Tracking Conformational Changes in Phosvitin throughout a Crowding‐Agent‐Based Titration

Abstract

The sensitivity of Raman optical activity (ROA) towards small conformational changes is explored by tracking the structural changes in an intrinsically disordered protein—phosvitin—induced by different concentrations of crowding agent. It is shown that ROA is capable of tracking small conformational changes involving β‐sheet and α‐helical secondary structural properties of the protein. Furthermore, it is indicated that the influences of the crowding agents employed, Ficoll 70 and dextran 70, on the structural properties of phosvitin differ significantly, with the structural changes induced by the presence of Ficoll 70 being more pronounced and already being visible at a lower concentration. The data also suggest that some spectral changes do not arise from a change in the secondary structure of the protein, but are related to differences in interaction between the phosphorylated residues of the protein and the sugar‐based crowding agent.

Employing complementary spectroscopies to study the conformations of an epimeric pair of side-chain stapled peptides in aqueous solution

“… 31 The latter two regions were shown to be sensitive to the secondary structure of proteins and peptides. 29,32,33 …”

“…Both spectra show distinct spectral patterns typical for structurally disordered peptides. [27][28][29] The broad band at $1680 cm À1 (amide I region) and the 1256 cm À1 Raman band, observed for both peptides, are diagnostic of a disordered structure or random protein/peptide conformation. 28 The highly similar spectral pattern of the two samples is expected for epimeric peptides.…”

“…31 The latter two regions were shown to be sensitive to the secondary structure of proteins and peptides. 29,32,33 The ROA spectra of 1 and 2 depict a rather featureless pattern in the backbone skeletal stretch, and broad positive bands at 1323 cm À1 and 1677 cm À1 , in the amide III and amide I regions, respectively. For proteins and peptides, this ROA pattern indicates a polyproline II (PPII) peptide backbone structure.…”

“…For proteins and peptides, this ROA pattern indicates a polyproline II (PPII) peptide backbone structure. 28,29,34 A PPII helix is a common conformational element, characterized by the backbone f and j torsion angles clustering around f ¼ À75 and j ¼ 145 in the Ramachandran plot, and is observed also for sequences that do not contain proline. 35 Negative contributions in the 1200-1300 cm À1 region of the ROA spectrum were previously reported for intrinsically disordered proteins with large portions of PPII structure, such a-synuclein.…”