Semisynthetic prion protein (PrP) variants carrying glycan mimics at position 181 and 197 do not form fibrils† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc02719b Click here for additional data file.

Semisynthesis and characterization of homogeneously mono- and di-PEGylated full length PrP variants to study the impact of PEGylation (as N-glycan mimics) on protein folding and aggregation.


S5
The results were aligned with the theoretical sequence to confirm in-frame fusion of the inserted PrP genes and MxeIntein.
Elution fractions containing the protein-thioester were combined; pooled and lyophilized. Subsequent identification was done via analytical RP-HPLC, ESI-MS and SDS-PAGE.

mercapto-aspartate
All EPL reactions were performed in ligation buffer (6 M Gdn-HCl, 100 mM NaPi buffer and 100 mM MPAA). 50 mM TCEP was used to prevent disulfide formation during EPL. Prior to the ligation, the buffer was degassed with argon for 15 min. Product containing collected fractions were pooled, lyophilized and stored at -80°C for further use. (typically between 0.05-0.2 mg/mL) and CD spectra were collected.

Circular Dichroism (CD)
Far UV-CD spectra were measured using a Chirascan Plus spectrometer (Applied Photophysics, UK) between 190-260 nm. Unless stated otherwise, ten spectra with an acquisition time of 10 s for each scan in a 1 mm quartz cell at 1 nm resolution were acquired at r.t. and averaged. Typical protein concentrations were 0.1 mg/mL in folding buffer. To determine the secondary structure, minima at 208 and 222 nm as well as maxima at 193 nm for α-helices were considered, whereas a negative peak at 218 nm and a positive peak at 195 nm were used for the calculation of the percentage of antiparallel β-sheets. The amount of random coil structure was calculated using the maximum peak at 210 nm and minimum peak at 195 nm. All calculations were carried out using CDNN software comparing at least 13 spectra from the database.

Aggregation assays with Thioflavin-T (ThT)
For aggregation assays a procedure modified from Baskakov et al.

3.
General procedures for introduction of modifications into PrP

Covalent attachment of acetyl groups into Dpr residues of PrP peptides. 4
Mtt temporary protecting groups on Dpr amine side chains were deprotected as described in 3.1. The N-terminus of the peptidyl resin was Boc protected. Acetic anhydride (20% v/v) and DIEA (20% v/v) was added to a mixture of DMF/DCM (1/1). This mixture (10 mL/g resin) was added to the peptidyl resin and reacted for 1 h. Upon completion, the resin was washed with DMF (3 mL, 1min) and dried in vacuo.

4.
General procedures for recombinant protein expression 4

.1. Recombinant Expression of wild type, full length PrP.
Wild type (wt), recombinant PrP was generated via hydrolysis of recombinant, full length PrP α-thioester as described previously by Becker and coworkers. 5

5.
Expressed Protein Ligation (EPL)      nm highlighted a tendency for β-sheets, whereas the typical minimum at 218 was not observed, suggesting a predominant random coiled structure ( Figure S10). S18