Abstract:
:The unfolding and refolding of alpha-helical proteins has been extensively studied, demonstrating formation of intermediate structures which retain the native-like alpha-helix but lack the tertiary structure. Studies on the folding of proteins consisting primarily of beta-sheet are interesting since, unlike the alpha-helix, the beta-sheet requires the formation of peptide hydrogen bonds between two or more polypeptide segments which may be far apart in the linear sequence. Here we have studied the unfolding of the beta-sheet-containing protein tumor necrosis factor-alpha (TNF-alpha). This protein exists as a symmetric trimer in solution. Murine TNF-alpha begins to melt at 60 degrees C and unfolds to a soluble structure with a transition midpoint of 66 degrees C. This reaction is irreversible. This unfolded form contains a considerable amount of (approximately 30%) alpha-helix, as determined by circular dichroism. Human TNF-alpha begins to melt at 60 degrees C and precipitates concurrently with unfolding, such that there is no soluble protein present by 70 degrees C. The secondary and tertiary structures of murine TNF-alpha unfold simultaneously, suggesting that unfolding from the native to the unfolded state occurs cooperatively. The thermal-induced denaturation is very insensitive to protein concentration, indicating that trimer to monomer conversion, if it occurs, is not rate-limiting. Trifluoroethanol induces alpha-helix in both human and murine TNF-alpha, further demonstrating the propensity of TNF-alpha to form alpha-helix. The different behavior of human versus murine TNF-alpha upon thermal unfolding is due to differences in the solubility of the unfolded protein, the murine form being more soluble. These results indicate that TNF-alpha can form alpha-helix when the long range interactions conferred by the native structure are removed during unfolding.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Narhi LO,Philo JS,Li T,Zhang M,Samal B,Arakawa Tdoi
10.1021/bi952766vsubject
Has Abstractpub_date
1996-09-03 00:00:00pages
11447-53issue
35eissn
0006-2960issn
1520-4995pii
bi952766vjournal_volume
35pub_type
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