Analysis of electrostatic interactions in the denatured state ensemble of the N‐terminal domain of L9 under native conditions

Abstract

The pH dependence of protein stability is defined by the difference in the number of protons bound to the folded state and to the denatured state ensemble (DSE) as a function of pH. In many cases, the protonation behavior can be described as the sum of a set of independently titrating residues; in this case, the pH dependence of stability reflects differences in folded and DSE pK(a)'s. pH dependent stability studies have shown that there are energetically important interactions involving charged residues in the DSE of the N-terminal domain of L9 (NTL9), which affect significantly the stability of the protein. The DSE of wild type NTL9 cannot be directly characterized under native conditions because of its high stability. A destabilized double mutant of NTL9, V3AI4A, significantly populates the folded state and the DSE in the absence of denaturant. The two states are in slow exchange on the nuclear magnetic resonance time scale, and diffusion measurements indicate that the DSE is compact. The DSE pK(a)'s of all of the acidic residues were directly determined. The DSE pK(a) of Asp8 and Asp23 are depressed relative to model compounds values. Use of the mutant DSE pK(a)'s together with known native state pK(a)'s leads to a significantly improved agreement between the measured pH dependent stability and that predicted by the Tanford-Wyman linkage relationship. An analysis of the literature suggests that DSE interactions involving charged residues are relatively common and should be considered in discussions of protein stability.

The Denatured State Ensemble Contains Significant Local and Long-Range Structure under Native Conditions: Analysis of the N-Terminal Domain of Ribosomal Protein L9

“…Mutation of these residues to alanine destabilizes the domain significantly, and the DSE of the double mutant is ∼70% populated at 12°C and pH 5.5, with two sets of resonances observed in the HSQC spectrum. 40 pH jump fluorescence-monitored stopped-flow measurements were used to directly determine the rate of exchange between the folded state and DSE. The exchange rate (k ex = k fold + k unfold ) is 15.7 s −1 , confirming that the system is in slow exchange on the NMR chemical shift time scale ( Figure S1 of the Supporting Information).…”

“…These observations are consistent with our previous hydrodynamic measurements of the DSE of V3A/I4A-NTL9 in native buffer and in different concentrations of urea, which showed that the DSE is compact in native buffer and becomes more extended in urea. 40 The secondary shift analysis shows that there is substantial α-helical propensity in the DSE of V3A/I4A-NTL9 populated in buffer. It is natural to inquire about the origin of these propensities and to ask whether the residual secondary structure is native-like or non-native-like, and if it is stabilized by local interactions.…”

“…NTL9 is a complicated case; this work documents the presence of nativelike DSE interactions, while earlier studies have shown that K12 is involved in energetically important non-native interactions in the DSE. 39,40,61 Furthermore, studies of wild-type NTL9 under denaturing conditions have revealed the presence of extensive, long, and midrange non-native contacts in the DSE. 20 Thus, the DSE of NTL9 contains both native and non-native interactions.…”

Nascent β -Hairpin Formation of a Natively Unfolded Peptide Reveals the Role of Hydrophobic Contacts

“…The probability for the distance to be within 5 Å (defined here as an ion contact) is generally <10%, whereas the probability for the distance to be within 8 Å is no more than 30%. Second, no significant differences are found between the distributions for NTL9 and NTL9 (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), suggesting that the presence of the b-sheet segments (residues 1-5 and [18][19][20][21][22] has no influence on the electrostatic interactions. Also, the distributions for the b-hairpin and unstructured states of NTL9 are similar, further suggesting the lack of correlation between electrostatic interactions and b-sheet formation.…”

“…It was found that even though both fragments are unstructured, the pK a of Asp 8 is shifted lower than the model value (4.0) by 0.16 and 0.34 units, respectively (13,14). Interestingly, these shifts are slightly smaller than the pK a of Asp 8 in a destabilizing mutant, V3AI4A, which was estimated to be 0.44 units (21). The observation that the pK a of Asp 8 is slightly depressed in NTL9 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) suggests the presence of other attractive interactions in addition to Lys 12 , and raises the question about specificity of electrostatic interactions in the unfolded state.…”