Anabaena apoflavodoxin hydrogen exchange: On the stable exchange core of the α/β(21345) flavodoxin‐like family

Abstract

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (k(ex) < 10(-1) min(-1)). According to their distribution in the structure, protein stability is highest on the beta-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other alpha/beta (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central beta-strands and in helix 5 is common to the cores.

How FMN Binds to Anabaena Apoflavodoxin

“…It is thus tempting to speculate that the binding of FMN starts by association at the phosphate-binding site. Conflicting with this hypothesis, solution studies on the structure of the Anabaena (20) and Azotobacter (26) apoflavodoxins suggest that the isoalloxazinebinding site is very flexible. This flexibility is also evidenced by the different x-ray structures observed for the Trp 57 loop in wild type and mutant Anabaena flavodoxins (7).…”

“…1 and Ref. 16), and in addition, an extensive characterization of the solution behavior of the two forms has been performed (7,(17)(18)(19)(20)(21)(22)(23)(24). The binding of FMN to apoflavodoxins from several species seems to occur in one step (5,25), although the molecular details are not known.…”

The ribosome regulates flavodoxin folding

“…In vitro, the folding of the C-terminal part of MG off precedes the folding of the N-terminal part and the MG gradually compacts due to progressive extension of its ordered core (74). (83), with α-helices in green and β-strands in red. FMN is shown in yellow.…”

“…In native apoflavodoxin from Anabaena PCC 7119 the cofactor-binding site is flexible and FMN binds preferentially to native apo-protein (82)(83)(84). Full release of cofactor happens upon unfolding of this 169-residue long-chain flavodoxin (85).…”

“…Full release of cofactor happens upon unfolding of this 169-residue long-chain flavodoxin (85). Both urea-and GuHCl-dependent equilibrium folding of apoflavodoxin are two-state (83,85,86). During kinetic folding an intermediate transiently accumulates, whose unfolding is rate-limiting (3).…”