Bacterial Inclusion Bodies Contain Amyloid-Like Structure
Figure 4
The Segments of Residues 85–95, 101–108, and 111–118 of MOG(ECD) Form a Cross-β-Sheet Structure in Inclusion Bodies of E. coli.
(A) Fast [15N,1H]-HMQC spectra of homogenously 15N-labeled monomeric MOG(ECD) in d6-DMSO containing 0.05 % TFA and 25 mM DTT, corresponding to fully protonated inclusion bodies (left), and to inclusion bodies that exchanged for 285 h in D2O (right). After 285 h, many cross peaks show a virtually complete loss of intensity, indicative of fast exchange. In contrast, a set of cross peaks labeled by their corresponding amino acid residue number are still present, indicative of slow exchange.
(B) Ribbon representation of the 3-D structure of soluble MOG(ECD) [28]. The green-colored segments correspond to residues 85–95, 101–108, and 111–118, which comprise slow exchange in inclusion bodies as shown in (C).
(C) Plots of the observed exchange rates kex/h, the relative population P(F) of the two exchange regimes observed, and the predictions of aggregation-prone segments against the amino acid sequence of MOG(ECD). The exchange rates of the major population are colored green. If the minor population is present more than 1/3, the corresponding exchange rates are shown in grey. Because of the size of the protein, considerable overlap is observed in the DMSO spectrum (see [A]), making the analysis of the exchange rates of some residues difficult. However, most of these overlap problems could be resolved by the assumption that sequential neighboring residues show a similar extent of exchange. The exchange rates that have been extracted following this procedure are colored in light green. In the third plot of (C), predicted aggregation-prone segments of MOG(ECD) are shown using two algorithms: 3DPROFILE [33] in gray and TANGO [32] in blue. Predictions of aggregation are shown for segments having energies ≤ −19.5 kcal/mol from 3DPROFILE, and values >0 from TANGO. The secondary structures of the soluble conformation shown in (B) are highlighted in red for helix and blue for β-sheet, respectively. The secondary structural elements predicted by the software Jpred [53] are highlighted by cyan arrows for β-sheet conformation and a yellow helix for helical structure, respectively. An amino acid sequence-resolved hydrophobicity score plot calculated by the software ProtScale [54,55] is shown at the bottom labeled with “‘H,” with positive values indicative of hydrophobicity.
(D) X-ray diffraction of inclusion bodies of MOG(ECD). The two observed bands at 4.7 Å and approximately 10 Å indicative of cross-β-sheet structure are labeled.
(E) Mutagenesis of MOG(ECD) and the influence of amino acid substitutions in the formation of inclusion bodies. Coomassie-stained SDS-polyacrylamide gels were obtained from soluble (s) and insoluble (i) fractions of lysates of E. coli cells expressing wild-type MOG(ECD) (WT) or variants as indicated. The 15-kDa molecular weight standard is labeled. The variants that are present in the insoluble fraction are colored blue in the amino acid sequence of (C). The same nomenclature, labeling, and layout are used as in Figure 2.