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| Home > Publications database > Isoleucine Side Chains as Reporters of Conformational Freedom in Protein Folding Studied by DNP-Enhanced NMR |
| Home > Publications database > Isoleucine Side Chains as Reporters of Conformational Freedom in Protein Folding Studied by DNP-Enhanced NMR |
| Journal Article | FZJ-2025-02519 |
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2025
ACS Publications
Washington, DC
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Please use a persistent id in citations: doi:10.1021/jacs.5c04159 doi:10.34734/FZJ-2025-02519
Abstract: Conformations of protein side chains are closely linked to protein function. DNP-enhanced solid-state NMR (ssNMR), which operates at cryogenic temperatures (<110 K), can be used to freeze-trap protein conformations, including the side chains. In the present study, we employed two-dimensional DNP-enhanced ssNMR to get detailed insights into backbone and side chain conformations of isoleucine. We used different amino acid selectively labeled model proteins for intrinsically disordered proteins (IDPs), denatured and well-folded proteins, and amyloid fibrils. 13C chemical shifts are closely correlated with secondary structure elements and χ1 and χ2 angles in isoleucine side chains. Thus, line shape analysis by integration of representative peak areas in 2D spectra provides an accurate overview of the distribution of backbone and side chain conformations. For the well-folded proteins GABARAP and bovine PI3-kinase (PI3K) SH3 domain, most Ile chemical shifts in frozen solution are well resolved and similar to those observed in solution. However, line widths of individual Ile residues are directly linked to residual mobility, and line broadening or even signal splitting appears for those Ile residues, which are not part of well-defined secondary structure elements. For unfolded PI3K SH3 and the IDP α-synuclein (α-syn), all Ile side chains have full conformational freedom, and as a consequence, inhomogeneous line broadening dominates the cryogenic spectra. Moreover, we demonstrate that conformational ensembles of proteins strongly depend on solvent and buffer conditions. This allowed different unfolded structures for chemical and acidic pH denaturation of the PI3K SH3 domain to be distinguished. In amyloid fibrils of α-syn and PI3K SH3, chemical shifts typical for β-strand like secondary structure dominate the spectra, whereas Ile residues belonging to the fuzzy coat still add the IDP-type line shapes. Hence, DNP-enhanced ssNMR is a useful tool for investigating side chain facilitated protein functions and interactions.
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