Detection of early unfolding events in a dimeric protein by amide proton exchange and native electrospray mass spectrometry

Abstract

Oligomeric proteins generally undergo unfolding through a dissociation/denaturation mechanism wherein the subunits first dissociate and then unfold. This mechanism can be detected by the fact that the proteins exhibit a concentration dependence of the denaturation curve. However, the concentration dependence does not answer the question of whether there are thermally induced conformational changes that facilitate subunit dissociation. To fully probe these mechanisms it is desirable to have an analytical approach that is capable of measuring both subunit dissociation and protein denaturation in a highly sensitive manner. In this article, we demonstrate that the combined use of native mass spectrometry to detect subunit mixing, and amide hydrogen/deuterium exchange to detect transient unfolding events can provide a very unique insight into the pre-melting transitions in a protein oligomer. Both methods keep an isotopic record of each transformation event, without the dependence on equilibrium of the unfolding reaction. Here, we use a combined form of H/D exchange/mass spectrometry and isotopic labeling/native electrospray mass spectrometry to study the pre-unfolding events of Bacillus subtilis NAD 1 synthetase, a symmetrical dimer protein, which plays a vital role in the lifecycle of the bacteria. In the experimental outcome provided, we were able to clearly illustrate that at elevated temperatures, the NAD synthetase dimer undergoes reversible dissociation without monomer unfolding, while at temperatures where monomer unfolding is observed to take place, the rate of dimer dissociation still yet exceeds the rate of unfolding. Information provided by combining these two mass spectrometric methods was found to be very robust, and allowed us to establish an NAD synthetase unfolding model, where primary dissociation occurs prior to the complete unfolding of the NAD 1 synthetase.

X-ray crystallographic validation of structure predictions used in computational design for protein stabilization

“…Another important protein stabilization mechanism is the improvement of intersubunit interactions. The disulfide bond G89C‐S91C' enhances interactions between the monomers and increases the energy required to dissociate the dimer . Other frequently reported stabilization mechanisms such as the removal of oxidation‐prone residues or the filling of internal cavities are not observed in the structures of LEH‐P and F1b .…”

“…Disulfide bond formation between residues 89 and 91' and 91 with 89' (Δ T M, app = 15.0 °C) creates two covalent links between the monomers, which reduces unfolding caused by dissociation into subunits . However, C89 and C91 in monomers A and B have a dual conformation.…”

“…However, the consensus method revealed no preference for lysine or arginine residues at positions 45, 76, or 92, while FRESCO introduced stabilizing surface‐located positive charges at these positions (Table ). It is likely that protein surface charges vary greatly among homologous protein, since they influence many protein characteristics besides stability, such as activity, solubility and protein‐protein interactions . Furthermore, the effect of mutations influencing surface‐charges will depend on the presence and distribution of other charged and polar groups on the protein surface.…”

“…Better modeling of intrinsic protein flexibility and improved conformational sampling for mutant structure prediction can also increase predictive quality. The identification of labile areas may be done by a variety of techniques such as electron density analysis, conformational plasticity analysis of homologous structures, B‐factor analysis, flexibility analysis by hydrogen deuterium exchange mass spectrometry or MD simulations . An approach that more selectively stabilizes flexible areas and that takes into account backbone changes is expected to further improve the quality of mutant libraries obtained by computational design.…”

“…The disulfide bond G89C-S91C' enhances interactions between the monomers and increases the energy required to dissociate the dimer. 46,47,57 Other frequently reported stabilization mechanisms such as the removal of oxidationprone residues or the filling of internal cavities are not observed in the structures of LEH-P and F1b. 58 In conclusion, three mechanisms seem to be responsible for the majority of the obtained stabilization interactions: (i) enhanced interactions between the flexible loop close to the N-terminus and the rest of the protein; (ii) improved interactions at the dimer interface; and (iii) removal of unsatisfied hydrogen bonding groups.…”