The Unfolded State of NTL9 Is Compact in the Absence of Denaturant

Abstract

Interest in the unfolded state of proteins has grown with the realization that this state can have considerable structure in the absence of denaturants. Natively unfolded proteins, mutations that unfold proteins under native conditions, and changes in pH that induce unfolding are attractive models for the unfolded state in the absence of denaturant. The unfolded state of the N-terminal domain of ribosomal protein L9 (NTL9) was previously shown to contain significant non-native electrostatic interactions [Cho, J. H., Sato, S., and Raleigh, D. P. (2004) J. Mol. Biol. 338, 827-837]. NTL9 has a mixed alpha-beta structure and folds via a two-state mechanism. We have generated a model of the unfolded state of NTL9 in the absence of denaturant by substitution of an alanine for phenylalanine 5 located in the core of this protein. The CD spectrum of the variant, denoted as F5A, exhibits significantly less structure than the wild type; however, the mean residue ellipticity of F5A at 222 nm (-8200 deg cm(2) dmol(-)(1)) is considerably larger than expected for a fully unfolded protein, indicating that residual secondary structure is populated. F5A also has more residual structure than the urea-unfolded wild type. The stability of F5A is estimated to be at least 1 kcal/mol unfavorable, showing that the unfolded state is populated to 84% or more. NMR pulsed-field gradient measurements yield a hydrodynamic radius of 16.1 A for wild-type NTL9 and 20.8 A for the F5A variant in native buffer. The physiologically relevant unfolded state of wild-type NTL9 is likely to be even more compact than F5A since the mutation should reduce the level of hydrophobic clustering in the unfolded state in the absence of denaturant. The hydrodynamic radius of F5A increases to 25.9 A in 8 M urea, and a value of 23.5 A is obtained for the wild type under similar conditions. The results show that the unfolded state of F5A in the absence of denaturant is more compact and contains more structure than the urea-unfolded form.

Nonnative Electrostatic Interactions Can Modulate Protein Folding: Molecular Dynamics with a Grain of Salt

“…This finding is consistent with the results of Raleigh et al, who found the K12M mutation to cause a significant stabilization effect (ΔΔG ∼ 2 kcal/mol) and attributed it to destabilization of the unfolded state. 33,53 Their analysis suggests that the destabilization of the unfolded state is due to the non-formation of a nonnative salt bridge between residues 8 and 12 that is formed (at least transiently) in wild-type NTL9. We find evidence for such interactions in our simulations examining the distance profile between residues 8 and 12.…”

“…A recent series of studies by Raleigh et al 28,33,50,53,[61][62][63][64] focused on NTL9 as a model protein (Fig. 1a) to investigate the effect of electrostatic interactions on protein stability.…”

“…33,40,47,48 It was shown that modulating nonnative electrostatic interactions within the unfolded-state ensemble can significantly change protein stability. 29,32,33,37,[49][50][51][52][53][54] Variations in crowding or confinement conditions [55][56][57] and attachment of oligosaccharides to proteins 58,59 are reported to have a direct effect on the free energy of the unfolded state. These examples illustrate that understanding the properties of the unfolded state may shed light on various biophysical characteristics of proteins.…”

“…1b), it was shown experimentally that eliminating or reversing certain charged residues that are extensively exposed to the solvent can significantly change the protein's stability and kinetics. Specifically, some of the mutations in NTL9 33,50,53 and in RNase Sa 29,32,54 principally influence the unfolded-state ensemble, while the folded state remains similar to the wild type. Using a novel, coarse-grained model that includes an electrostatic potential specifically calibrated for this purpose, we reproduced the charge neutralization and charge reversal mutations in silico and correlated the thermodynamic and kinetic results from our simulations with experimental data.…”

Electrostatic effects in unfolded staphylococcal nuclease

“…Interest in the U state has increased following the recognition that the structural basis of protein stability cannot be understood without understanding both the folded and the unfolded states (Tanford 1968). It has also been spurred by the discovery of possible organization in the U state (Gillespie and Shortle 1997;Pappu et al 2000;Shortle and Ackerman 2001;Shi et al 2002;Fitzkee and Rose 2005;Anil et al 2006) and by the recognition of a biological role for intrinsically disordered proteins and protein aggregates (Dunker et al 2001;Chiti and Dobson 2006). As interest in the U state has developed, computational models of unfolded proteins have matured.…”