Probing protein folding using hydrogen/deuterium exchange-mass spectrometry

A new method and a modified method were developed to calculate folding energies of proteins based on the hydrogen deuterium exchange of globally protected amide protons. The modified method was electro spray ionization mass spectrometry (ESI-MS) based and resembles the SUPREX method. This method is called the kinetic method. A novel method was also developed which is called the protein equilibrium population snapshot (PEPS) method. Both these methods assume that the protein of interest follows a two state folding mechanism.;The HX ESI-MS based kinetic method, which is the modified SUPREX method, measures hydrogen deuterium exchange at different guanidine hydrochloride (GdHCl) concentrations as a function of time. This method utilizes ESI-MS, contrary to the usual MALDI-MS approach adopted by the SUPREX method. It measures the folding energies of proteins, which follow an EX2 type of exchange mechanism. Accurate folding energies were obtained for Ubiquitin as expected as it follows an EX2 exchange mechanism. Wild type staph nuclease and various quadruple staph nuclease mutants showed consistently higher values with good correlation. This can be attributed to deviation from EX2 exchange. Like SUPREX, this method is expected to work best for proteins, which follow EX2 exchange kinetics.;The protein equilibrium population snapshot (PEPS) method has been successfully used to accurately determine the folding energies of wild type staph nuclease and two of its mutants, which follow the EX1 mechanism of hydrogen deuterium exchange. This method measures the population distribution of open and closed states of a protein with hydrogen deuterium exchange as a function of denaturant concentration, under equilibrium. This method. when applied to Ubiquitin, which follows EX kinetics, also generated accurate folding energies comparable with literature values from NMR and fluorescence studies. So it is determined that the novel PEPS method is capable of measuring folding energies of a protein, regardless of the exchange mechanism is EX1 or EX2. This method is also based on fewer assumptions than the kinetic method.