Title : Observation of Protein Unfolding during pH Ramp Evoked by Lipase-Catalyzed Ester Hydrolysis - Shiu_2023_J.Am.Soc.Mass.Spectrom__ |
Author(s) : Shiu RT , Prabhu GRD , Elpa DP , Urban PL |
Ref : J Am Soc Mass Spectrom , : , 2023 |
Abstract :
Studies of protein folding often involve offline experimental methods such as titrating protein samples with denaturants or equilibrating them in the presence of denaturants. Here, we demonstrate an online analytical approach in which the protein structure is perturbed by a pH ramp evoked by immobilized lipase-catalyzed ester hydrolysis. Changes in the tertiary structure of the protein in response to a pH ramp (from approximately 6.3 to 2.8) are monitored using electrospray ionization mass spectrometry and spectrofluorometry. Interestingly, we discovered a side reaction of ammonium and formate leading to the production of cyanide that occurred during the ionization process. We also found that only certain protein analytes were bound to the formed cyanide species. Nevertheless, this problem was readily overcome by carefully selecting a specific ester substrate. Overall, the alterations in the charge-state distribution and fluorescence intensitycaused by the lipase-induced pH rampreveal conformational transitions in different proteins. In line with previous reports, the acid-induced denaturation of holo-myoglobin occurs through a two-step mechanism, which is supported by identification of protein-unfolding intermediates and the loss of noncovalent protein ligand (heme). The resultsobtained using the developed catalytic methodare also consistent with the results of equilibrium-based experiments, while sample preparation steps are substantially reduced. The proposed approach simplifies the identification of the pH range that has the greatest impact on the protein structure. Thus, it has the potential to be a useful tool for studying protein conformational transitions in the course of pH changes. |
PubMedSearch : Shiu_2023_J.Am.Soc.Mass.Spectrom__ |
PubMedID: 37620995 |
Shiu RT, Prabhu GRD, Elpa DP, Urban PL (2023)
Observation of Protein Unfolding during pH Ramp Evoked by Lipase-Catalyzed Ester Hydrolysis
J Am Soc Mass Spectrom
:
Shiu RT, Prabhu GRD, Elpa DP, Urban PL (2023)
J Am Soc Mass Spectrom
: