Solid‐State NMR of PEGylated Proteins

Abstract

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.

Characterization of the Conjugation Pattern in Large Polysaccharide–Protein Conjugates by NMR Spectroscopy

“…As already observed in the case of PEGylation, [30] these signals correspond to the amide groups generated by functionalization of the e-amino group of lysines (H z -N z )w ith the MenC polysaccharide moieties.T he resonances of the non-functionalized lysines would appear at very low 15 Nchemical shift values (around 30 ppm), but are usually wiped out by exchange.O nt he contrary,w hen they become amides,e xchange is slowed down and the intrinsic flexibility of the sidechain justifies the appearance of the H z -N z cross-peaks.T hese signals are markers that can provide direct information on the number of lysine residues involved in the formation of new bonds with the MenC polysaccharide. However,t he assignment of each H z -N z couple to the corresponding H-N of the backbone is adifficult task because of the high overlap between H z -N z signals,a nd the lack of connections between the two H-N couples across the side chain.…”

“…[27] However,a lso for mass spectrometry the evaluation of the conjugation degree at the different sites in polydisperse populations of isomers is ac hallenge.R ecently,w eh ave shown that structural information on large PEGylated proteins,s uch as E. coli l-Asparaginase-II (138 kDa, ANSII, see the Supporting Information), can be obtained by solid-state NMR spectroscopy. [30] Several excellent reports on capsular polysaccharides,b acterial cell walls and their maturation have been obtained by the same technique. [31][32][33] ANSII is not used as carrier protein in glycoconjugate vaccines,h owever,i ty ields highly resolved SSNMR spectra, [30] and approximately 74 %o ft he resonances have been assigned (unpublished data).…”

“…[30] Several excellent reports on capsular polysaccharides,b acterial cell walls and their maturation have been obtained by the same technique. [31][32][33] ANSII is not used as carrier protein in glycoconjugate vaccines,h owever,i ty ields highly resolved SSNMR spectra, [30] and approximately 74 %o ft he resonances have been assigned (unpublished data). We have thus selected ANSII as am odel system for this investigation.…”

“…Fort he solid-state NMR investigation, ANSII (either free or its MenC conjugate) was freeze-dried, packed into the magic-angle spinning (MAS) rotors,t hen rehydrated to restore the resolution. [30,[34][35][36] Thes pectra collected on the pelleted ANSII-MenC conjugate (Figure 2) are largely superimposable to those of the native and PEGylated forms of the enzyme (see Figure S2 in the Supporting Information) making possible the assessment of the preservation of the protein fold after conjugation with the MenC polysaccharide. [37][38][39][40] Ther esonances of the protein residues were easily reassigned starting from the assignment of the native form of ANSII, by using ac arbon-detection approach ( Figure S3).…”

Biosilica and bioinspired silica studied by solid-state NMR

“…The introduction of NMR of sedimented solutes [69][70][71] has lifted the needs for crystallization in the case of soluble proteins, yielding access to soluble but non-crystallizing targets such as PEGylated proteins [130]. However, as seen by NMR, biomaterials are complex entities: the protein and the inorganic matrix interplay to define physicochemical characteristics of the composite, in terms of mobility, hydration, etc.…”