CONFORMATIONAL STABILITY OF APOFLAVODOXIN

Flavodoxins are alpha/beta proteins that mediate electron transfer rea ctions. The conformational stability of apoflavodoxin from Anabcena PC C 7119 has been studied by calorimetry and urea denaturation as a func tion of pH and ionic strength. At pH > 12, the protein is unfolded. Be tween pH 11 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism CCD) and high-field H-1 NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfol ding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is un usually low. Near its isoelectric point (3.9), the protein is highly i nsoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conf ormation with the properties of a molten globule. Although apoflavodox in at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transit ion midpoint depends on the concentration of protein, ruling out a sim ple two-state process at acidic pH. Apoflavodoxin constitutes a promis ing system for the analysis of the stability and folding of alpha/beta proteins and for the study of the interaction between apoflavoprotein s and their corresponding redox cofactors.