Characterization of dry globular proteins and protein fibrils by synchrotron radiation vacuum UV circular dichroism

Abstract

Circular dichroism using synchrotron radiation (SRCD) can extend the spectral range down to approximately 130 nm for dry proteins, potentially providing new structural information. Using a selection of dried model proteins, including alpha-helical, beta-sheet, and mixed-structure proteins, we observe a low-wavelength band in the range 130-160 nm, whose intensity and peak position is sensitive to the secondary structure of the protein and may also reflect changes in super-secondary structure. This band has previously been observed for peptides but not for globular proteins, and is compatible with previously published theoretical calculations related to pi-orbital transitions. We also show that drying does not lead to large changes in the secondary structure and does not induce orientational artifacts. In combination with principal component analysis, our SRCD data allow us to distinguish between two different types of protein fibrils, highlighting that bona fide fibrils formed by lysozyme are structurally more similar to the nonclassical fibrillar aggregates formed by the SerADan peptide than with the amyloid formed by alpha-synuclein. Thus, despite the lack of direct structural conclusions, a comprehensive SRCD-based database of dried protein spectra may provide a useful method to differentiate between various types of supersecondary structure and aggregated protein species.

Structural and Functional Characterization of Human Peripheral Nervous System Myelin Protein P2

“…as a film dried onto the surface of the CaF 2 cuvette. Such measurements on other proteins were recently reported and found to correlate reasonably well with solution data [48]. In the case of P2, the spectra are nearly identical in the region available for solution measurements (Figure 1B), indicating the protein retains its native fold upon the drying procedure.…”

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

“…In this case, stirring the sample in the cuvette is mandatory. Another approach is to measure CD on dried samples [123]. Extremely unstable or rapidly aggregating proteins can change their conformation within minutes.…”

A thermodynamic analysis of fibrillar polymorphism

“…The spectra were recorded at the SRCD facility at the UV1 beamline [26] on the storage ring ASTRID at Institute for Storage Ring Facilities (ISA), University of Aarhus, Denmark as previously described [27]. An MgF 2 plate was thoroughly cleaned with milliQ water and dried before a baseline spectrum was collected.…”