Thermal unfolding of G-actin monitored with the DNase I-inhibition assay stabilities of actin isoforms.
Actin is one of the proteins that rely on chaperonins for proper folding. This paper shows that the thermal unfolding of G-actin, as studied by CD and ultraviolet difference spectrometry, coincides with a loss in DNase I-inhibiting activity of the protein. Thus, the DNase I inhibition assay should be useful for systematic studies of actin unfolding and refolding. Using this assay, we have investigated how the thermal stability of actin is affected by either Ca2 + or Mg2 + at the high affinity divalent cation binding site, by the concentration of excess nucleotide, and by the nucleotide in different states of phosphorylation (ATP, ADP.Pi, ADP. Vi, ADP.AlF4, ADP.BeFx, and ADP). Actin isoforms from different species were also compared, and the effect of profilin on the thermal stability of actin was studied. We conclude that the thermal unfolding of G-actin is a three-state process, in which an equilibrium exists between native actin with bound nucleotide and an intermediate free of nucleotide. Actins in the Mg-form were less stable than the Ca-forms, and the stability of the different isoforms decreased in the following order: rabbit skeletal muscle alpha-actin = bovine cytoplasmic gamma-actin > yeast actin > cytoplasmic beta-actin. The activation energies for the thermal unfolding reactions were in the range 200-290 kJ.mol- 1, depending on the bound ligands. Generally, the stability of the actin depended on the degree with which the nucleotide contributed to the connectivity between the two domains of the protein.
Schüler
H
H
Department of Cell Biology, The Wenner-Gren Institute, Stockholm University, Sweden.
Lindberg
U
U
Schutt
C E
CE
Karlsson
R
R
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002412:Cations; D002417:Cattle; D003285:Contractile Proteins; D003593:Cytoplasm / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000378:metabolism*; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010590:Phalloidine / Q000737:chemistry / Q000378:metabolism; D051304:Profilins; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D013696:Temperature
D000199:Actins; D002412:Cations; D003285:Contractile Proteins; D008840:Microfilament Proteins; D009711:Nucleotides; D051304:Profilins; D020033:Protein Isoforms; D010590:Phalloidine; D003850:Deoxyribonuclease I