Abstract
Maize ferredoxin-NADP(+) reductase (FNR) consists of flavin adenine dinucleotide (FAD) and NADP(+) binding domains with a FAD molecule bound noncovalently in the cleft between these domains. The structural changes of FNR induced by dissociation of FAD have been characterized by a combination of optical and biochemical methods. The CD spectrum of the FAD-depleted FNR (apo-FNR) suggested that removal of FAD from holo-FNR produced an intermediate conformational state with partially disrupted secondary and tertiary structures. Small angle x-ray scattering indicated that apo-FNR assumes a conformation that is less globular in comparison with holo-FNR but is not completely chain-like. Interestingly, the replacement of tyrosine 95 responsible for FAD binding with alanine resulted in a molecular form similar to apo-protein of the wild-type enzyme. Both apo- and Y95A-FNR species bound to Cibacron Blue affinity resin, indicating the presence of a native-like conformation for the NADP(+) binding domain. On the other hand, no evidence was found for the existence of folded conformations in the FAD binding domains of these proteins. These results suggested that FAD-depleted FNR assumes a partially folded structure with a residual NADP(+) binding domain but a disordered FAD binding domain.
Highlights
Erties similar to those of kinetic folding intermediates have been found under mild denaturing conditions in equilibrium [7, 8]
Structure and Stability of the Intermediates—The present study showed that two apoenzymes of ferredoxin-NADPϩ reductase (FNR) produced by different methods assume partially folded structures rather than being fully unfolded (Tables I and II)
The reconstitution of holo-FNR was demonstrated by adding flavin adenine dinucleotide (FAD) to apo-FNR, suggesting that the partially folded species is a productive intermediate rather than a misfolded state
Summary
Protein Preparation—Recombinant maize leaf FNR [18] and ferredoxin [19] were prepared using an Escherichia coli expression system. The fraction containing Y95A-FNR was further purified on a column of HiTrap Blue (Amersham Biosciences), which was equilibrated with 50 mM Tris-HCl buffer (pH 7.5), by developing with a linear gradient of 0 –1 M NaCl in the same buffer. The resulting transition curves were analyzed by nonlinear least squares curve fitting, assuming a two-state transition [24] as shown in the following equation In this equation, [Signal] is either the ellipticity at 222 nm or fluorescence at 530 nm, a and c are the intercepts, and b and d are the slopes of the base lines for native and unfolded species, respectively, R is the gas constant, and T is a temperature. The protein solutions containing various amounts of FAD were incubated for 3 days at 4 °C before fluorescence measurements
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
