How cellular functions are regulated through protein phosphorylation events that promote or inhibit protein–protein interactions (PPIs) is key to understanding regulatory molecular mechanisms. Whilst phosphorylation can orthosterically or allosterically influence protein recognition, phospho-driven changes in the conformation of recognition motifs are less well explored. We recently discovered that clathrin heavy chain recognizes phosphorylated TACC3 through a helical motif that, in the unphosphorylated protein, is disordered. However, it was unclear whether and how phosphorylation could stabilize a helix in a broader context. In the current manuscript, we address this challenge using poly-Ala-based model peptides and a suite of circular dichroism and nuclear magnetic resonance spectroscopies. We show that phosphorylation of a Ser residue stabilizes the α-helix in the context of an Arg(i−3)pSeri Lys(i+4) triad through charge-reinforced side chain interactions with positive co-operativity, whilst phosphorylation of Thr induces an opposing response. This is significant as it may represent a general method for control of PPIs by phosphorylation; basic kinase-substrate motifs are common with 55 human protein kinases recognizing an Arg at a position −3 from the phosphorylated Ser, whilst the Arg(i−3)Seri Lys(i+4) is a motif found in over 2000 human proteins.

The determinants of α-helix stability are key in understanding the formation and strength of α-helix mediated protein–protein interactions (PPIs) [1], and therefore in enabling peptide drug-discovery [2,3]. Prior studies established helix propensities of individual amino acids [4,5], the role of helix capping [6,7] and effects of interaction between side chains [8–11]; however, the role of phosphorylation is less well explored.

Classically, phosphorylation occurs within solvent-exposed loops or disordered regions [12]. Such phosphorylation events play a regulatory role in the PPI network of substrate proteins; primed with a new highly charged phosphate group as a recognition feature, such regions can bind to a different protein to activate or deactivate a subsequent cellular process [13]. More recently, phosphorylation has been shown to affect secondary/tertiary structure, e.g. the intrinsically disordered 4E-BP2 undergoes a disorder-to-helix transition upon binding to eIF4E, but on multisite phosphorylation, folds instead into a four-stranded β-domain [14]. Ser phosphorylation has been shown to increase helicity in model peptides [15], notably at the N-terminus [16,17], a feature exploited in design of phosphorylation-stabilized tertiary helix structures [18,19]. Secondary structure has also been shown to be destabilized by phosphorylation [20,21] with internal phosphorylation of Thr and Ser residues helix destabilizing [15–17]. Herein we describe the effects of phosphorylation on helix stability using model poly-Ala-based peptides, into which we grafted a motif inspired by studies on the Aurora A/TACC3/CHC pathway, an unusual example of a PPI centred on a phosphorylated helix [22].

Aurora A-mediated phosphorylation of TACC3, a member of the transforming acidic coiled coil (TACC) family of centrosomal and microtubule-associated proteins, promotes complex formation with clathrin heavy chain (CHC) protein resulting in molecular ‘bridges’ across parallel microtubules to ensure accurate chromosome alignment and segregation during mitosis. The TACC3/CHC complex is α-helix mediated [22]; the TACC3 residue phosphorylated by Aurora A, Ser558, is in a basic kinase-substrate motif, flanked by basic Arg555 and Lys562 residues in i − 3 and i + 4 positions rendering them suitably placed to stabilize helicity via interactions with the phosphate (Figure 1). The TACC3-inspired Arg(i−3)pSeri Lys(i+4) motif was grafted into a well-studied poly-Ala-based model peptide [23,24]. Poly-Ala peptides are good models due to their intrinsic α-helix-forming propensity; sequence modifications can be related to the introduction/deletion of potential interactions between side chains [9,25]. Peptides were designed to investigate the α-helix stabilizing influence of phosphate salt bridging. The phosphorylation state was altered, Arg and Lys side chains were systematically moved in and out of pSer registry, and the effect of substitution of Ser for Thr was studied (8).

### TACC3 structure in the TACC3/CHC complex.

Figure 1.
TACC3 structure in the TACC3/CHC complex.

X-ray crystal structure for the TACC3/CHC interaction (PDB ID: 5ODS; TACC3 cyan, CHC forest green), together with the TACC3 sequence; the Arg(i−3)pSeri Lys(i+4) motif is highlighted with side chains shown as sticks.

Figure 1.
TACC3 structure in the TACC3/CHC complex.

X-ray crystal structure for the TACC3/CHC interaction (PDB ID: 5ODS; TACC3 cyan, CHC forest green), together with the TACC3 sequence; the Arg(i−3)pSeri Lys(i+4) motif is highlighted with side chains shown as sticks.

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### Basic residues in flanking positions enhance the helicity of peptides incorporating a pSer residue

A range of peptides (110, Table 1) were designed and prepared using solid phase synthesis as N-terminal acetamides and C-terminal amides (see Supplementary Information for analytical characterization data). The relative helical propensity for each of the peptides was established using far UV circular dichroism (CD) spectroscopy [26]. Figure 2a compares the CD spectrum of the phosphorylated and unphosphorylated peptides 1 and 2. 1 exhibits a CD profile characteristic of an α-helix with negative bands at 222 and 208 nm, and a positive band at ∼190 nm (Figure 2a, see Supplementary Figure S1 for data demonstrating spectra are concentration independent). For peptide 2, reduced helicity is indicated from smaller bands at 222 and 190 nm, and a shift of the peak at 208 nm to lower wavelength. Estimation of the % helicity using the mean residue ellipticity (MRE) value at 222 nm gave 29% for 1 and 22% for 2 (Table 1), indicating that phosphorylation enhances helicity with i − 3 Arg and i + 4 Lys basic side chains. Variant pSer to Ala-control peptides were more helical as would be expected given the increased helical propensity of Ala (peptides 9 and 10, Table 1, Supplementary Figure S2) [4]. The variation in MRE222 with temperature indicated a broad transition from part-helical to coil structures and weak folding co-operativity as expected for short peptides of this length (Supplementary Figure S3). At ∼70°C, all peptides converged at ∼4000 deg cm2 dmol−1 indicating complete loss in helical content at these temperatures.

#### CD spectroscopy for model peptides.

Figure 2.
CD spectroscopy for model peptides.

(a) CD spectra for peptides 12 showing the effect of phosphorylation on the Arg(i−3)Seri Lys(i+4) motif. (b) CD spectra for 1, 3, 5 and 6 illustrating the effect of moving one or two basic groups out of ‘helical-registry’ with the pSer. (c) CD spectra for peptides 78 showing the effect of phosphorylation on the Arg(i−3)Thri Lys(i+4) motif. Experiments were carried out at 5°C in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate and 1 mM sodium citrate) at pH 7. Peptide concentrations were in the range 50–100 µM.

Figure 2.
CD spectroscopy for model peptides.

(a) CD spectra for peptides 12 showing the effect of phosphorylation on the Arg(i−3)Seri Lys(i+4) motif. (b) CD spectra for 1, 3, 5 and 6 illustrating the effect of moving one or two basic groups out of ‘helical-registry’ with the pSer. (c) CD spectra for peptides 78 showing the effect of phosphorylation on the Arg(i−3)Thri Lys(i+4) motif. Experiments were carried out at 5°C in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate and 1 mM sodium citrate) at pH 7. Peptide concentrations were in the range 50–100 µM.

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Table 1
Sequences, MRE222 data, helicity estimates and free energies values calculated for poly-Ala model peptides 1–10

1Residues with positive charges are highlighted in blue; residues with negative charges are highlighted in red; 2% Helicity values shown were calculated based on MRE222 values recorded at 5°C (see Experimental section); 3ΔGx values are estimates of the Gibbs free energy change for the folded peptides in solution relative to their unfolded states; 4ΔΔG values are the change in the Gibbs free energy change from differences in ΔGx values, here given relative to the unphosphorylated version of the peptide (2, 4 or 8); 5ΔΔG values relative to the least helical pSer-containing peptide 3; 6ΔΔG values relative to the Ala-control peptide (9 or 10) with the same R and K spacing. N is the number of independent measurements.

The effect on peptide helicity of moving Arg and Lys out of helical-registry was explored (Figure 2b). In a peptide where no helix compatible side chain interactions with the phosphate are possible, α-helicity was lowest (3, 7%), as expected given the propensity of pSer to be destabilizing in the centre of an α-helix [17]. Indeed, the unphosphorylated version (4) has higher helicity, matching that of peptide 2 (Table 1, Supplementary Figure S2). The similarity of the helicity of peptides 4 and 2 (and peptides 9 and 10, Table 1, Supplementary Figure S2) indicates that the position of Lys and Arg has little effect on the overall helicity when there is no phosphoryl group with which to interact. Having an Arg residue i − 3 or Lys residue i + 4 to the pSer increased the α-helix-forming propensity (5 and 6, both 13%). CD analyses on the pThr-containing peptide 7 indicated low helicity (2%) whereas the Thr analogue elicited higher helicity (8, 18%) similar to unphosphorylated Ser peptide 2 (Figure 2c). The additional methyl group in pThr compared with pSer will likely reduce the helix compatible side chain dihedral angle pairings that allow the phosphoryl group to interact with flanking basic residues. More generally, pThr shows a proclivity to adopt a compact conformation due to intraresidue phosphate–amide hydrogen bonding, inter-residue n → π* interactions between consecutive carbonyls, and an eclipsed conformation of the C–OP bond arising from an n → σ* interaction. Such a conformation is incompatible with a helical conformation and is evidently not countered by the benefit of side chain hydrogen bonding as for pSer (peptide 1) [27].

### Side chain interactions that enhance pSer peptide helicity are pH dependent and positively cooperative

The effect of pH on helicity for peptide 1 provided useful insights into the contribution of charge-based interactions between side chains (Figure 3). Protonating the phosphate by reducing the pH below the pKa of PO32− (pH < 5 forming singly charged PO3H) or deprotonating the Lys or Arg groups at high pH would be anticipated to reduce or eliminate charge-reinforced interactions between side chains. The estimated helicity of peptide 1 at low pH is ∼16% and at high pH is ∼6%, similar values to those of peptide 5/6 (with a single potential X+–PO32− interaction) and peptide 3 (no interaction), respectively. For comparison, the more limited effect of changing pH on peptide helicity for other selected peptides is shown in Supplementary Figure S4.

We used the measured % helicity values to estimate the Gibbs free energy change of the α-helical peptides in solution relative to their unfolded states, ΔG (Table 1) [11]. This allowed us to make quantitative estimates of the change in the Gibbs free energy change for helix formation, ΔΔG, associated with phosphorylation or formation of charge-reinforced side chain interactions involving the phosphate. ΔΔG values measured relative to unphosphorylated peptides (Table 1) show that phosphorylation can have a wide-ranging influence, being beneficial (−0.8 kJ mol−1) in the case of Arg(i3)Seri Lys(i+4), highly destabilizing (5.7 kJ mol−1) in the case of Arg(i3)Thri Lys(i+4) and moderately destabilizing for Arg(i5)Seri Lys(i+5) where no side chain interactions are possible (3.1 kJ mol−1). By relating the ΔG of 5 to that of 3 (Table 1), the formation of such an interaction between the phosphate group and i − 3 Arg side chain has a ΔΔG of −1.6 kJ mol−1. Comparison of ΔG values for 6 and 3 show that potential interaction between the phosphate and the i + 4 Lys side chain has a similar ΔΔG (−1.7 kJ mol−1). These values are in agreement with typical values for the formation of charge-reinforced interactions between side chains in helices [28,29], e.g. Glu–Lys (i, i ± 3 or i, i ± 4) interactions under similar conditions gave rise to ΔΔG values of 1.2–1.7 kJ mol−1 [29]. For both 5 and 6, the ability to form interactions between side chains is insufficient to offset the destabilizing effect of introducing a pSer in the middle of the helical sequence (4 vs. 3, ΔΔG = −3.1 kJ mol−1) [16]. In contrast, 1, where both interactions can form, would be expected to have a reported ΔΔG (relative to 3) of ∼−3.3 kJ mol−1 if the contributions were additive (only marginally higher than the ΔΔG for 2). However, with a ΔΔG of −4.0 kJ mol−1, this indicates that the effects of two charge-reinforced interactions with phosphate are positively cooperative.

#### The effect of pH on the helicity of peptide 1.

Figure 3.
The effect of pH on the helicity of peptide 1.

Low and high pH reduce peptide 1 helicity indicating that charge-reinforced side chain interactions stabilize the helix. Experiments were carried out at 5°C in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate and 1 mM sodium citrate). Peptide concentrations were in the range 50–100 µM. The pH was altered by the addition of small amounts of 0.1 M HCl or 0.05 M NaOH, with volume changes factored-in to concentration and MRE calculations.

Figure 3.
The effect of pH on the helicity of peptide 1.

Low and high pH reduce peptide 1 helicity indicating that charge-reinforced side chain interactions stabilize the helix. Experiments were carried out at 5°C in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate and 1 mM sodium citrate). Peptide concentrations were in the range 50–100 µM. The pH was altered by the addition of small amounts of 0.1 M HCl or 0.05 M NaOH, with volume changes factored-in to concentration and MRE calculations.

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Calculating ΔΔG values relative to Ala-control peptides (Table 1) shows that the replacement of Ala10 with Ser decreases helix stability by 1.8–1.9 kJ mol−1 (2 and 4). Phosphorylation to pSer rescues approximately half of the original stabilizing energy (0.8 kJ mol−1) when both positively charged residues are in register (compare 1 and 2) or further destabilizes the helix by 3.2 kJ mol−1 when they are out of register (compare 3 and 4).

### NMR chemical shifts show increased helicity in pSer peptides with in-helical-range basic residues

To provide additional structural insight to complement the CD analyses, a suite of four NMR spectra was recorded for each peptide to assign 1H, 13C and 15N resonances. Residue assignments in a 1H–15N HMQC spectrum were achieved using a combination of 1H–1H TOCSY/NOESY spectra, informed by a 1H–13C HSQC spectrum and systematic peak position shifts between spectra for different peptides. Natural abundance 1H–15N HMQC spectra for peptides 1 and 2 are shown in Figure 4a; phosphorylation leaves H–N peak positions for residues 1–5 largely unaffected whilst a characteristic downfield shift is observed for the backbone H–N of pSer with respect to Ser as marked by the purple arrow [30]. Neighbouring residues also exhibit shifts with the phosphorylated peptide 1 showing greater peak dispersion than the unphosphorylated peptide 2, suggesting increased secondary structure content. Patterns in Cα shifts also provide a useful guide to local secondary structure. Figure 4b shows the Hα–Cα region of 1H–13C HSQC spectra for two examples, peptide 1 and peptide 3 with Arg and Lys in- and out-of-helical-range, respectively. Systematic downfield Cα shifts and upfield Hα shifts observed for peptide 1 compared with peptide 3 indicate increased helical content. To account for changes in sequence between peptides, secondary shifts (Δδ) (observed shifts (δ) — expected shifts for that residue in a random coil conformation (δRC)) [31–33] were calculated using random coil values that were recently updated to include values for phosphorylated residues (Figure 5) [34]. Plots show the effect of altering the position of the basic residues (Figure 5a) and the effect of phosphorylation (Figure 5b). Data for control Ala peptides 9 and 10, as well as unphosphorylated peptide 4 are shown in Supplementary Figure S5. These data largely mirror the CD data and show the weighting of the more helical residues towards the N-terminus. It was noted that the pThr Cα secondary shift values are higher than expected for a residue in a random coil configuration, indicating that pThr occupies an arrangement unlike that in the QQpTQQ peptide used to generate the values [34]. Example spectra showing the expected effect of pH on pSer and pThr Hα–Cα correlations are given in Supplementary Figure S6.

#### NMR spectroscopy of peptides.

Figure 4.
NMR spectroscopy of peptides.

(a) A comparison of part of the 1H–15N HMQC spectra for peptides 1 (brown) and 2 (blue), showing the effect of phosphorylation on main-chain HN peaks of the Arg(i−3)Seri Lys(i+4) motif peptide; (b) A comparison of regions of 1H–13C HSQC spectra for peptides 1 (brown) and 3 (magenta) showing Arg Hα-Cα correlations. These spectra highlight the difference between a phospho-peptide that is capable (1) or incapable (3) of forming inter-turn interactions between the phosphate group and neighbouring basic residues (Arg and Lys). Spectra were recorded on a 600-MHz Bruker Avance spectrometer equipped with a quadruple resonance QCI-P cryoprobe, or a 950-MHz Bruker Ascend Aeon spectrometer equipped with a 5 mm TXO cryoprobe. Experiments were performed in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate, and 1 mM sodium citrate, pH 6.5–7) at 5°C. Peptide concentrations were in the range 0.5–1.0 mM.

Figure 4.
NMR spectroscopy of peptides.

(a) A comparison of part of the 1H–15N HMQC spectra for peptides 1 (brown) and 2 (blue), showing the effect of phosphorylation on main-chain HN peaks of the Arg(i−3)Seri Lys(i+4) motif peptide; (b) A comparison of regions of 1H–13C HSQC spectra for peptides 1 (brown) and 3 (magenta) showing Arg Hα-Cα correlations. These spectra highlight the difference between a phospho-peptide that is capable (1) or incapable (3) of forming inter-turn interactions between the phosphate group and neighbouring basic residues (Arg and Lys). Spectra were recorded on a 600-MHz Bruker Avance spectrometer equipped with a quadruple resonance QCI-P cryoprobe, or a 950-MHz Bruker Ascend Aeon spectrometer equipped with a 5 mm TXO cryoprobe. Experiments were performed in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate, and 1 mM sodium citrate, pH 6.5–7) at 5°C. Peptide concentrations were in the range 0.5–1.0 mM.

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#### Residue-specific Cα secondary shifts at 5°C and pH 6.5–7.

Figure 5.
Residue-specific Cα secondary shifts at 5°C and pH 6.5–7.

Secondary shift values are plotted for each residue for (a) peptides 1, 3, 5 and 6, showing the effect of changing the position of the basic residues, and (b) peptides 1, 2, 7 and 8, showing the effect of phosphorylation. Error bars represent the range in possible shift measurements from 1H–13C HSQC spectra — particularly important for the overlapping Ala Hα–Cα peaks. Shifts recorded from clean/distinct peaks are shown with a baseline error of 0.05 ppm (related to the peak width). Secondary shifts from spectra for peptides 1 and 7 recorded at pH values between 6 and 7.5, showed no significant variation. Spectra were recorded on a 600-MHz Bruker Avance spectrometer equipped with a quadruple resonance QCI-P cryoprobe, or a 950-MHz Bruker Ascend Aeon spectrometer equipped with a 5 mm TXO cryoprobe. Experiments were performed in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate and 1 mM sodium citrate). The pH was altered by the addition of small amounts of 0.1 M HCl or 0.05 M NaOH. Peptide concentrations were in the range 0.5–1.0 mM.

Figure 5.
Residue-specific Cα secondary shifts at 5°C and pH 6.5–7.

Secondary shift values are plotted for each residue for (a) peptides 1, 3, 5 and 6, showing the effect of changing the position of the basic residues, and (b) peptides 1, 2, 7 and 8, showing the effect of phosphorylation. Error bars represent the range in possible shift measurements from 1H–13C HSQC spectra — particularly important for the overlapping Ala Hα–Cα peaks. Shifts recorded from clean/distinct peaks are shown with a baseline error of 0.05 ppm (related to the peak width). Secondary shifts from spectra for peptides 1 and 7 recorded at pH values between 6 and 7.5, showed no significant variation. Spectra were recorded on a 600-MHz Bruker Avance spectrometer equipped with a quadruple resonance QCI-P cryoprobe, or a 950-MHz Bruker Ascend Aeon spectrometer equipped with a 5 mm TXO cryoprobe. Experiments were performed in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate and 1 mM sodium citrate). The pH was altered by the addition of small amounts of 0.1 M HCl or 0.05 M NaOH. Peptide concentrations were in the range 0.5–1.0 mM.

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### Interaction effects shown through specific side chain resonance analyses

Confirmation of intra-helix side chain interactions was obtained using additional NMR experiments focussed on side chain resonances. Performing experiments at a low temperature of 5°C maximizes the helical content and when coupled with use of low pH (below pH 7) also reduces proton exchange enough for Arg Hε–Nε side chain groups to be visible in 1H–15N HMQC spectra [35]. Each peptide has a single Arg residue resulting in a single Hε–Nε correlation (Figure 6a). The Arg Hε shift appears at ∼7.25 ppm for all peptides with the exception of peptide 5 (7.5 ppm) and peptide 1 (7.8 ppm), both of which have Arg in the i − 3 position relative to the phosphorylated residue — a position suitable for interactions between side chains across a helical turn — and have been shown to be helical. The magnitude of the downfield shift with respect to peptides that have no potential to form such interactions correlates with the overall helicity of the peptide and thus peptide 1 has the largest downfield shift, peptide 5 has an intermediate value and peptide 7 is unmoved. The Hε shift for peptide 1 is similar to those reported for Arg residues in a single-α-helix domain that had three potential Glu salt bridge partners (7.7–7.9 ppm) [36]. In line with the CD analyses, on decreasing pH, the Hε resonance in peptide 1 experiences an upfield shift indicating a loss of interaction as the phosphate starts to become protonated (Figure 6b), whereas the Hε–Nε correlation in peptide 3 does not change (Figure 6c). Under the standard conditions (T = 5°C, pH 6.5–7) the Arg Hη–Nη and Lys Hζ–Nζ were not visible in 1H–15N HMQC spectra, although at lower pH [37], the Lys Hζ–Nζ correlation could be observed and the Hζ resonance for 1 was slightly downfield in comparison to 3 (Figure 6d). In addition, 1H–13C HSQC and 1H–1H TOCSY spectra revealed that the diastereotopic Arg Hδ protons were resolved in a pH-dependent manner for 1 alone, indicating a more restricted side chain conformation for this peptide (Figure 6e–f). For comparison, 1H–1H TOCSY spectra showing Arg Hε–Hδ correlations for peptides 3, 5, 6 and 7 are given in Supplementary Figure S7. The two Arg Hγ resonances for 1 also become non-equivalent on increasing the pH (Supplementary Figure S8). However, this effect appears to be related to the helicity of the peptide, rather than interaction with the phosphoryl group, since distinct Hγ resonances are also observed for helical, nonphosphorylated peptides 2 and 9.

#### NMR correlation spectroscopy to investigate side chain interactions.

Figure 6.
NMR correlation spectroscopy to investigate side chain interactions.

(a) The region of the 1H–15N HMQC spectra showing Arg Hε–Nε correlations at pH 7 for various peptides that have Arg at position 5 or 7. (b,c) The region of the 1H–15N HMQC spectra showing pH-dependent chemical shifts in the Arg Hε–Nε correlation for peptides 1 (b) and 3 (c). (d) The region of the 1H–15N HMQC spectra showing a small chemical shift difference in the Lys Hζ–Nζ correlations for peptides 1 and 3 at pH 5. (e) The region of 1H–13C HSQC spectra for peptides 1, 3 and 5, pH 7, showing Arg Hδ–Cδ correlations and Lys Hε–Cε correlations. Note the non-equivalence of the two Hδ for peptide 1 only. (f) The region of 1H–1H TOCSY spectra for peptides 1, 3 and 5 at pH values 5–7, showing Arg Hε–Hδ and Lys Hζ–Hε correlations. Again, the two Hδ for peptide 1 are not equivalent at high pH. Spectra were recorded on a 600-MHz Bruker Avance spectrometer equipped with a quadruple resonance QCI-P cryoprobe at 5°C in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate and 1 mM sodium citrate). The pH was altered by the addition of small amounts of 0.1 M HCl or 0.05 M NaOH. Peptide concentrations were in the range 0.5–1.0 mM.

Figure 6.
NMR correlation spectroscopy to investigate side chain interactions.

(a) The region of the 1H–15N HMQC spectra showing Arg Hε–Nε correlations at pH 7 for various peptides that have Arg at position 5 or 7. (b,c) The region of the 1H–15N HMQC spectra showing pH-dependent chemical shifts in the Arg Hε–Nε correlation for peptides 1 (b) and 3 (c). (d) The region of the 1H–15N HMQC spectra showing a small chemical shift difference in the Lys Hζ–Nζ correlations for peptides 1 and 3 at pH 5. (e) The region of 1H–13C HSQC spectra for peptides 1, 3 and 5, pH 7, showing Arg Hδ–Cδ correlations and Lys Hε–Cε correlations. Note the non-equivalence of the two Hδ for peptide 1 only. (f) The region of 1H–1H TOCSY spectra for peptides 1, 3 and 5 at pH values 5–7, showing Arg Hε–Hδ and Lys Hζ–Hε correlations. Again, the two Hδ for peptide 1 are not equivalent at high pH. Spectra were recorded on a 600-MHz Bruker Avance spectrometer equipped with a quadruple resonance QCI-P cryoprobe at 5°C in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate and 1 mM sodium citrate). The pH was altered by the addition of small amounts of 0.1 M HCl or 0.05 M NaOH. Peptide concentrations were in the range 0.5–1.0 mM.

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1D 31P spectra provided further evidence for differences between peptides in phosphate shielding and altered pSer/pThr side chain dihedral propensities. 31P NMR spectra of phospho-peptides contain two peaks: one from the 1 mM free phosphate in the buffer and one from pSer/pThr (Figure 7). pSer 31P resonances appear as a triplet (sometimes unresolved) due to 3JPH coupling to the two β protons, pThr 31P resonances appear as a doublet through coupling to the single β proton. These spectra are highly sensitive to changes in pH. The buffer phosphate peak provides a guide to sample pH through calibration of peak position using standard CD buffer samples at pH 5, 6, 7 and 8. At low pH (pH 5, Figure 7a), the doublet from peptide 7 appears upfield of the buffer phosphate peak, whereas the triplets from the pSer-containing peptides 1, 3, 5 and 6 appear downfield. The ordering of the peptide 31P chemical shifts (7 < 3 < 6 ∼ 5 < 1) at pH 5 follows the helicity. This suggests a de-shielding effect as the phosphate is involved in interactions with more neighbouring basic groups, although the (likely related) effect of altered PO32−/PO3H equilibrium could be a more significant factor. At high pH (pH ∼ 8, Figure 7b) the peak positions are all downfield of the buffer phosphate and they are closer together in chemical shift (7 < 1 < 3 ∼ 5 ∼ 6). The fine structure of the peaks is clearer at pH 8 allowing the 3JPH coupling constants to be measured (Figure 7c). There is an inverse correlation between peptide helicity and 3JPH, with non-helical peptide 7 having the highest 3JPH (9 Hz) and peptide 1 having the lowest (5.3 Hz). These values match very well with those found by Pandey et al. [27] for pThr- and pSer-containing peptides; the high value for peptide 7 suggests a well-defined P–O–C–H dihedral and ordered pThr side chain, whereas the lower values for peptides 3, 5 and 1 are consistent with a greater degree of pSer side chain freedom. The reduced value for 1 compared with 3, 5 and 6 is interesting and suggests altered dihedral propensities for the most helical pSer-containing peptide.

#### 31P NMR spectra for selected peptides (1, 3, 5, 6 and 7) and CD buffer standards at pH 5, 6, 7 and 8.

Figure 7.
31P NMR spectra for selected peptides (1, 3, 5, 6 and 7) and CD buffer standards at pH 5, 6, 7 and 8.

(a) At pH 5. (b) Just below pH 8. (c) Zoomed-in view of the peak fine-structure at pH ∼ 8 with spectra shown realigned to facilitate comparison. Spectra were recorded on a 600-MHz Bruker Avance spectrometer equipped with a quadruple resonance QCI-P cryoprobe at 5°C in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate and 1 mM sodium citrate). The pH was altered by the addition of small amounts of 0.1 M HCl or 0.05 M NaOH. Peptide concentrations were in the range 0.5–1.0 mM.

Figure 7.
31P NMR spectra for selected peptides (1, 3, 5, 6 and 7) and CD buffer standards at pH 5, 6, 7 and 8.

(a) At pH 5. (b) Just below pH 8. (c) Zoomed-in view of the peak fine-structure at pH ∼ 8 with spectra shown realigned to facilitate comparison. Spectra were recorded on a 600-MHz Bruker Avance spectrometer equipped with a quadruple resonance QCI-P cryoprobe at 5°C in CD buffer (10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate and 1 mM sodium citrate). The pH was altered by the addition of small amounts of 0.1 M HCl or 0.05 M NaOH. Peptide concentrations were in the range 0.5–1.0 mM.

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Scheme 1.

The 10 peptides used in the study are arranged to highlight their relationships. Differences in sequence from the Arg(i−3)pSeri Lys(i+4) motif of peptide 1 are highlighted in blue, bold text. Peptides are grouped according to whether the positively-charged residues are in or out of register with their positions in peptide 1.

Scheme 1.

The 10 peptides used in the study are arranged to highlight their relationships. Differences in sequence from the Arg(i−3)pSeri Lys(i+4) motif of peptide 1 are highlighted in blue, bold text. Peptides are grouped according to whether the positively-charged residues are in or out of register with their positions in peptide 1.

Close modal

An interesting feature of the TACC3–CHC binding interface in the crystal structure is that it does not exhibit the canonical recognition of phosphorylation, in which a cluster of basic residues on the binding partner clamps down on the phosphate group. Instead, the phosphorylated serine is positioned between two basic residues along one side of a helix in TACC3, and hydrophobic residues on another side of the helix line up to complement a groove on CHC. We hypothesized that the role of a phosphoserine in this arrangement is to increase the helical propensity of this disordered region, and we set out to explore the conformational effects of phosphorylation in a model peptide system that is decoupled from intermolecular considerations. Here we have shown that phosphorylation of Ser residues stabilizes the α-helix in the context of an Arg(i3)pSeri Lys(i+4) triad through charge-reinforced side chain interactions with positive co-operativity. This differs to other systems where multiple interactions between side chains are feasible, e.g. Glu–Lys–Glu which shows anti-cooperativity [38], and provides an exception to the general principle that internal phosphorylation is helix destabilizing [39]. Basic kinase-substrate motifs are common; an Arg at position −3 to the phosphorylated residue is a key selectivity determining residue within the recognition motif of 55 human protein kinases, including Aurora A and PKA [40], and there are over 2000 Arg(i3)Seri Lys(i+4) motifs in human proteins (identified using Expasy ScanProsite). In contrast, the corresponding Thr variants are not stabilized: Ser is slightly less helix destabilizing than Thr [4,5,11], whilst the latter favours a β-strand conformation [41] and branching at β-carbon is helix destabilizing [42]. These features likely reduce the number of side chain conformation pairs in which the pThr phosphoryl group is able to interact with the Arg and Lys while maintaining a helical structure (when compared with pSer). Moreover, recent studies have shown Thr phosphorylation promotes intraresidue hydrogen-bonding between the phosphate and the backbone amide, which would disrupt helix propagation in a manner analogous to the effect of Pro [27]. Taken together these results highlight the secondary structural sequence context of phosphorylation and highlight the potential for it to affect PPIs not only through orthosteric [13] or allosteric [43] changes in recognition features and switching off recognition by changing conformation [14], but also through phospho-driven stabilization of an α-helical recognition motif.

### Peptide solutions

CD buffer’ containing 1 mM sodium borate, 1 mM sodium citrate, 1 mM sodium phosphate and 10 mM NaCl, pH 7 [15], was used to facilitate easy alteration of peptide solution pH through the addition of small amounts of HCl or NaOH. This buffer has low absorbance in the region of wavelengths (190–260 nm) used for CD measurements. Solid lyophilized peptides were dissolved to give 1–2 mM solutions based on mass using CD buffer resulting in mildly acidic solutions (pH 6–7). Solution absorbance measurements at 280 nm were used to calculate more accurately the peptide concentration, based on a Tyr extinction coefficient of 1280 M−1 cm−1 [26,45], with 1 mM concentrations based on mass typically resulting in 0.7–1.0 mM solutions based on absorbance. Initial (∼1 ml) sample pH measurements were made using a three-point-calibrated meter equipped with a microelectrode probe. In situ pH measurements (e.g. during CD experiments) were made by careful removal of small volumes of sample to test with pH strips. In situ estimates of pH during NMR experiments were made using the buffer phosphate chemical shift by comparison with CD buffer-only samples calibrated at pH 5, 6, 7 and 8.

### CD measurements

CD spectra were recorded using an APP Chirascan CD spectropolarimeter and 1 mm pathlength quartz cuvettes. Sample concentrations were typically 50–100 µM and once diluted to this degree had a pH matching the buffer alone (pH 7). Spectra were recorded at 5°C over wavelengths ranging from 260 to 180 nm, with data collected every 1 nm (1 nm s−1), and each spectrum was recorded in duplicate. Temperature ramping experiments were recorded from 5 to 70–80°C with data recorded every ∼1°C using a settling time between measurements of 120 s. A background spectrum for CD buffer alone was recorded for each data collection session, and background ellipticity values were subtracted from raw sample ellipticity values ($θ$) when calculating MREs using:
where $Mw$ is the peptide molecular weight, and n is the number of total peptide bonds, in each case taken to be 16 accounting for the acetyl capping group.
Several methods are available to estimate the helicity based on MRE values [46–48]. Estimates of peptide helicity were made using the relationship used by Baker et al. [49] as follows:
$%helix=100×(MRE222−MREcoil)−42500(1−(3n))$
where $MRE222$ is the MRE value at 222 nm, $MREcoil=640−45T$ (with T in °C) =  415 deg cm2 dmol−1 res−1 at 5°C and n is the number of backbone amide bonds including the N-terminal acetyl (as above). For a selection of peptides, MRE values were calculated from data collected over a wider (100×) range of concentrations (roughly 7–700 µM, Supplementary Figure S1). No change in MRE values was observed suggesting that peptide self-association does not occur.

### Analysis of energetics

A simple estimate of the Gibbs free energy change (ΔG) of the helical (α) peptide in solution relative to its unfolded (u) state, can be calculated under a two-state, all-or-nothing approximation from the % helicity values determined experimentally by CD spectroscopy [11]. The equilibrium constant Kα between the folded and unfolded conformations in solution is defined by
$Kα=fα/(1−fα)$
where fα is the fraction of helical and (1 − fα) = fu, the fraction of unfolded peptide. The Gibbs free energy change on going from the unfolded state to the helical state is
$ΔGx=−RTln(Kα).$
The change in the Gibbs free energy change on folding (ΔΔG) is calculated, where appropriate, relative to the unphosphorylated peptide (ΔΔG = ΔGx − ΔGunphos) or the equivalent motif Ala-control peptide (ΔΔG = ΔGx − ΔG9/10). ΔΔG values were also calculated relative also to the least helical pSer-containing peptide 3 (ΔΔG = ΔGx − ΔG3) to highlight how the introduction of potential sites for non-covalent interaction into the sequence influences conformational energetics; the i + 5 spacings in peptide 3 mean that it has no potential for inter-side chain interaction for a helical conformation.

### NMR experiments

NMR spectra were recorded on a 600-MHz Bruker Avance spectrometer equipped with a quadruple resonance QCI-P cryoprobe, or a 950-MHz Bruker Ascend Aeon spectrometer equipped with a 5 mm TXO cryoprobe. For each peptide, natural abundance 1H–15N HMQC and 1H–13C HSQC (constant-time) spectra were recorded for 0.5–1.0 mM samples at 5°C. The low temperature maximizes the helical content of each peptide, and also (when combined with using pH between 6 and 7) slows proton exchange enough to allow the Arg Hε–Nε correlation to be visible. Clean spectra 1H–15N HMQC took ∼12 h to record. To reduce experiment time (to ∼6 h), 1H–13C HSQC spectra were recorded with reduced spectral width in 13C (typically 20 ppm centred on 56 ppm), and the spectrum folded out to show the position of the non-Hα–Cα correlations. 1H–1H TOCSY and 1H–1H NOESY spectra were recorded and used to assign resonances for each peptide through residue-specific shifts and strong (i, i − 1) NOE connectivity. Spectra were initially processed using NMRPipe/NMRDraw [50], and resonance assignments were carried out using CCPNmr Analysis [51]. Systematic patterns of peak shifts in spectra for different but related peptides were used to aid assignment. Full assignment of backbone resonances was possible (with the exception of uncharacterized amide carbonyl carbons). 13C resonance assignment in 1H–13C HSQC spectra required a unique attached 1H resonance and/or the peak position to occur in a residue-specific region of the spectrum. In a few instances, Ala Cα and Cβ resonances could not be independently assigned so two positions — marking the potential range in Cα/Cβ shift — were recorded. Secondary shifts (Δδ) = observed shifts (δ) — expected shifts for that residue in a random coil conformation (δRC), were calculated using recently published δRC values for phosphorylated residues [34] available via the website:

(https://www1.bio.ku.dk/english/research/bms/sbinlab/randomchemicalshifts2/). 1D 31P spectra were recorded on the 600-MHz spectrometer using the 30° flip angle pulse sequence.

### Analysis of Arg(i−3)Seri Lys(i+4) motifs in human proteome

A search for Arg(i3)Seri Lys(i+4) motifs in human proteins was carried out using the Expasy ScanProsite tool (https://prosite.expasy.org/scanprosite/) using the motif pattern ‘R-{P}(2)-S-{P}(3)-K', excluding isoforms, and filtering to taxonomy ‘Homo sapiens'. This generated 2226 hits in 1966 sequences, based on release 2021_04 of 29-Sep-21: 565 928entries) of the UniProtKB/Swiss-Prot database.

The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary materials. Raw data were generated at the University of Leeds and CD datasets and NMR chemical shift lists are available in the University of Leeds Data Repository via the DOI: 10.5518/1104.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by the EPSRC (EP/N013573/1) and BBSRC (BB/V003577/1). R.S.D. is supported by a studentship from the MRC Discovery Medicine North (DiMeN Doctoral Training Partnership (MR/N013840/1). A.J.W. held a Royal Society Leverhulme Trust Senior Fellowship (SRF/R1/191087). Facilities for NMR spectroscopy and CD spectroscopy were funded by the University of Leeds (ABSL award) and Wellcome Trust (108466/Z/15/Z, 094232/Z/10/Z).

Richard Bayliss: Conceptualization, supervision, funding acquisition, writing — review and editing. Matthew Batchelor: Investigation, writing — original draft. Robert S. Dawber: Investigation, writing — original draft. Andrew J. Wilson: Conceptualization, supervision, funding acquisition, writing — review and editing.

We thank Nasir Khan and Arnout Kalverda for their support and assistance in this work.

• CD

circular dichroism

•
• CHC

clathrin heavy chain

•
• MREs

mean residue ellipticities

•
• PPIs

protein–protein interactions

•
• SPPS

solid-phase peptide synthesis

•
• TACC

transforming acidic coiled coil

1
Bullock
,
B.N.
,
Jochim
,
A.L.
and
Arora
,
P.S.
(
2011
) Assessing helical protein interfaces for inhibitor design.
J. Am. Chem. Soc
.
133
,
14220
14223
2
Wang
,
H.
,
Dawber
,
R.S.
,
Zhang
,
P.
,
Walko
,
M.
,
Wilson
,
A.J.
and
Wang
,
X.
(
2021
) Peptide-based inhibitors of protein–protein interactions: biophysical, structural and cellular consequences of introducing a constraint.
Chem. Sci.
12
,
5977
5993
3
Muttenthaler
,
M.
,
King
,
G.F.
,
,
D.J.
and
Alewood
,
P.F.
(
2021
). Trends in peptide drug discovery.
Nat. Rev. Drug Discov.
20
,
309
325
4
Nick Pace
,
C.
and
Martin Scholtz
,
J.
(
1998
) A helix propensity scale based on experimental studies of peptides and proteins.
Biophys. J.
75
,
422
427
5
Miller
,
S.E.
,
Watkins
,
A.M.
,
Kallenbach
,
N.R.
and
Arora
,
P.S.
(
2014
) Effects of side chains in helix nucleation differ from helix propagation.
111
,
6636
6641
6
Doig
,
A.J.
,
Chakrabartty
,
A.
,
Klingler
,
T.M.
and
Baldwin
,
R.L.
(
1994
) Determination of free energies of N-capping in α-helixes by modification of the Lifson-Roig helix-coil theory to include N- and C-capping.
Biochemistry
33
,
3396
3403
7
Aurora
,
R.
and
Rosee
,
G.D.
(
1998
) Helix capping.
Protein Sci.
7
,
21
38
8
Shi
,
Z.S.
,
Olson
,
C.A.
and
Kallenbach
,
N.R.
(
2002
) Cation-π interaction in model α-helical peptides.
J. Am. Chem. Soc.
124
,
3284
3291
9
Olson
,
C.A.
,
Shi
,
Z.S.
and
Kallenbach
,
N.R.
(
2001
) Polar interactions with aromatic side chains in α-Helical peptides:  Ch···O H-Bonding and cation−π Interactions.
J. Am. Chem. Soc.
123
,
6451
6452
10
Tsou
,
L.K.
,
Tatko
,
C.D.
and
Waters
,
M.L.
(
2002
) Simple cation–π interaction between a phenyl ring and a protonated amine stabilizes an α-helix in water.
J. Am. Chem. Soc.
124
,
14917
14921
11
Lyu
,
P.
,
Liff
,
M.
,
Marky
,
L.
and
Kallenbach
,
N.
(
1990
) Side chain contributions to the stability of alpha-helical structure in peptides.
Science
250
,
669
673
12
Iakoucheva
,
L.M.
,
,
P.
,
Brown
,
C.J.
,
O'Connor
,
T.R.
,
Sikes
,
J.G.
,
,
Z.
et al (
2004
) The importance of intrinsic disorder for protein phosphorylation.
Nucleic Acids Res.
32
,
1037
1049
13
Nishi
,
H.
,
Hashimoto
,
K.
and
Panchenko
,
A.R.
(
2011
) Phosphorylation in protein–protein binding: effect on stability and function.
Structure
19
,
1807
1815
14
Bah
,
A.
,
Vernon
,
R.M.
,
Siddiqui
,
Z.
,
Krzeminski
,
M.
,
Muhandiram
,
R.
,
Zhao
,
C.
et al (
2015
) Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch.
Nature
519
,
106
109
15
Errington
,
N.
and
Doig
,
A.J.
(
2005
) A phosphoserine–lysine salt bridge within an α-helical peptide, the strongest α-helix side-chain interaction measured to date.
Biochemistry
44
,
7553
7558
16
Andrew
,
C.D.
,
Warwicker
,
J.
,
Jones
,
G.R.
and
Doig
,
A.J.
(
2002
) Effect of phosphorylation on α-helix stability as a function of position.
Biochemistry
41
,
1897
1905
17
Elbaum
,
M.B.
and
Zondlo
,
N.J.
(
2014
) OGlcNAcylation and phosphorylation have similar structural effects in α-helices: post-translational modifications as inducible start and stop signals in α-helices, with greater structural effects on threonine modification.
Biochemistry
53
,
2242
2260
18
Szilák
,
L.
,
Moitra
,
J.
and
Vinson
,
C.
(
1997
) Design of a leucine zipper coiled coil stabilized 1.4 kcal mol−1 by phosphorylation of a serine in the e position.
Protein Sci.
6
,
1273
1283
19
Signarvic
,
R.S.
and
,
W.F.
(
2003
) De novo design of a molecular switch: phosphorylation-dependent association of designed peptides.
J. Mol. Biol.
334
,
1
12
20
Kumar
,
A.
,
Gopalswamy
,
M.
,
Wolf
,
A.
,
Brockwell
,
D.J.
,
Hatzfeld
,
M.
and
Balbach
,
J.
(
2018
) Phosphorylation-induced unfolding regulates p19INK4d during the human cell cycle.
115
,
3344
3349
21
Harrington
,
L.
,
Fletcher
,
J.M.
,
Heermann
,
T.
,
Woolfson
,
D.N.
and
Schwille
,
P.
(
2021
) De novo design of a reversible phosphorylation-dependent switch for membrane targeting.
Nat. Commun.
12
,
1472
22
Burgess
,
S.G.
,
Mukherjee
,
M.
,
Sabir
,
S.
,
Joseph
,
N.
,
Gutiérrez-Caballero
,
C.
,
Richards
,
M.W.
et al (
2018
) Mitotic spindle association of TACC3 requires Aurora‐A‐dependent stabilization of a cryptic α‐helix.
EMBO J.
37
,
e97902
23
Scholtz
,
J.M.
,
York
,
E.J.
,
Stewart
,
J.M.
and
Baldwin
,
R.L.
(
1991
) A neutral, water-soluble, α-helical peptide: the effect of ionic strength on the helix -coil equilibrium.
J. Am. Chem. Soc.
113
,
5102
5104
24
Shalongo
,
W.
,
,
L.
and
Stellwagen
,
E.
(
1994
) Distribution of helicity within the model peptide Acetyl(AAQAA)3amide.
J. Am. Chem. Soc.
116
,
8288
8293
25
Huyghues-Despointes
,
B.M.P.
,
Martin Scholtz
,
J.
and
Baldwin
,
R.L.
(
1993
) Helical peptides with three pairs of Asp–Arg and Glu–Arg residues in different orientations and spacings.
Protein Sci.
2
,
80
85
26
Greenfield
,
N.J.
(
2006
) Using circular dichroism spectra to estimate protein secondary structure.
Nat. Protoc.
1
,
2876
2890
27
Pandey
,
A.K.
,
Ganguly
,
H.K.
,
Sinha
,
S.K.
,
Daniels
,
K.E.
,
Yap
,
G.P.A.
,
Patel
,
S.
et al (
2020
) An inherent structural difference between serine and threonine phosphorylation: phosphothreonine prefers an ordered, compact, cyclic conformation.
bioRxiv
2020.2002.2029.971382
28
Scholtz
,
J.M.
,
Qian
,
H.
,
Robbins
,
V.H.
and
Baldwin
,
R.L.
(
1993
) The energetics of ion-pair and hydrogen-bonding interactions in a helical peptide.
Biochemistry
32
,
9668
9676
29
Smith
,
J.S.
and
Scholtz
,
J.M.
(
1998
) Energetics of polar side-chain interactions in helical peptides: salt effects on ion pairs and hydrogen bonds.
Biochemistry
37
,
33
40
30
Selenko
,
P.
,
Frueh
,
D.P.
,
Elsaesser
,
S.J.
,
Haas
,
W.
,
Gygi
,
S.P.
and
Wagner
,
G.
(
2008
) In situ observation of protein phosphorylation by high-resolution NMR spectroscopy.
Nat. Struct. Mol. Biol.
15
,
321
329
31
Wishart
,
D.S.
,
Sykes
,
B.D.
and
Richards
,
F.M.
(
1991
) Relationship between nuclear magnetic resonance chemical shift and protein secondary structure.
J. Mol. Biol.
222
,
311
333
32
Wishart
,
D.S.
and
Sykes
,
B.D.
(
1994
) The 13C Chemical-Shift Index: A simple method for the identification of protein secondary structure using 13C chemical-shift data.
J. Biomol. NMR
4
,
171
180
33
Wishart
,
D.S.
,
Sykes
,
B.D.
and
Richards
,
F.M.
(
1992
) The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy.
Biochemistry
31
,
1647
1651
34
Hendus-Altenburger
,
R.
,
Fernandes
,
C.B.
,
Bugge
,
K.
,
Kunze
,
M.B.A.
,
Boomsma
,
W.
and
Kragelund
,
B.B.
(
2019
) Random coil chemical shifts for serine, threonine and tyrosine phosphorylation over a broad pH range.
J. Biomol. NMR
73
,
713
725
35
Pascal
,
S.M.
,
Yamazaki
,
T.
,
Singer
,
A.U.
,
Kay
,
L.E.
and
Forman-Kay
,
J.D.
(
1995
) Structural and dynamic characterization of the phosphotyrosine binding region of an Src homology 2 domain–phosphopeptide complex by NMR relaxation, proton exchange, and chemical shift approaches.
Biochemistry
34
,
11353
11362
36
Batchelor
,
M.
,
Wolny
,
M.
,
Baker
,
E.G.
Paci
,
E.
,
Kalverda
,
A.P.
and
Peckham
,
M.
(
2019
) Dynamic ion pair behavior stabilizes single α-helices in proteins.
J. Biol. Chem.
294
,
3219
3234
37
Zandarashvili
,
L.
,
A.
and
Iwahara,
J.
(
2013
) NMR studies on the dynamics of hydrogen bonds and ion pairs involving lysine side chains of proteins. In
Advances in Protein Chemistry and Structural Biology
(
Christov
,
C.Z.
ed.), vol.
93
, pp.
37
80
,
38
Iqbalsyah
,
T.M.
and
Doig
,
A.J.
(
2005
) Anticooperativity in a Glu–Lys–Glu salt bridge triplet in an isolated α-helical peptide.
Biochemistry
44
,
10449
10456
39
Szilák
,
L.
,
Moitra
,
J.
,
Krylov
,
D.
and
Vinson
,
C.
(
1997
) Phosphorylation destabilizes α-helices.
Nat. Struct. Biol.
4
,
112
114
40
,
D.
,
Vieitez
,
C.
,
Rajeeve
,
V.
,
Selkrig
,
J.
,
Cutillas
,
P.R.
and
Beltrao
,
P.
(
2021
) Sequence and structure-based analysis of specificity determinants in eukaryotic protein kinases.
Cell Rep.
34
,
108602
41
Minor
,
D.L.
and
Kim
,
P.S.
(
1994
) Measurement of the β-sheet-forming propensities of amino acids.
Nature
367
,
660
663
42
Lyu
,
P.C.
,
Sherman
,
J.C.
,
Chen
,
A.
and
Kallenbach
,
N.R.
(
1991
) Alpha-helix stabilization by natural and unnatural amino acids with alkyl side chains.
88
,
5317
5320
43
Skinner
,
J.J.
,
Wang
,
S.
,
Lee
,
J.
,
Ong
,
C.
,
Sommese
,
R.
,
Sivaramakrishnan
,
S.
et al (
2017
) Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome.
114
,
13453
13458
44
Kaiser
,
E.
,
Colescott
,
R.L.
,
Bossinger
,
C.D.
and
Cook
,
P.I.
(
1970
) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides.
Anal. Biochem.
34
,
595
598
45
Kelly
,
S.M.
,
Jess
,
T.J.
and
Price
,
N.C.
(
2005
) How to study proteins by circular dichroism.
Biochim. Biophys. Acta - Proteins Proteom.
1751
,
119
139
46
Lacroix
,
E.
,
Viguera
,
A.R.
and
Serrano
,
L.
(
1998
) Elucidating the folding problem of α-helices: local motifs, long-range electrostatics, ionic-strength dependence and prediction of NMR parameters.
J. Mol. Biol.
284
,
173
191
47
Sommese
,
R.F.
,
Sivaramakrishnan
,
S.
,
Baldwin
,
R.L.
and
Spudich
,
J.A.
(
2010
) Helicity of short E-R/K peptides.
Protein Sci.
19
,
2001
2005
48
Shepherd
,
N.E.
,
Hoang
,
H.N.
,
Abbenante
,
G.
and
Fairlie
,
D.P.
(
2005
) Single turn peptide alpha helices with exceptional stability in water.
J. Am. Chem. Soc.
127
,
2974
2983
49
Baker
,
E.G.
Bartlett
,
G.J.
,
Crump
,
M.P.
,
Sessions
,
R.B.
,
Linden
,
N.
,
Faul
,
C.F.J.
et al (
2015
). Local and macroscopic electrostatic interactions in single α-helices.
Nat. Chem. Biol.
11
,
221
228
50
Delaglio
,
F.
,
Grzesiek
,
S.
,
Vuister
,
G.W.
,
Zhu
,
G.
,
Pfeifer
,
J.
and
Bax
,
A.
(
1995
). NMRPipe: A multidimensional spectral processing system based on UNIX pipes.
J. Biomol. NMR
6
,
277
293
51
Vranken
,
W.F.
,
Boucher
,
W.
,
Stevens
,
T.J.
,
Fogh
,
R.H.
,
Pajon
,
A.
,
Llinas
,
M.
et al (
2005)
The CCPN data model for NMR spectroscopy: Development of a software pipeline.
Proteins
59
,
687
696