Stability and reconstitution of the soluble variant surface glycoprotein (sVSG) from Trypanosoma brucei.
Soluble variant surface glycoprotein (sVSG) is the form of the coat protein of Trypanosoma brucei released by cleavage of its lipid anchor. As shown by ultracentrifugal analysis, the protein of the variant clone MITat 1.2 is a stable dimer of (117 +/- 6)-kDa molecular mass. Its quaternary structure remains unaltered in the concentration range from 0.01 to approximately 50 mg/mL. Further extrapolation to the in situ concentration on the cell surface points to no significant protein association beyond the dimer, because after correction for solution viscosity sedimentation velocity is independent of the protein concentration. The sedimentation constant, s20,w = 5.1 X 10(-13) s, together with the particle weight confirms the high anisotropy of the dimer. Circular dichroism and fluorescence spectra show the typical properties of an alpha-helical protein (51% alpha-helix) with fluorophores buried in the hydrophobic interior of the protein. Denaturation at extremes of pH leads to the monomer still maintaining a relatively compact structure. Increased concentrations of urea and guanidine hydrochloride cause randomization with cooperative transitions at 1.7 and 0.7 M, respectively. The yield of reconstitution of the denatured protein reaches 87% under optimum conditions. The final product is indistinguishable from the native protein in its spectral, hydrodynamic, and immunochemical properties. Immunological analysis included polyclonal antibodies as well as monoclonal antibodies raised against epitopes in the surface of the complete trypanosome, as well as cryptic epitopes exposed only on sVSG in solution. The kinetics of reconstitution involve sequential uni-bimolecular processes, corresponding to consecutive folding and subunit association. About 38% of the fluorescence of the native protein is recovered within the mixing time (secondary structure formation).(ABSTRACT TRUNCATED AT 250 WORDS)
Rehaber
P
P
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, West Germany.
Staudacher
N
N
Seckler
R
R
Bülow
R
R
Overath
P
P
Jaenicke
R
R
D000818:Animals; D000940:Antigenic Variation; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D014346:Trypanosoma brucei brucei / Q000032:analysis / Q000276:immunology*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification
D014643:Variant Surface Glycoproteins, Trypanosoma