Conformational changes accompanying proteolytic cleavage of human complement protein C3b by the regulatory enzyme factor I and its cofactor H. Spectroscopic and enzymological studies.
The conformational basis for the loss of C3b functional site expression following its conversion to iC3b by the regulatory proteins factor H and factor I has been examined by a number of spectroscopic techniques including the fluorescence of the extrinsic probe 8-anilino-1-naphthalenesulfonate, circular dichroism, and UV absorption difference spectroscopy. These techniques all detected significant conformational alterations accompanying the proteolytic cleavage event. To a substantial extent, the observed spectral changes tended to reverse those seen in the original conversion of native C3 to its functionally active C3b form. Similar changes were also seen when the thioester-cleaved, but peptide chain-intact, form of C3 was acted upon by the regulatory proteins. The most dramatic effect was seen in the 8-anilino-1-naphthalenesulfonate fluorescence system where the fluorescence enhancement observed upon conversion of C3 to C3b, which presumably reflects an increase in surface hydrophobicity, is lost upon further cleavage of the molecule to iC3b. This marked signal change forms the basis of a simple spectrofluorometric assay with which to study the enzymatic properties of factor I for its true substrate: the complex formed between C3b and factor H. Using this assay, factor I was found to display very tight binding of its substance, the Km at 25 degrees C being on the order of 0.025-0.1 microM, but it was also found to show a low rate of cleavage, the turnover number being less than 10 min-1 at this temperature.
D002942:Circular Dichroism; D003176:Complement C3 / Q000378:metabolism; D003179:Complement C3b / Q000378:metabolism*; D003180:Complement C3b Inactivator Proteins / Q000378:metabolism*; D017242:Complement Factor H; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
C493495:CFH protein, human; D003176:Complement C3; D003180:Complement C3b Inactivator Proteins; D003179:Complement C3b; D017242:Complement Factor H; D005340:Fibrinogen