Quaternary structure of aldolase leads to differences in its folding and unfolding intermediates.
Pulsed hydrogen exchange mass spectrometry has been used to investigate folding of rabbit muscle aldolase, an alpha/beta-barrel protein exhibiting the classic TIM structure. Aldolase unfolded in GdHCl refolded as the denaturant concentration was reduced by dialysis. Samples withdrawn during dialysis were pulse-labeled with deuterium to identify unfolded regions in structural forms highly populated during the folding process. Intact, labeled aldolase was digested into fragments, which were analyzed by HPLC electrospray ionization mass spectrometry to detect the H/D exchange along the aldolase backbone. For some concentrations of GdHCl, bimodal distributions of deuterium were found for most peptic fragments, indicating that regions represented by these fragments were either unfolded or folded in the intact polypeptide prior to labeling. The extent of folding was determined from these mass spectra, as well as by CD (220 nm) and enzymatic activity. These results show that folding to the active form involves three domains and two intermediates. Approximately 110 residues fold to highly compact forms in each step. These results also show that each folding domain includes widely separated regions of the backbone. When compared with the results of a previous study of aldolase unfolding, these results show that the folding and unfolding domains include most of the same residues. However, three short segments change domains depending on whether the process is folding or unfolding. These changes are attributed to the very stable quaternary structure of rabbit muscle aldolase.
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA.
Hai
H
Pan
David L
DL
Smith
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium; D004795:Enzyme Stability; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry*; D019791:Guanidine; D006859:Hydrogen / Q000737:chemistry; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011817:Rabbits
D010446:Peptide Fragments; D006859:Hydrogen; D003903:Deuterium; D005634:Fructose-Bisphosphate Aldolase; D019791:Guanidine