Characterization of folding intermediates of TIM barrel proteins by hydrogen exchange and mass spectrometry

How the amino acid sequence of a protein specify its three dimensional structure is referred as protein folding problem. It is the major unsolved problem in structural biology. TIM barrel is one of the most frequently encountered and the most versatile structures among the enzymes. Characterization of the folding intermediates of TIM barrel proteins is critical to solve the folding problem for TIM barrel proteins and it potentially offers insights to the folding of other protein architectures as well.;A new method, based on hydrogen/deuterium exchange, protein fragmentation and mass spectrometry was developed to identify and characterize the structures of folding intermediates at high resolution. In this method, the protein can be fragmented by an acid protease after the protein undergoes H/D exchange. The deuterium level and isotope pattern present in each fragment is determined by HPLC and mass spectrometry.;Equilibrium and kinetic folding intermediates of four TIM barrel proteins have been detected and characterized by this method. The results from rabbit muscle aldolase, a tetrameric TIM barrel protein, showed that folding to the native state involves three steps and two intermediates. Approximately 110 residues fold to highly compact forms in each step. These results also showed that each folding domain includes widely separated regions of the backbone. Interestingly, two intermediates are also populated in the equilibrium and kinetic folding experiments of monomeric S. aureus aldolase, which has high sequence homology with rabbit muscle aldolase. The structures of two intermediates are similar but not the same as those observed in the rabbit muscle aldolase, suggesting that quaternary structure affects folding/unfolding pathways. The sequential folding of two domains was observed in the folding of triosephosphate isomerase (TIM), the prototype in TIM barrel family. The C-terminal four (alpha/beta) units folds much faster than the N-terminal four (alpha/beta) units. Two intermediates are characterized in the equilibrium unfolding of a subunit of bacterial luciferase. The domain that unfolds at lower concentrations of denaturant is located at the C-terminus of the protein including the last two (alpha/beta) units.;All these results suggest that several different models are required to describe the folding of TIM barrel proteins, despite their high level of structural and functional similarity.