Changes in protein conformation and stability accompany complex formation between human C1 inhibitor and C1-s.
The fluorescence spectrum of C1 inhibitor (C1-Inh) in aqueous buffer has a maximum at 324 nm which shifts to 358 nm in 6.0 M guanidinium chloride (GdmC1), indicating that fluorescent tryptophans are buried in the native protein. When titrated with GdmC1, the fluorescence intensity, polarization, and emission maximum of C1-Inh and C1-s exhibited clear transitions which were more prominent than those of the enzyme-inhibitor complex. Two of the variables (intensity and emission maximum) suggest biphasic unfolding of C1-Inh. Differential absorption measurements and sodium iodide quenching of intrinsic fluorescence were consistent with a net increase in the exposure of tryptophans and tyrosines upon complex formation. This reaction, i.e., complex formation, was also accompanied by an increase in the ability to enhance the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate. Fluorescence assays of heat denaturation showed transitions at 40 and 52 degrees C for C1-s and at 60 degrees C for C1-Inh whereas there was no detectable melting transition for the complex. Similarly, differential scanning calorimetric measurements revealed transitions at 42, 52, and 62 degrees C for C1-s and one transition at 60 degrees C for C1-Inh, with no major transitions detectable for the complex. The ratio of the calorimetric enthalpy to the apparent van't Hoff enthalpy for thermal unfolding of C1-Inh was 1.6. Taken together, these results suggest that C1-Inh and C1-s are each composed of at least two independently unfolding domains and that complex formation, which involves conformational change, yields a protein substantially more stable than either component alone.
D002151:Calorimetry; D003166:Complement Activating Enzymes / Q000378:metabolism*; D003174:Complement C1 Inactivator Proteins / Q000378:metabolism*; D003173:Complement C1s; D004355:Drug Stability; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D003174:Complement C1 Inactivator Proteins; D006146:Guanidines; D046911:Macromolecular Substances; D003166:Complement Activating Enzymes; D003173:Complement C1s; D019791:Guanidine