NMR Structures of Salt-Refolded Forms of the 434-Repressor DNA-Binding Domain in 6 M Urea

Abstract

The N-terminal 63-residue fragment of the phage 434-repressor, 434(1-63), has a well-defined globular fold in H 2 O solution, and is unfolded in 6 M urea at pH 7.5. In this study, 434(1-63) has been refolded by adding either 1.7 M NaCl or 0.47 M NaTFA to the solution in 6 M urea, and the NMR structures of both refolded forms have been determined. The two refolded forms have similar free energies of unfolding and are ∼16 kJ/mol less stable than the protein in H 2 O solution. 434(1-63) refolded with NaCl exhibits NMR chemical shifts very similar to and a three-dimensional structure nearly identical to those of 434(1-63) in H 2 O solution. The protein refolded with NaTFA also has a similar global fold, but it shows local differences near Phe44, of which two different orientations of the aromatic ring are compatible with the experimental data. This local conformational polymorphism attracted our interest because hydrophobic contacts between two subdomains of residues 1-36 and 45-63 are mediated by the Phe44 side chain. Anion binding experiments suggest that this polymorphism is caused by binding of TFAanions to a cluster of positively charged Arg and Lys residues located in the loop connecting the two subdomains, with apparent binding constants for TFA -(K app ) on the order of 30 mM -1 .

Molecular Mechanism Underlying the Thermal Stability and pH-induced Unfolding of CHABII

“…It is possible that, as recently proposed, a large population of proteins still maintained a nativelike tertiary topology under a variety of denaturing conditions. [64][65][66][67][68][69] The broadening and disappearance of NMR peaks observed for many partially folded or even completely unfolded proteins might result from a slight increase of conformational dynamics on the microseconds to milliseconds time-scales, rather than from the highly disordered tertiary topology.…”

Urea production capacity in the wood frog (Rana sylvatica) varies with season and experimentally induced hyperuremia

“…Although this effect might explain our findings for saline-treated frogs, salt stabilization was not expressed in urea-treated frogs, which instead maintained levels of CPS I activity during dehydration. Speculation suggests that the much higher uremia in these frogs could account for the disparity, as elevated urea is known to counteract such salt inhibition (Dotsch et al, '95;Cerasoli et al, 2003;Pervushin et al, 2004).…”

NMR Analysis of a Kinetically Trapped Intermediate of a Disulfide-Deficient Mutant of the Starch-Binding Domain of Glucoamylase

“…These results imply that the aromatic cluster on the surface is structurally less organised after rapid cooling, resulting in the formation of the intermediate. 37 Previous studies have indicated that folding intermediates often lack uniquely structured hydrophobic cores. [10][11][12][13][14][15][16][17] However, the present study suggests that this is not the case for the mutant SBD.…”