A Unique Tool for Cellular Structural Biology: In-cell NMR

Abstract

Conventional structural and chemical biology approaches are applied to macromolecules extrapolated from their native context. When this is done, important structural and functional features of macromolecules, which depend on their native network of interactions within the cell, may be lost. In-cell nuclear magnetic resonance is a branch of biomolecular NMR spectroscopy that allows macromolecules to be analyzed in living cells, at the atomic level. In-cell NMR can be applied to several cellular systems to obtain biologically relevant structural and functional information. Here we summarize the existing approaches and focus on the applications to protein folding, interactions, and post-translational modifications.

Keywords: cell compartmentalization; intracellular processing; molecular cell biology; nuclear magnetic resonance (NMR); post-translational modification (PTM); protein folding; structural biology.

FIGURE 1.

Overview of the existing sample preparation approaches for in-cell NMR. a–c, isotopically labeled proteins can be expressed (green) in bacterial cells (a) and yeast cells (b) by transforming the cells with expression vector(s) encoding the protein(s) of interest; proteins can be expressed in insect and human cells (c) by transfecting the cells with constitutive expression vectors. Isotopically enriched nutrients are added after induction/transfection. d–f, purified labeled proteins (blue) can be inserted in human cells by Cu,Zn-SOD1 CPP-mediated delivery (d), or by permeabilizing the cells either with pore-forming toxins (e) or via electroporation (f). g, proteins can be inserted in X. laevis oocytes by microinjection.

FIGURE 2.

Effect of the molecular crowding on different proteins. a, in-cell ¹H-¹⁵N correlation spectra of the B1 domain of staphylococcal protein G (GB1, left), mercuric ion reductase (NmerA, center), and a fusion of two GB1 domains (dGB1, right) in E. coli. Although dGB1 is larger than NmerA, it interacts less with the environment, giving rise to sharper NMR signals. a.a., amino acids. Adapted with permission from Ref. . Copyright (2011) American Chemical Society. b, in-cell ¹H-¹⁵N correlation spectra of the Trp-Trp-binding module (WW) domain of the peptidyl-prolyl isomerase Pin1 in X. laevis oocytes. Unmodified WW (left) constantly interacts with the environment, resulting in signal loss; phosphorylation of Ser-16 (mimicked by S16E mutation, center) and interaction with a substrate (right) abrogate the nonspecific interactions, and the fast-tumbling WW domain can be detected by NMR. Adapted with permission from Ref. . Copyright (2013) American Chemical Society.

FIGURE 3.

Applications of in-cell NMR to study protein function. a, in E. coli, the interaction between ¹⁵N-labeled Pup and unlabeled proteasomal ATPase (Mpa) causes the resonances from some residues of Pup to broaden (left, black spectrum). The broadened residues mapped on a structural model (right, in red) show the mode of interaction. Reprinted from Ref. under CC BY license. b, time-resolved in-cell NMR of CK2-dependent phosphorylation of a ¹⁵N-labeled SV40 large T antigen peptide injected in X. laevis oocytes shows that Ser-111 and Ser-112 are sequentially phosphorylated with a two-step mechanism. pSer111, phospho-Ser-111; pSer112, phospho-Ser0112. Reprinted by permission from Macmillan Publishers Ltd., Nat. Struct. Mol. Biol. (49). Copyright (2008). c, the chaperone-dependent formation of an intramolecular disulfide bond (S–S) is observed by in-cell NMR on [¹⁵N]cysteine-labeled SOD1 expressed in human cells. In the absence of supplemented copper, partial disulfide formation occurs (left); when copper is supplemented, Cu-CCS-dependent disulfide formation is fully observed. Reprinted by permission from Macmillan Publishers Ltd., Nat. Chem. Biol. (26). Copyright (2013).

FIGURE 4.

Disease-related mutations cause maturation defects in SOD1. fALS-linked mutations in the SOD1 gene impair zinc binding of the apo protein and cause the intracellular accumulation of an unstructured species detected by NMR in human cells (left), which is potentially a precursor of the cytotoxic aggregated SOD1 species that are a hallmark of the disease. Co-expression of CCS in the presence of excess copper rescues the mutant protein, preventing misfolding and allowing it to reach the mature, folded form (right). Reprinted by permission from Macmillan Publishers Ltd.,: Nat. Commun. (52). Copyright (2014).