Does Chemical Cross-Linking with NHS Esters Reflect the Chemical Equilibrium of Protein-Protein Noncovalent Interactions in Solution?

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December 23, 2010

Authors

Stefanie Mädler1, Markus Seitz2, John Robinson2, and Renato Zenobi1

Organizations

Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland
Institute of Organic Chemistry, University of Zurich, Zurich, Switzerland

Abstract

Chemical cross-linking in combination with mass spectrometry has emerged as a powerful tool to study noncovalent protein complexes. Nevertheless, there are still many questions to answer. Does the amount of detected cross-linked complex correlate with the amount of protein complex in solution? In which concentration and affinity range is specific cross-linking possible? To answer these questions, we performed systematic cross-linking studies with two complexes, using the N-hydroxysuccinimidyl ester disuccinimidyl suberate (DSS): (1) NCoA-1 and mutants of the interacting peptide STAT6Y, covering a KD range of 30 nM to >25 μM, and (2) α-thrombin and basic pancreatic trypsin inhibitor (BPTI), a system that shows a buffer-dependent KD value between 100 and 320 μM. Samples were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). For NCoA-1· STAT6Y, a good correlation between the amount of cross-linked species and the calculated fraction of complex present in solution was observed. Thus, chemical cross-linking in combination with MALDI-MS can be used to rank binding affinities. For the mid-affinity range up to about KD ≈ 25 μM, experiments with a nonbinding peptide and studies of the concentration dependence showed that only specific complexes undergo cross-linking with DSS. To study in which affinity range specific cross-linking can be applied, the weak α-thrombin · BPTI complex was investigated. We found that the detected complex is a nonspecifically cross-linked species. Consequently, based on the experimental approach used in this study, chemical cross-linking is not suitable for studying low-affinity complexes with K_D >> 25 μM.

CovalX Technology Used (Click each option to learn more)

HM1

Outcomes

NCoaA-1 PAS-B domain was dialyzed against phosphate buffer (10 mM Na2HPO4/NaH2PO4/150 mM NaCl, pH 8.0) before the 70 μM final solution was diluted to a range of 10 to 30 μM with the phosphate buffer. Alanine or cyclohexylalanine mutants of a linear peptide containing STAT6 residues L794 to G814 were obtained by solid-phase peptide synthesis, dissolved in water and then diluted using the phosphate buffer to the same range.

10 μL of 10 μM NCoaA-1 protein solution was incubated with 10 μL of 10 μM peptide solution for 10 minutes at 2 °C. 10 μL of 10, 20 or 30 μM coactivator protein solutions were incubated with equally concentrated peptides mutated with alanine (position 794, 796, or 797). 10 μL of 10,20,30 or 70 μM coactivator protein solution incubated with equal concentrations of the nonbinding peptide oxidized bovine insulin chain A were used as negative controls. 10 μL of each solution was mixed with DSS (50-fold molar excess) dissolved in dimethylformamide (DMF) at 2,4, or 6 mg/mL in a ratio of 10/1. The samples were incubated between 2 and 8 °C for four hours. 1 μL of DMF was added to the remaining solution as a control. Both solutions were quenched with 5 μL of 50 mM NH4HCO3 and purified using a 0.6 μl pipette tip with C4 resin. If the final protein concentration was above 5 μM, the reaction mixtures and control were diluted so that the final protein concentration was comparable to the concentration of the first experiment before the addition of the resin in the pipette tip. The α-thrombin from the α-thrombin•BPTI complex had a final concentration of proteins of approximately 20 μM and was incubated for 15 minutes with BPTI (equimolar, 2 or fold amount) in various buffers (0 mM Na2HPO4/NaH2PO4 pH 8 no additional salt, 145 mM tetramethylammonium chloride/ mM NaCl or 150 mM NaCl). 9 μL of the solution was mixed with DSS (30 fold molar excess) at room temperature in a 10/1 volume ratio for various times.

The NCoA-1•STAT6Y complex was mixed with the matrix (sinapic acid dissolved at 10 mg/ml in water/acetonitrile/TFA (49.95/49.95/0.1, v/v/v) after being quenched and purified in a 1/1 volume ratio. The α-thrombin•BPTI complex was mixed directly with the matrix using the same ratio. 1 μL of each final mixture was spotted onto a stainless steel MALDI plate before being allowed to dry under ambient conditions. Two different MALDI mass spectrometers, each modified with the CovalX HM1 detection system, were used to measure the complexes.