His-tagged ClpX purified from E. coli was observed as two separated migration bands in conventional Laemmli SDSPAGE (A). The separation pattern did not change by reduction.
Next the His-tagged ClpX was analyzed by Mn2+–Phos-tag SDS-PAGE(B).
The protein was detected as clear multiple bands, including highly up-shifted bands , and the
banding pattern suggested that phosphorylation-dependent variants with different phosphorylation states were present.
However, the recombinant protein was never confirmed to be actually phosphorylated, as demonstrated by the following three independent experimental results .
(1) The banding pattern did not change even after the protein had been treated with several types of phosphatase.
(2) After SDS-PAGE, no ECL signals from the protein on the blotting membrane were detected by Western blotting analysis with Phos-tag biotin,which permits the detection of phosphoproteins on a membrane.
(3) No phosphopeptides were detected in an MS analysis of the protein.
Next, we constructed a N1–61truncated mutant of the Histagged ClpX protein (ClpX_delta N61). When we compared the banding pattern
The mutant was detected as a single nonshifted band in both normal SDS-PAGE and Mn2+–Phos-tag SDS-PAGE. We therefore confirmed that the N-terminal region of ClpX was responsible for producing the multiple phosphorylation-independent structural variants, even under denaturing conditions, and that some of the variants were detected as highly up-shifted bands in Phos-tag SDS-PAGE despite being nonphosphorylated.
We revealed that the N47–61 region is responsible for the exaggerated retardation in Phos-tag SDS-PAGE migration and for the appearance of multiple bands in the ClpX full-length protein.
To identify the site responsible for the appearance of multiple bands of ClpX, we next performed a systematic Alascanning mutation analysis in the region N47–61. The Ala substituted mutants I47A, I48A, R49A, E50A, E51A, I52A, K53A, E54A, V55A, P57A, H58A, R59A, E60A, and R61A
were constructed in the His-tagged ClpX full-length protein and were analyzed by using the normal SDS-PAGE (A) and Mn2+ (B)and Zn2+–Phos-tag SDS-PAGE (C) respectively. In the normal SDS-PAGE, the upper band of the wild-type ClpX (WT) and major bands of its Ala-substituted mutants showed almost the same degrees of migration. In the I47A, I48A, R49A, I52A, K53A, V55A, H58A, R59A, E60A, and R61A mutants, some minor up-shifted bands were observed. In Mn2++–Phos-tag
SDS-PAGE, all the mutants except E51A and E54A were detected as multiple migration bands, indicating that the multiple phosphorylation-independent structural variants are present, even under denaturing conditions, in the same manner as the WT protein. The E51A and E54A mutants
were detected as single nonshifted bands, indicating that the Glu-51 and Glu-54 residues are the sites responsible for the appearance of multiple migration bands of ClpX, including
some highly up-shifted bands. In Zn2+–Phos-tag SDS-PAGE , the WT protein and the mutants except for E51A were detected as multiple bands as a result of structural variations.
The banding pattern of each sample was different from that in Mn2+–Phos-tag SDS-PAGE, showing that there are some differences in the affinities of the various Phos-tag complexes, even with the same targets. The E51A mutant was detected as a single band in both Phostag
methods, suggesting that the Glu-51 residue is critical with respect to the generation of multiple structural variantsin the ClpX full-length protein under the experimentaln conditions.