Figure 1.
Amyloid-Like Character of Three Bacterial Inclusion Bodies
CR birefringence and Thio T binding of inclusion bodies are indicative of amyloid formation. Under cross-polarized light with 10× magnification, CR birefringence is observed for BMP2(13–74), ESAT-6, and MOG(ECD). Larger images together with the corresponding pictures from the bright-field microscope are shown in Figure S1. The histogram shows the relative Thio T binding of BMP2(13–74), ESAT-6, and MOG(ECD) in comparison to aged α-synuclein (α-Syn) fibrils. The intensity of the Thio T fluorescence is shown in arbitrary units (AU).
Figure 2.
The Short Segment of Residues 7–23 of ESAT-6 Forms Cross-β-Sheet Structure in Inclusion Bodies of E. coli
(A) Fast [15N,1H]-HMQC spectra of dissolved, monomeric 15N-labeled ESAT-6 in d6-DMSO containing 0.05 % TFA and 25 mM DTT, corresponding to fully protonated inclusion bodies (left), and to inclusion bodies exchanged for 311 h in D2O (right). After 311 h, many cross peaks show a complete loss of intensity indicative of fast exchange. In contrast, cross peaks of residues 8–25 and 36–43 are still present, indicative of slow exchange. Sequence-specific chemical-shift assignment of protein backbone amide cross peaks are indicated by a single-letter amino acid code and the corresponding residue number.
(B) Ribbon representation of the 3-D structure of soluble ESAT-6 in complex with CFP-10 [25]. The green-colored segment corresponds to residues 7–23, which experiences 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 with P = 1 for 100% occupancy and P = 0 for 0% occupancy, and the predictions of aggregation-prone segments against the amino acid sequence of ESAT-6. 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. Although some of the residues 36–43 show slow exchange in the HMQC spectrum in (A), their slow-exchanging population is present less than 1/3 and hence not shown in (C). In the third plot of (C) labeled with an “A,” predicted aggregation-prone segments of ESAT-6 are shown using two distinct algorithms: 3DPROFILE [33] in gray, and TANGO [32] (the latter is not shown, since no aggregation-prone segment was predicted). For 3DPROFILE, predictions are shown for segments having energies ≤ −23 kcal/mol. For both algorithms, outstanding relative values (≤ −23 in 3DPROFILE, and >0 in TANGO) within a segment of several amino acid residues are indicative of an aggregation-prone segment. 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 lowest panel labeled with “H,” with positive values indicative of hydrophobicity.
(D) X-ray diffraction of inclusion bodies of ESAT-6. The two reflections at 4.7 Å and approximately 10 Å consistent with cross-β-sheet structure are labeled.
(E) Mutagenesis of ESAT-6 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 ESAT-6 (WT) or ESAT-6 variants as indicated (i.e., point mutations are indicated by a one-letter code, Δ7–23 stands for the deletion variant, which lacks the slow-exchanging residues 7–23, and “replaced” stands for the mutant for which residues 7–23 were replaced with the helical segment of residues 25–41 of ESAT-6. The 10-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), and the single amino acid residue variants that are absent in the insoluble fractions are colored red in the amino acid sequence of (C), respectively.
Figure 3.
The Short Segment of Residues 62–67 of BMP2(13–74) Forms Cross-β Structure in Inclusion Bodies of E. coli
(A) Fast [15N,1H]-HMQC spectra of homogenously 15N-labeled monomeric BMP2(13–74) 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 185 h in D2O (right). After 185 h, many cross peaks show a complete loss of intensity, indicative of fast exchange. In contrast, cross peaks of residues 62–70 are still present, indicative of slow exchange.
(B) Ribbon representation of the 3-D structure of soluble BMP2. The green-colored segment corresponds to residues 62–67 that comprise slow exchange in inclusion bodies as shown in (A) and (C). The protein fragment BMP2(13–74) comprising residues 13–74 is highlighted in grey. The N-terminal 12 residues and the C-terminal 40 residues of BMP2 are colored black.
(C) Plots of the observed exchange rates kex/h, the relative population P(F) of the two exchange regimes observed (Figure S2), and the predictions of aggregation-prone segments against the amino acid sequence of BMP2(13–74). 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. In the third plot of (C) labeled with “A,” predicted aggregation-prone segments of BMP2(13–74) are shown using two algorithms: 3DPROFILE [33] in gray and TANGO [32] in blue. Predictions of aggregation are shown for segments having energies ≤ −23 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 BMP2(13–74), indicative of cross-β-sheet structure.
(E) Mutagenesis of BMP2(13–74). Coomassie-stained SDS-polyacrylamide gels were obtained from soluble (s) and insoluble (i) fractions of lysates of E. coli cells expressing wild-type BMP2(13–74) (WT) or variants as indicated. The identities of the bands were verified by mass spectrometry analysis (see Material and Methods). The 6-kDa molecular weight standard is labeled. The same nomenclature, labeling, and layout are used as in Figure 2.
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.
Figure 5.
EM of Inclusion Bodies of BMP2(13–74) with Amyloid-Like Fibrils upon Incubation
(A) Freshly purified inclusion bodies. They are electron-dense, round aggregates with a diameter approximately 0.5 μm. In some inclusion bodies, short amyloid-like fibrils can be observed.
(B) Inclusion bodies after in vitro incubation at 37 °C for 12 h. Fibrils are present from the purified inclusion bodies.
(C) Freshly purified inclusion bodies from E. coli cells that overexpressed BMP2(13–74) at 37 °C for 12 h. Fibrils are present from the purified inclusion bodies.
Scale bars indicate 1 μm.