The interplay between transient α-helix formation and side chain rotamer distributions in disordered proteins probed by methyl chemical shifts

Abstract

The peptide backbones of disordered proteins are routinely characterized by NMR with respect to transient structure and dynamics. Little experimental information is, however, available about the side chain conformations and how structure in the backbone affects the side chains. Methyl chemical shifts can in principle report the conformations of aliphatic side chains in disordered proteins and in order to examine this two model systems were chosen: the acid denatured state of acyl-CoA binding protein (ACBP) and the intrinsically disordered activation domain of the activator for thyroid hormone and retinoid receptors (ACTR). We find that small differences in the methyl carbon chemical shifts due to the γ-gauche effect may provide information about the side chain rotamer distributions. However, the effects of neighboring residues on the methyl group chemical shifts obscure the direct observation of γ-gauche effect. To overcome this, we reference the chemical shifts to those in a more disordered state resulting in residue specific random coil chemical shifts. The (13)C secondary chemical shifts of the methyl groups of valine, leucine, and isoleucine show sequence specific effects, which allow a quantitative analysis of the ensemble of χ(2)-angles of especially leucine residues in disordered proteins. The changes in the rotamer distributions upon denaturation correlate to the changes upon helix induction by the co-solvent trifluoroethanol, suggesting that the side chain conformers are directly or indirectly related to formation of transient α-helices.

Figure 1

Constant time ¹H-¹³C HSQC spectra of the methyl regions of acid denatured ACBP (A) and ACTR (B). The experiment was run without urea (black) and in the presence of 6M urea (red).

Figure 2

Correlation between chemical shifts from the geminal methyl groups of Leu side chains of ACBP and ACTR with and without urea. The ¹H^δ chemical shifts are correlated (R = 0.85) (A) and the ¹³C^δ chemical shifts (B) are anticorrelated (R = −0.62). The anticorrelation of the ¹³C chemical shifts is better for ACTR alone (R = −0.74). The anticorrelation of the ¹³C chemical shifts suggests that variations in the χ₂-distributions affect the methyl chemical shifts.

Figure 3

TFE titration of ACTR suggests interplay between backbone and side chain conformations. ACTR was titrated with the helix inducing co-solvent TFE, which results in increasing populations of the transient helices as demonstrated by C^α (A) and C′ (B) chemical shifts. The side chain distributions in the disordered state changes approximately linearly with increasing TFE concentration (C). The largest changes are roughly in the regions where transient helices are formed (D) suggesting that there is an interplay between the backbone and the side chain conformations. The populations of the trans rotamer P_t is calculated based on the difference in ¹³C chemical shifts between the two geminal methyl groups. At the highest TFE concentrations, the chemical shifts in the helical segments are missing due to exchange broadening, suggesting formation of relatively long-lived interactions stabilized by TFE.

Figure 4

Methyl chemical shift perturbations from sequential neighbors. ¹H (A) and ¹³C (B) methyl secondary chemical shifts of Ile were obtained for a series of Ac-GGXIGG-NH₂ and Ac-GGIXGG-NH₂ peptides. The X residue is indicated on the X-axis. ¹H (C) and ¹³C (D) methyl secondary chemical shifts were determined in the peptide series Ac-GGFG_nIGG-NH₂ for separations up to 6 residues. The secondary chemical shifts were calculated using the chemical shifts from the peptide Ac-GGIGG-NH₂ in 1M urea as random coil reference.

Figure 5

Urea titration of ACTR. The backbone chemical shifts of ACTR are observed with increasing urea concentration for the first transiently formed helix (residues 1045–1055). The helical population decreases rapidly at low concentrations of urea, but levels of at higher concentration. The secondary chemical shifts do not reach zero, which suggests a small helical population in the urea denatured state consistent with other studies of denatured proteins. Random coil chemical shifts are based on work from Ref. 46.

Figure 6

Methyl secondary chemical shifts were determined by intrinsic statistical coil referencing for ACBP (A,C) and ACTR (B,D). The chemical shifts determined for the highly disordered state in 6M urea are used as the intrinsic statistical coil chemical shifts and are subtracted from the chemical shifts of the partially folded states to give secondary chemical shifts. V77 has a particularly large ¹H secondary chemical shift, which suggests a ring current effect. The insert shows the conformations of V77 and Y73 the NMR structure of ACBP (PDB:1NTI). The cylinders show the locations of the α-helices in the folded state of ACBP and the complex between ACTR and CBP. The shading of the cylinders represents the approximate populations of the helices in the free state with fully formed helices being solid black.

Figure 7

Perturbations of the χ₂-rotamer distributions between the partially folded states and the highly disordered state in urea for ACBP (A) and ACTR (B). The population of the trans rotamer of the χ₂-angle was estimated from the difference between δ(C^δ1) and δ(C^δ2) for leucine or from δ(C^δ) for isoleucine using the linear relationships reported previously.,

Figure 8

Correlation between changes in the rotamer distribution in solvent conditions stabilizing (TFE) and destabilizing α-helices (urea) suggests that the subtle rearrangements in the side chain rotamer distributions are mainly caused by helix formation