Alteration in folding efficiency and conformation of recombinant human tumor necrosis factor-alpha by replacing cysteines 69 and 101 with aspartic acid 69 and arginine 101.
An analog of human tumor necrosis factor-alpha (TNF-alpha) was created involving the replacement of Cys69 with Asp and Cys101 with Arg. The solution structure and behavior of this analog were compared with the native protein. The analog exhibited a greatly decreased folding efficiency following dilution from urea, but essentially identical circular dichroic spectra in both the folded and unfolded states. The Stokes radius of the native and analog TNF-alpha in the folded state were identical, with the analog exhibiting a slight broadening of the eluting peak. The fluorescence emission spectrum of the native protein exhibits a plateau from 320 to 328 nm, while the spectrum of the analog consisted of a single peak with a maximum at 335 nm. The analog also had a 1.4-fold increase in the fluorescence intensity. Limited proteolysis of the analog resulted in only one of the two peptides seen following digestion of the native protein, and this product was less stable than the equivalent native protein fragment. The analog exhibited a 10-fold lower cytolytic activity than the native protein. These results demonstrated that the disulfide bond is not necessary for folding and activity, but are consistent with the analog having a looser, more flexible structure in solution than the native TNF-alpha.
Arakawa
T
T
Amgen Inc., Amgen Center, Thousand Oaks, CA 91320.
Visger
J V
JV
McGinley
M
M
Rohde
M F
MF
Fox
G M
GM
Narhi
L O
LO
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D006801:Humans; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha