title: Reversible and irreversible unfolding of multi-domain proteins.
abstract: In contrast to single-domain proteins unfolding of larger multi-domain proteins is often irreversible. In a comparative case study on three different multi-domain proteins (phosphoglycerate kinase: PGK and two homologous alpha-amylases: TAKA and BLA) we investigated properties of unfolded states and their ability to fold back into the native state. For this purpose guanidine hydrochloride, alkaline pH, and thermal unfolded states were characterized. Structural alterations upon unfolding and refolding transitions were monitored using fluorescence and CD spectroscopy. Static and dynamic light scattering was employed to follow aggregation processes. Furthermore, proper refolding was also investigated by enzyme activity measurements. While for PGK at least partial reversible unfolding transitions were observed in most cases, we found reversible unfolding for TAKA in the case of alkaline pH and GndHCl induced unfolding. BLA exhibits reversible unfolding only under conditions with high concentrations of protecting osmolytes (glycerol), indicating that aggregation of the unfolded state is the main obstacle to achieve proper refolding for this protein. Structural properties, such as number and size of domains, secondary structure contents and compositions within domains, and domain topology were analyzed and considered in the interpretation of differences in refolding behavior of the investigated proteins.
authors.affiliation: Forschungszentrum Jülich, IBI-2, Biologische Strukturforschung, D-52425 Jülich, Germany.
authors.forename: K H
authors.initials: KH
authors.lastname: Strucksberg
authors.forename: T
authors.initials: T
authors.lastname: Rosenkranz
authors.forename: J
authors.initials: J
authors.lastname: Fitter
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology; D001407:Bacillus / Q000201:enzymology; D002021:Buffers; D005990:Glycerol / Q000494:pharmacology; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009994:Osmolar Concentration; D010735:Phosphoglycerate Kinase / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology*; D013696:Temperature; D044366:Transition Temperature; D000516:alpha-Amylases / Q000737:chemistry*
chemical_list: D002021:Buffers; D010735:Phosphoglycerate Kinase; D000516:alpha-Amylases; D019791:Guanidine; D005990:Glycerol