Direct Effects of Phosphorylation on the Preferred Backbone Conformation of Peptides: A Nuclear Magnetic Resonance Study

Abstract

Control of protein activity by phosphorylation appears to work principally by inducing conformational change, but the mechanisms so far reported are dependent on the structural context in which phosphorylation occurs. As the activity of many small peptides is also regulated by phosphorylation, we decided to investigate possible direct consequences of this on the preferred backbone conformation. We have performed 1H nuclear magnetic resonance (NMR) experiments with short model peptides of the pattern Gly-Ser-Xaa-Ser, where Xaa represents Ser, Thr, or Tyr in either phosphorylated or unphosphorylated form and with either free or blocked amino and carboxy termini. The chemical shifts of amide protons and the 3JNH-Halpha coupling constants were estimated from one-dimensional and two-dimensional scalar correlated spectroscopy (COSY) spectra at different pH values. The results clearly indicate a direct structural effect of serine and threonine phosphorylation on the preferred backbone dihedrals independent of the presence of charged groups in the surrounding sequence. Tyrosine phosphorylation does not induce such a charge-independent effect. Additionally, experiments with p-fluoro- and p-nitro-phenylalanine-containing peptides showed that the mere presence of an electronegative group on the aromatic ring of tyrosine does not produce direct structural effects. In the case of serine and threonine phosphorylation a strong dependence of the conformational shift on the protonation level of the phosphoryl group could be observed, showing that phosphorylation induces the strongest effect in its dianionic, i.e., physiological, form. The data reveal a hitherto unknown mechanism that may be added to the repertoire of conformational control of peptides and proteins by phosphorylation.

Tyrosine Phosphorylation of the Well Packed EphrinB Cytoplasmic β-Hairpin for Reverse Signaling

“…3c). Previously, it was shown that, unlike serine and threonine phosphorylations, tyrosine phosphorylation had no direct effect on the backbone conformation of model peptides (20). Therefore, the current observation implies that tyrosine phosphorylations of ephrinB2 301-322 , regardless of their locations in the hairpin (Fig.…”

Control of Intrinsically Disordered Stathmin by Multisite Phosphorylation

“…The microtubule-polymerization inhibition and tubulin binding activities, the secondary structure content, and the thermal stability of the quadruple mutant 4A were indistinguishable from the wild-type, justifying the validity of the approach (Table 1). To produce the mutant molecules, two silent nucleotide mutations, G66C and G141C, were introduced by PCR into the human pET-16b (Novagen) stathmin clone (7) generating SacI and XhoI recognition sites between the codons for Ser 16 and Ser 25 and Ser 38 and Ser 63 , respectively. This new plasmid provided the basis for subsequent cloning of all mutants by using synthetic oligonucleotide adapter primers at either NdeI/SacI, SacI/XhoI, or XhoI/ BbvCI recognition sites.…”

“…ND, not determined. 38 . The isothermal titration calorimetry data of p16,25,38,63 could not be evaluated because binding was too weak.…”

“…In vivo, the activity of stathmin is down-regulated by posttranslational phosphorylation in response to a number of signals on four serine residues, Ser 16 , Ser 25 , Ser 38 , and Ser 63 (26 -29). In mitotic cells, for example, phosphorylation by an unknown kinase-phosphatase system allows creating local stathmin activity gradients, a process essential for regulating microtubule dynamics and spindle formation (30 -33).…”

Thermodynamic Signatures of Macromolecular Complexes ‒ Insights on the Stability and Interactions of Nucleoplasmin, a Nuclear Chaperone

“…The most active mutant, NP13D, is also the most unstable (Tm  55.2°C, H  17.9 kcal/mol). Taking into account that at neutral pH aspartic acid has one negative charge, while a phosphoryl group would display an average negative charge of -1.5 [81], 13 Asp would be a reasonable approximation of 7-10 phosphates per monomer in egg NP; however, the fact that this mutant is less stable than egg NP reflects that the conformational properties of phosphorylated NP may not be exactly reproduced [9]. The mutations-induced destabilization of NP is also readily observed by chemical unfolding experiments [9].…”