Comparison of solution properties of human and rat ciliary neurotrophic factor.
The solution structure and stability of rat and human ciliary neurotrophic factor (CNTF) were examined by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopy and sedimentation equilibrium analyses. The secondary structure of both proteins, as assessed by CD and FTIR, consists primarily of alpha-helix, consistent with CNTF being a member of the four-helical bundle family of cytokines and neurokines, with rat CNTF containing slightly less helix (about 10% less) and slightly more disordered structure. The environment of the tyrosine and tryptophan residues, assessed by intrinsic fluorescence emission spectroscopy, appears to be the same in both proteins. Binding of anilinonaphthalene sulfonate is identical for both proteins, indicating that these two proteins have similar surface hydrophobicities in the native state. The thermal stability of the human CNTF is significantly less than that of the rat CNTF, yet their stabilities to guanidine HCl-induced denaturation are equivalent. This apparent discrepancy in stability between the two proteins may be explained by solubility differences upon thermal unfolding. Although the human protein precipitates as it is denatured by heat, the rat protein does not. It thus appears that the unfolded state of human CNTF is less soluble and more prone to aggregation than that of the rat protein upon heating, although their conformational stability is similar. Both proteins remain largely folded at pH 3.0. Sedimentation equilibrium analysis demonstrates that both rat and human CNTF exist primarily as monomers; however, significant dimer formation occurs as the protein concentrations are increased to greater than 3 mg/mL, particularly in the presence of ammonium sulfate.
Narhi
L O
LO
Amgen Inc., Amgen Center, Thousand Oaks, California, USA. LNARHI@amgen.com
Rosenfeld
R
R
Shimamoto
G
G
Lee
R
R
Hawkins
N
N
Li
T
T
Philo
J
J
Wen
J
J
Arakawa
T
T
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D020934:Ciliary Neurotrophic Factor; D002942:Circular Dichroism; D019281:Dimerization; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009414:Nerve Growth Factors / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014461:Ultracentrifugation
D000817:Anilino Naphthalenesulfonates; D020934:Ciliary Neurotrophic Factor; D005456:Fluorescent Dyes; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; C027132:1-anilino-8-naphthalenesulfonate