The conformational stability of a non-covalent dimer of a platelet-derived growth factor-B mutant lacking the two cysteines involved in interchain disulfide bonds.
Native platelet-derived growth factor-B (PDGF-B) forms a covalent dimer through interchain disulfide bonds. In a previous study, an analog of PDGF-B was produced by replacing cysteine 43 and 52, which are involved in the interchain disulfide bonds, with serine. It was revealed that this analog protein has the dimeric molecule weight at pH 4 to 7, forming a non-covalent dimer in solution, and its mitogenic activity is similar to the native covalent dimer. However, the analog protein was more labile to pepsin digestion and low pH treatment, indicating that the interchain disulfides contribute to the stability of the protein. It is interesting to see if the conformation of the protein is affected by elimination of the interchain disulfide bonds, and if the interchain disulfides play any role in the stability of the protein. Circular dichroism and Fourier transform infrared spectroscopic analyses of the analog showed that it has a conformation similar to the wild type at pH 7.5, but is unfolded at pH 2.5, while the native PDGF-B disulfide-linked dimer shows an apparently unaltered conformation at pH 2.5. The analog is also less stable to sodium dodecylsulfate and guanidine HCl-induced denaturation at neutral pH. These results indicate that the non-covalent interactions are sufficient for proper folding and dimer formation at neutral pH, but that the interchain disulfide bonds greatly stabilize the native conformation of PDGF-B.
Prestrelski
S J
SJ
Amgen Inc., Amgen Center, Thousand Oaks, California.
Arakawa
T
T
Duker
K
K
Kenney
W C
WC
Narhi
L O
LO
D002942:Circular Dichroism; D003553:Cystine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004355:Drug Stability; D019791:Guanidine; D006146:Guanidines; D009154:Mutation; D010982:Platelet-Derived Growth Factor / Q000737:chemistry* / Q000235:genetics*; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics*; D020574:Proto-Oncogene Proteins c-sis; D012967:Sodium Dodecyl Sulfate; D017550:Spectroscopy, Fourier Transform Infrared
D004220:Disulfides; D006146:Guanidines; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D012967:Sodium Dodecyl Sulfate; D003553:Cystine; D011505:Protein-Tyrosine Kinases; D019791:Guanidine