Isoleucine Side Chains as Reporters of Conformational Freedom in Protein Folding Studied by DNP-Enhanced NMR

https://doi.org/10.1021/jacs.5c04159

 

요약

1. Isoleucine의 side chain을 folding 센서로 활용: 단백질 접힘 과정에서 isoleucine의 γ1–χ1, χ2 회전각 분포가 folding 전후에 어떻게 달라지는지를 고체상 DNP-enhanced NMR으로 분석하여, side chain이 접힘 상태에서의 자유도를 반영하는 효과적인 지표임을 확인.
2. 고해상 DNP-NMR로 folding 중간체 분석 가능성 제시: Dynamic Nuclear Polarization (DNP)을 통해 고감도 측정이 가능해졌으며, 이는 기존 NMR보다 folding의 시간적·공간적 해상도를 높이는 데 기여.
3. 접힘 전후의 회전각 자유도 차이 정량화: 접힘 전의 unfolded 상태에선 side chain 회전각이 균일한 분포를 보이는 반면, 접힘 후에는 특정 conformer로 구속되는 양상이 뚜렷하게 나타남.
4. 이소류신 외에도 다른 hydrophobic side chain 확장 가능성 제안: Isoleucine은 대표적인 hydrophobic residue로서 구조 민감성이 뛰어나며, 이 접근법은 다른 아미노산에도 확장 가능함을 시사.
5. 접힘 상태에 따른 단백질의 energy landscape 해석 보완:side chain 회전 자유도의 정량화는 단백질의 conformational entropy 및 folding landscape 해석에 중요한 정보를 제공함.

 

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.