-cell. (b) The relative volumes of the G52, G66, and G85 peaks in the in vitro 1H–15N CRINEPT–HMQC–TROSY spectrum of total RNA-bound wild-type Trx are plotted vs the CRINEPT transfer delay times. An endogenous tryptophan indole amide peak in the in vitro spectra is used as a reference. The optimal CRINEPT transfer delay is 3.5 ms, which corresponds to a molecular mass of ~0.3 MDa. (c) Overlay of in-cell 1H–15N CRINEPT–HMQC–TROSY spectra of 15 μM REDPRO-labeled purified wild-type Trx treated with 15 mg/mL total E. coli RNA in the presence (black) and absence (red) of RNase A. The indole NH of W29 exhibits a downfield shift in the RNA-bound and RNase A-treated NMR spectra. The indole NH of W32, along with backbone amide peaks of E31, C33, C36, K37, I39, A40, and I42 (boxed), is broadened due to the protein–RNA interaction that is similar to the quinary interaction observed in-cell. (d) The relative volumes of the G52, G66, and G85 peaks in the in vitro 1H–15N CRINEPT–HMQC–TROSY spectrum of RNase A-treated total RNA-bound wild-type Trx are plotted vs the CRINEPT transfer delay times. The optimal CRINEPT transfer delay is ~5.4 ms, which corresponds to a molecular mass of ~12 kDa.