Random coil chemical shifts for serine, threonine and tyrosine phosphorylation over a broad pH range

Abstract

Phosphorylation is one of the main regulators of cellular signaling typically occurring in flexible parts of folded proteins and in intrinsically disordered regions. It can have distinct effects on the chemical environment as well as on the structural properties near the modification site. Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins (IDPs) and the reliability of the analysis depends on an appropriate choice of random coil model. Random coil chemical shifts and sequence correction factors were previously determined for an Ac-QQXQQ-NH2-peptide series with X being any of the 20 common amino acids. However, a matching dataset on the phosphorylated states has so far only been incompletely determined or determined only at a single pH value. Here we extend the database by the addition of the random coil chemical shifts of the phosphorylated states of serine, threonine and tyrosine measured over a range of pH values covering the pKas of the phosphates and at several temperatures (www.bio.ku.dk/sbinlab/randomcoil). The combined results allow for accurate random coil chemical shift determination of phosphorylated regions at any pH and temperature, minimizing systematic biases of the secondary chemical shifts. Comparison of chemical shifts using random coil sets with and without inclusion of the phosphoryl group, revealed under/over estimations of helicity of up to 33%. The expanded set of random coil values will improve the reliability in detection and quantification of transient secondary structure in phosphorylation-modified IDPs.Electronic supplementary materialThe online version of this article (10.1007/s10858-019-00283-z) contains supplementary material, which is available to authorized users.

Di-phosphorylated BAF shows altered structural dynamics and binding to DNA, but interacts with its nuclear envelope partners

“…These signals correspond to residues located in three regions of BAF: region from aa 6 to aa 33, forming helices α1 and α2, region from aa 43 to aa 53, forming helix α4 at the interface between BAF monomers, and region from aa 76 to aa 89, forming the C-terminal helix α6 (Figure 2B ). We also noticed that the only serines and threonines resonating in the 1 H– 15 N spectral region where phosphorylated serines and threonines are typically found are Thr3 and Ser4 ( 48 ). In the published crystal structures of BAF, Ser4 is the first residue of helix α1.…”

A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin

“…Two lines of evidence confirmed the disordered nature of the peptides (further pinpointing the findings from far-UV CD ( Figure S1 ) and the 1D- 1 H-NMR spectra ( Figure S2 )). First, the sequence-corrected conformational shifts (Δδ) of H α protons [ 40 , 41 , 42 , 43 , 44 ] were within the commonly accepted range for random-coil peptides (Δδ ≤ 0.1 ppm) ( Tables S1–S8 ). It is interesting to note at this stage that, in the phosphorylated Thr68 of the pT68 peptide, the signals from the H β protons were downfield shifted when compared to those of the wt peptide (4.58 versus 4.15 ppm, respectively), as well as the chemical shift of the amide proton: 8.62 versus 8.33, respectively ( Tables S1 and S3 ), as it has been reported to occur for phosphorylated threonines [ 43 , 44 ], thus confirming the phosphorylation of this particular threonine and not of the other one in the sequence, Thr54.…”

“…The 1 H resonances were assigned by standard sequential assignment processes [ 40 ]. The chemical shift values of H α protons in random-coil regions were obtained from tabulated data, corrected by neighboring residue effects [ 41 , 42 ] and taking into account the phosphorylation of Thr68 [ 43 , 44 ] for the corresponding peptide.…”

Site-specific phosphorylation of Huntingtin exon 1 recombinant proteins enabled by the discovery of novel kinases

“…After assigning the backbone resonances of our unmodified Httex1 construct in the conditions of interest (pH 7, 25°C, Table S2), we could use this information to interpret the differences between the HSQC spectra of unmodified and both phosphorylated forms. For Httex1-23Q 13 C/ 15 N pS13/pS16 (Figure 7-A), we observed strong downfield perturbations for S13 and S16 chemical shifts in the 1 H dimension, as documented for phosphorylated serine [63,64], along with smaller but sizeable perturbations of other residues within the Nt17 region including the first two glutamines of the polyQ tract (Q18 and Q19). However, we did not observe significant perturbations for the terminal glutamines (Q39 and Q40), nor for residues in the proline-rich or C-terminal regions.…”

“…This indicator is insensitive to offsets or calibration problems in the 13 C dimension; a positive value above +1 indicates alpha helical propensity, and a negative value under -1 indicates beta sheet propensity. For phosphorylated serine, the CA and CB chemical shifts reported by [63] were used as random coil references; for the other amino acids, we used the random coil shifts from [68]. In vitro phosphorylation of Httex1-43Q by GCK or TBK1.…”