Abstract
Protein–protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8–Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure.
Highlights
The use of photo-initiated cross-linking of protein nanoprobes in combination with mass spectrometry (MS) is an experimental technique suitable for the determination of the assembly of protein complexes in their native states at reasonable time-scales using relatively small quantities of protein [1]
Because only partial incorporation of the photo-labile methionine analog in place of naturally present methionine within the protein sequence was suggested, the MALDI-TOF analysis of the trypsinized protein nanoprobe was used to verify sufficient photo-labile analog incorporation. This was confirmed by the detection of signals indicating both tryptic peptides containing methionine and signals corresponding to tryptic peptides with methionine analog
Two recombinant photo-labile 14-3-3ζ protein nanoprobes, WT and S58D, were successfully prepared, and partial incorporation of a photo-labile analog of methionine into both proteins was demonstrated by MS analysis
Summary
The use of photo-initiated cross-linking of protein nanoprobes in combination with mass spectrometry (MS) is an experimental technique suitable for the determination of the assembly of protein complexes in their native states at reasonable time-scales using relatively small quantities of protein [1]. This approach is an alternative to chemical cross-linking, but more powerful due to the absence of limitations in reaction specificity and restrictions on reaction conditions that are inherent to chemical cross-linkers [1]. The deduction of which two amino acid residues participate in the covalent coupling of two cross-linked peptides is based on the acquisition of the tandem mass spectra (MS/MS), which provides structural information about the analyzed species
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