In-cell NMR in E. coli to monitor maturation steps of hSOD1

Abstract

In-cell NMR allows characterizing the folding state of a protein as well as posttranslational events at molecular level, in the cellular context. Here, the initial maturation steps of human copper, zinc superoxide dismutase 1 are characterized in the E. coli cytoplasm by in-cell NMR: from the apo protein, which is partially unfolded, to the zinc binding which causes its final quaternary structure. The protein selectively binds only one zinc ion, whereas in vitro also the copper site binds a non-physiological zinc ion. However, no intramolecular disulfide bridge formation occurs, nor copper uptake, suggesting the need of a specific chaperone for those purposes.

Figure 1. In-cell NMR spectra of apo-hSOD1 and of cell lysate.

(A) In-cell ¹H-¹⁵N SOFAST-HMQC spectrum of E. coli cells expressing hSOD1 in defect of Zn(II). Two different thresholds are shown (black, purple). At lower threshold (purple) some weak and broad signals are visible, while only signals in the unfolded region of the spectrum are visible at higher threshold (black). Signals belonging to other cellular components are overlaid in red. These are always present in all in-cell spectra. (B) Overlay of the ¹H-¹⁵N SOFAST-HMQC spectrum of a cell lysate without addition of Zn(II) (black), and the ¹H-¹⁵N HSQC of an in vitro sample of E,E-hSOD1^SH-SH (red).

Figure 2. Trp 32 side chain of denatured in vitro apo-hSOD1.

(A) ¹H-¹⁵N HSQC of an in vitro sample of E,E-hSOD1^SH-SH denatured with 0.5 M guanidinium chloride. (B) Zoom of the (A) spectrum showing the signal of Trp 32 (black). Overlaid are the in-cell NMR spectrum of apo-hSOD1 (orange) and the in vitro spectrum of non-denatured E,E-hSOD1^SH-SH.

Figure 3. In-cell NMR spectra of hSOD1 with added Zn(II).

(A) Overlay of the in-cell ¹H-¹⁵N SOFAST-HMQC spectrum of E. coli cells expressing hSOD1 in presence of Zn(II) (black), and 1H-15N HSQC spectrum of an in vitro sample of E,Zn-hSOD1^S-S (green). (B) Zoom of the (A) spectrum showing the peaks of Gly 61 and Thr 135 in-cell (black), in vitro E,Zn-hSOD1^S-S (green) and in vitro Zn,Zn-hSOD1^S-S (yellow).

Figure 4. In-cell and in vitro NMR spectra of ¹⁵N cysteine-labelled E,E-hSOD1 and E,Zn-hSOD1.

(A) Overlay of ¹H-¹⁵N SOFAST-HMQC in-cell spectrum (black) of ¹⁵N cysteine-labelled E,E-hSOD1, oxidized E,E-hSOD1 purified from the lysate (red), same sample reduced with DTT (blue). (B,C) Detailed views of (A) showing Cys 146 and Cys 6 amide cross-peaks. (D) Overlay of ¹H-¹⁵N SOFAST-HMQC in-cell spectrum (black) of ¹⁵N cysteine-labelled E,Zn-hSOD1, oxidized E,Zn-hSOD1 purified from the lysate (red), same sample reduced with DTT (blue). (E,F) Detailed views of (D) showing Cys 146 and Cys 6 amide cross-peaks. When DTT is added to the sample of E,Zn-hSOD1^S-S, E,Zn-hSOD1^SH-SH is detected, together with E,E-hSOD1^SH-SH and other species (indicated with an asterisk).