Chemical cross-linking and high resolution mass spectrometry for mNKR-P1C protein structure modeling
Daniel Rozbesky1,2, Petr Man1,2, Karel Bezouska1,2 and Petr Novak1,2
1Institute of Microbiology, Academy of Science of the Czech Republic, Vídeňská 1083, Prague 4, Czech Republic
2Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, Prague 2, Czech Republic
pnovak@biomed.cas.cz
Determination of the three-dimensional structures of proteins has traditionally been realized by X-ray crystallography and NMR spectroscopy. Although these techniques provide high resolution atomic data, they have some limitations. Both NMR spectroscopy and X-ray crystallography require large amounts of pure analyte and are time-consuming techniques. Mass spectrometry combined with chemical cross-linking offers alternative approach to identify the protein fold. This method is fast, is general and uses small amounts of material.
Our aim was to gain insight into low-resolution structure of NKR-P1C protein, important activating receptor which plays a key role in eliminating virally infected and tumor cells, using unambiguous distance constraints derived from the chemical cross-linking data in combination with computational methods.
We used homobifunctional cross-linking reagents disuccinimidyl suberate (DSS) and disuccinimidyl glutarate (DSG), which react with primary amino groups, and heterobifunctional "zero-length" cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), which couples carboxyl groups to primary amines. After cross-linking reaction, the SDS-PAGE of the cross-linking reaction mixture was performed and the band of cross-linked protein was excised and subjected to in gel digestion by endoproteinases Asp-N, Glu-C and trypsin. The peptide mixtures from the enzymatic digest were separated by μHPLC system coupled online to an Apex-ULTRA FT ICR mass spectrometer equipped with 9.4 T superconducting magnet. Cross-linking products were identified using MS3D software.
In order to build the protein structure model of NKR-P1C receptor, we have prepared recombinant NKR-P1C protein encompassing amino acids Ser89 – Ser223 and performed optimization of conditions for the chemical cross-linking reactions. Mass spectra of the crude product mixtures from the cross-linking reactions showed that 3× molar excess of DSS and DSG and 10× molar excess of EDC to the protein concentration gave the highest relative yield of protein containing a single internal cross-link. Optimal duration was chosen so that all unreacted cross-linkers were hydrolyzed. After μLC-ESI-FT MS analysis, the experimentally obtained monoisotopic masses were compared with calculated masses of peptides and cross-linking products employing MS3D software. Masses corresponding to cross-linked intramolecular peptides were found. These cross-links provided new and very valuable throughspace distance information. It is the type of throughspace distance constraint that is the key to build the protein structure model of the NKR-P1C receptor.
This study was supported by grants from the Grant Agency of the Czech Republic (P207/10/1040 and P207/10/1934), the Academy of Sciences of the Czech Republic (Institutional Research Concept AV0Z50200510) and from the Grant Agency of Charles University (403211/2011 and Project UNCE #42).