title: The importance of protonation in the investigation of protein phosphorylation using Raman spectroscopy and Raman optical activity.
abstract: The effect of protonation on amino acid monomers and protein phosphorylation was studied by means of a combination of Raman scattering and Raman optical activity (ROA). In the past, identifying spectral variations in phosphorylated proteins arising from either the phosphate stretch or amide vibrational modes has proven to be challenging mainly due to the loss of amide and P═O band intensity in the presence of phosphate. By contrast, we have developed a novel strategy based on the careful monitoring of the sample pH and thereby modified the protonation state, such that these difficulties can be overcome and phosphate-derived vibrations are readily visualized with both Raman and ROA. Variations in pH-dependent spectral sets of phosphorylated amino acid monomers serine and threonine demonstrated that the protonation state could be determined by the intensity of the monobasic (-OPO(3)H(-)) phosphate stretch band occurring at ~1080 cm(-1) versus the dibasic (-OPO(3)(2-)) band measured at ~980 cm(-1) in both Raman and ROA. Furthermore, by adjustment of the pH of aqueous samples of the phosphoprotein α-casein and comparing this result with dephosphorylated α-casein, spectral variations in phosphate stretch bands and amide bands could be easily determined. Consequently, structural variations due to both protonation and dephosphorylation could be distinguished, demonstrating the potential of Raman and ROA for future investigations of phosphoprotein structure and interactions.
authors.affiliation: School of Chemistry, Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. Lorna.Ashton@manchester.ac.uk
authors.forename: Lorna
authors.initials: L
authors.lastname: Ashton
authors.forename: Christian
authors.initials: C
authors.lastname: Johannessen
authors.forename: Royston
authors.initials: R
authors.lastname: Goodacre
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002364:Caseins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009904:Optical Rotation; D010766:Phosphorylation; D011522:Protons; D013059:Spectrum Analysis, Raman; D014732:Vibration
chemical_list: D000596:Amino Acids; D002364:Caseins; D011522:Protons