The denaturant-induced~un!folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1 H-15 N heteronuclear single-quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR~un!folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule-like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1 H-15 N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative. Keywords: apoflavodoxin; cooperative and noncooperative unfolding; equilibrium~un!folding; guanidinium hydrochloride; molten globule; NMR; protein aggregation; residue levelFlavodoxin is an interesting model to study protein folding and stability. In contrast to most protein folds, the flavodoxin-like fold is shared by many~i.e., nine! superfamilies~Brenner, 1997!. These nine superfamilies exhibit little or no sequence similarity, and comprise a broad range of unrelated proteins with different functions such as catalases, chemotactic proteins, lipases, esterases, and flavodoxins. They are all characterized by a five-stranded parallel b-sheet surrounded by a-helices at either side of the sheet. Studies on these structurally but not sequentially related proteins are pre-eminently suitable to study protein folding and stability. By studying the folding of flavodoxin, we expect to obtain a better understanding of the fundamental rules describing the folding of proteins with a flavodoxin-like fold.Flavodoxins are a group of small flavoproteins that function as low-potential one-electron carriers and contain a noncovalently bound FMN cofactor~Mayhew & Tollin, 1992!. The protein investigated by us is flavodoxin II from Azotobacter vinelandii~strain ATCC 478!, henceforth designated flavodoxin. The protein consists of 179 amino acid residues and belongs to the class of "longchain" flavodoxins~Tanaka et al., 1977!. Upon removal of the flavin, apoflavodoxin is generated. Despite the removal of the FMN cofactor, large parts of the tertiary structure of holo-and apoflavodoxin are strictly conserved as is reflected among others in the identity of NMR chemical shifts~Fig. 1!. Native apoflavodoxin has a stable well-ordered core, b...

The coil-globule transition has been studied for A-B copolymer chains both by means of lattice Monte Carlo (MC) simulations using bond fluctuation algorithm and by a numerical self-consistent-field (SCF) method. Copolymer chains of fixed length with A and B monomeric units with regular, random, and specially designed (proteinlike) primary sequences have been investigated. The dependence of the transition temperature on the AB sequence has been analyzed. A proteinlike copolymer is more stable than a copolymer with statistically random sequence. The transition is more sharp for random copolymers. It is found that there exists a temperature below which the chain appears to be in the lowest energy state (ground state). Both for random and proteinlike sequences and for regular copolymers with a relatively long repeating block, a molten globule regime is found between the ground state temperature and the transition temperature. For regular block copolymers the transition temperature increases with block size. Qualitatively, the results from both methods are in agreement. Differences between the methods result from approximations in the SCF theory and equilibration problems in MC simulations. The two methods are thus complementary.

We present numerical self-consistent field calculations in a two-gradient cylindrical coordinate system for a ͑translationally restricted͒ quenched polyelectrolyte star which is electrostatically attracted to an oppositely charged surface with homogeneous surface charge density at a given ionic strength of a 1:1 electrolyte. The results prove that without any additional driving force for adsorption, electrostatic attraction alone can give significant overcompensation of the surface charge provided that the ionic strength is below some critical value. This is demonstrated for the case that the charge density on the surface is lower than the ͑projected͒ charge density in the star. In the regime of charge overcompensation, the thickness of the adsorbed layer is of the order of the star size in solution. The adsorbed layer is laterally inhomogeneous and the outer part of the adsorption profile is locally neutralized by the small counterions.

No abstract