Researcher(s)
- Connor Kosinski, Biochemistry, University of Delaware
Faculty Mentor(s)
- Neal Zondlo, Chemistry & Biochemistry, University of Delaware
Abstract
Understanding the effects of phosphorylation on serine and threonine may provide insight into the aggregation and progression of Alzheimer’s disease. Within the tau protein, a decrease in enzyme activity leads to hyperphosphorylation, an occurrence when various phosphorylation sites become saturated. Hyperphosphorylation at Thr/Ser/Pro, Ser/Pro, and Thr/Pro motifs within the tau protein is a characteristic of disease progression.1 Proline is a unique amino acid, due to its ring structure, where 5.4% of proline residues exhibit the cis-amide conformation, while 0.03% of non-proline residues occupy the cis-amide conformation.2 Under physiological conditions, phosphorylation prior to proline slows the rate of cis/trans-amide isomerization.3 Phosphorylation induces stabilization of the trans conformation via an intraresidue phosphate-amide hydrogen bond and an n→π* interaction between the Ser-Pro amide and the carbonyl of serine upon phosphorylation of the residue preceding proline.4 Peptidyl-prolyl isomerase (Pin1) facilitates the amide bond rotation between trans/cis while phosphorylated. This catalysis is important for Alzheimer’s disease because Pin1 allows the cis conformation to be available for dephosphorylation. More rapid cis/trans isomerism due to prolyl isomerases allows phosphorylation regulation via conformer-specific protein phosphatases e.g. PP2A and SSu72. Previous data suggests a role for a C–H/Ophos interaction in stabilizing the cis-amide conformation.5 These effects are observed via Ac-TSPX-NH2 sequences, specifically TSPN, derived from the tau403-406 protein. These motifs are also present in the RNA polymerase II C-terminal domain (RNA PolII CTD), where phosphorylation sites are related to protein functions regulating DNA transcription within humans. The consensus repeat sequence, YSPTSPS, has phosphorylation sites at Thr-4 and Ser-5 preceding Pro-6, both of which are biologically relevant. Thr-4 phosphorylation is responsible for transcription elongation, termination, and post-transcriptional splicing. Ser-5 phosphorylation regulates transcription, mRNA capping and splicing, and non-coding RNA transcription.6 Structural change, in the repeat sequence, is induced by phosphorylation, modulate RNA pol II activity. To study the impact of phosphorylation on cis-trans isomerism in these contexts, Fmoc-L-4,4-difluoroproline (Fmoc-Dfp-OH) was synthesized in 2-steps from commercially available Boc-L-4,4-difluoroproline. This product is incorporated as a proline surrogate in the biologically relevant TSPN and YSPTSPS sequences. This 19F-NMR probe displays two distinct cis and two distinct trans peaks in its 19F spectra. By quantifying the integrals of these peaks, the calculation of Ktrans/cis is possible. This equilibrium constant provides further structural information regarding the relative populations of trans/cis Ser-Pro amide bonds within the peptide. pH-dependent NMR spectroscopy can be used to view the effect of protonation state on Ktrans/cis in phosphorylated TSPN and YSPTSPS peptides derived from tau protein and RNA PolII CTD. Analysis of this data provides insight into the relative populations of cis and trans and their roles towards the biological function of proteins and the impact of phosphorylation in general.