In vitro folding pathway of phage P22 tailspike protein.
The intracellular chain folding and association pathway of the thermostable, trimeric phage P22 tailspike endorhamnosidase has been the subject of a previous detailed study employing temperature-sensitive folding mutants. Recently, reconstitution of native tailspikes from completely unfolded polypeptides has been accomplished, providing a model system to compare protein folding pathways in vivo and in vitro. The in vitro reconstitution pathway of the protein after dilution from guanidine hydrochloride or acid-urea solutions at 10 degrees C was characterized by spectroscopic and hydrodynamic techniques, and may be summarized as an ordered sequence of folding, association, and folding reactions. Multiphasic folding of monomers was indicated by changes in circular dichroism and fluorescence, with a rate constant of k = 1.6 X 10(-3) s-1 for the slowest phase observed spectroscopically. Trimerization of structured monomers was followed by size-exclusion HPLC and was completed within 1.5 h at a protein concentration of 20 micrograms/mL. Although at this time trimers did not exchange subunits, they were readily dissociable by dodecyl sulfate in the cold. Formation of native, detergent-resistant trimers was only completed after 3 days of reconstitution at 10 degrees C. The reconstitution pathway of the tailspike protein closely resembles its intracellular maturation path. Thus, the in vitro reconstitution system, as a valid model of chain folding and association in vivo, should provide the tools to localize the steps or intermediates on the pathway that are the targets of temperature-sensitive folding mutations.
Fuchs
A
A
Universität Regensburg, Institut für Biophysik und Physikalische Biochemie, Federal Republic of Germany.
Seiderer
C
C
Seckler
R
R
D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D011489:Protein Denaturation; D012483:Salmonella Phages / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry* / Q000302:isolation & purification; D017376:Viral Tail Proteins
D003902:Detergents; D046911:Macromolecular Substances; D014764:Viral Proteins; D017376:Viral Tail Proteins