Detection of early unfolding events in a dimeric protein by amide proton exchange and native electrospray mass spectrometry.
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(+) 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(+) synthetase.
Mobley
James A
JA
Department of Surgery/Urology, Laboratory of Clinical Proteomics, University of Alabama, Birmingham, Alabama 35294, USA.
Poliakov
Anton
A
D019732:Amide Synthases / Q000737:chemistry*; D001412:Bacillus subtilis / Q000201:enzymology*; D011489:Protein Denaturation; D017510:Protein Folding; D055503:Protein Multimerization; D011522:Protons; D021241:Spectrometry, Mass, Electrospray Ionization
D011522:Protons; D019732:Amide Synthases; C038479:NAD+ synthase