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.2008 Apr;1778(4):937-44.
doi: 10.1016/j.bbamem.2007.11.013. Epub 2007 Dec 14.

Solution NMR of signal peptidase, a membrane protein

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Solution NMR of signal peptidase, a membrane protein

Monika Musial-Siwek et al. Biochim Biophys Acta.2008 Apr.

Abstract

Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC-TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly 2H, 13C, 15N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.

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Fig. 1
Fig. 1
Analysis ofE. coli SPase I stability and activity under different conditions. (A) Silver-stained SDS-PAGE of purified SPase I indicating the enzyme is about 95% pure. (B) Analysis of SPase I activity on silver stained SDS-PAGE. Different concentrations, as indicated, of the substrate (GST-SP-AP-His) were incubated with 1 μM SPase I over 2 h at 37 °C. The location of SPase I, GST-SP-AP-His, and the cleavage product, GST-SP, is indicated on the gel. (C) Comparison of the degradation of SPase I in Triton X-100, OG, and DPC detergent micelles via silver stained SDS-PAGE. Incubation time and temperature are indicated. The band corresponding to SPase I is marked and two of the degradation products are indicated by ● and ○. While, some discoloring is present on the scanned image in the last lane, the bands still can be distinguished. (D) Analysis of SPase I activity conducted as in (B). SPase I was incubated over 2 days in DPC micelles at different temperatures, as indicated, prior to the activity analysis. The band corresponding to SPase I is marked and two of the degradation products are indicated by ● and ○. (E) Analysis of SPase I activity conducted as in (B). SPase I was incubated over 7 days in DPC micelles at different temperatures, as indicated, prior to the activity analysis. The band corresponding to SPase I is marked and two of the degradation products are indicated by ● and ○.
Fig. 2
Fig. 2
1H,15N-TROSY-HSQC spectrum at 600 MHz (1H) of 0.3 mM uniformly2H,13C,15N-labeled SPase I in DPC micelles at pH 6.3 and 22 °C. Peaks that were identified, as described in the text, are labeled.
Fig. 3
Fig. 3
Sequential assignment of SPase I residues. Some of the strip plots acquired from the HNCACB experiment are shown with residues/numbers indicated above the plots and chemical shifts shown at the bottom of the plot for1H and15N and on the side for13C. The Cα (orange) and Cβ (blue) have opposite signs. Correlated peaks are connected with lines.
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References

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