Shigella flexneri Spa15 crystal structure verified in solution by double electron electron resonance.
Bottom Line: One of these effectors is IpgB1, a mimic of the human Ras-like Rho guanosine triphosphatase RhoG.This distance is explained by determining the crystal structure of the spin-labeled Spa15 where labels are seen to be buried in hydrophobic pockets.The double electron electron resonance experiment on the Spa15 complex with IpgB1 shows that IpgB1 does not bind Spa15 in the same way as is seen in the homologous Salmonella sp. chaperone:effector complex InvB:SipA.
Affiliation: Inorganic Chemistry Laboratory, University of Oxford, OX1 3QR, UK.Show MeSH
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Mentions: Spin labeling of Spa15 was achieved at 100%. This was shown by continuous-wave EPR calibration with the quantitative standard TEMPOL and verified by mass spectrometry (data not shown). DEER experiments at 50 K with 200 μM samples reproducibly identified the major distance between labels to be 4.5 nm (± 0.1 nm) (Fig. 1a and b). A fast dephasing T2 relaxation required overnight measurement despite the use of deuterated solvent. Since there was just one labeled site per monomer, the dominance of this peak indicated it as the intra-dimer label distance. A feature of the DEER spectrum was the inclusion of distances at either side of the 4.5-nm peak, which changed with sample and concentration (Fig. 1c). Other researchers have observed satellite signals to be a result of the constraining of a label, a situation identified by a variant DEER trace in an experiment upon changing the frequency difference between observer and pump frequency pulses.30 This possibility was ruled out, since such DEER experiments yielded identical signals between 50 and 80 MHz (data not shown). The increase in side peaks relative to the major intra-dimer peak at higher concentration led us to conclude that these side peaks were due to aggregation, a product of the preparative conditions. This was supported by multi-angle laser light scattering, which indicated that at the concentration level used in EPR, protein aggregates began to be evident (data not shown).
Affiliation: Inorganic Chemistry Laboratory, University of Oxford, OX1 3QR, UK.