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

TL;DR

This study uses DNP-enhanced NMR to explore how isoleucine side chains reflect protein structure and mobility in different states.

Contribution

The study introduces DNP-enhanced ssNMR as a novel method to analyze conformational freedom in protein side chains.

Findings

Isoleucine side chain conformations correlate with secondary structure elements and mobility.

Cryogenic NMR reveals distinct line shapes for folded, unfolded, and amyloid states of proteins.

Solvent and pH conditions significantly influence conformational ensembles of proteins.

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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