Limited proteolysis analysis of the C-terminal domain of wild-type μ4.
Recombinant C-terminal domain of wild-type μ4 (A), μ4-D190A (B), or μ4-R283D (C) were incubated with proteinase K at 25°C at an enzyme:substrate ratio of 1∶100, and after the times indicated on top of the panel the digestion was stopped by addition of PMSF. The reaction products were analyzed by SDS-PAGE and gels stained with Coomassie Brilliant Blue. In this condition similar stable fragments are produced from all μ4 variants. Samples from a similar gel shown in (A) were electroblotted onto a PVDF membrane. The three bands shown in lane 7 were excised and processed for N-terminal sequencing by Edman degradation, and the resulting amino acid sequences are shown on the right. (D) Amino acid sequence of the recombinant C-terminal domain of human μ4 (residues 160-453; accession number in parenthesis), with the N-terminal sequence of the fragments shown in (A) highlighted in different colors. (E) Surface model of the μ4 C-terminal domain with amino acids of the proteolytic fragments colored as in A and D. The regions digested are colored in grey, corresponding to a structured loop (S–L), an unstructured loop (U–L), and unstructured N-terminal residues (N–T) (represented as grey lines). (F) The same μ4 variants were processed as in A to C, but incubated with proteinase K at 50°C at the indicated times on top of the panel. In this case the three μ4 variants have different levels of sensitivity to proteinase K. The position of molecular mass markers is indicated on the left.