A Chemical Biology Primer for NMR Spectroscopists

Abstract

Among structural biology techniques, NMR spectroscopy offers unique capabilities that enable the atomic resolution studies of dynamic and heterogeneous biological systems under physiological and native conditions. Complex biological systems, however, often challenge NMR spectroscopists with their low sensitivity, crowded spectra or large linewidths that reflect their intricate interaction patterns and dynamics. While some of these challenges can be overcome with the development of new spectroscopic approaches, chemical biology can also offer elegant and efficient solutions at the sample preparation stage. In this tutorial, we aim to present several chemical biology tools that enable the preparation of selectively and segmentally labeled protein samples, as well as the introduction of site-specific spectroscopic probes and post-translational modifications. The four tools covered here, namely cysteine chemistry, inteins, native chemical ligation, and unnatural amino acid incorporation, have been developed and optimized in recent years to be more efficient and applicable to a wider range of proteins than ever before. We briefly introduce each tool, describe its advantages and disadvantages in the context of NMR experiments, and offer practical advice for sample preparation and analysis. We hope that this tutorial will introduce beginning researchers in the field to the possibilities chemical biology can offer to NMR spectroscopists, and that it will inspire new and exciting applications in the quest to understand protein function in health and disease.

Keywords: genetic code expansion; intein; post-translational modification; protein engineering; segmental labeling.

Figure 1.

Reactive versatility of cysteines. Oxidation reactions can be used to attach spectroscopic probes such as MTSL (A) or ubiquitin (B) to proteins [30, 53]. Maleimide based reactions can be used to target proteins with DNP polarization agents (C), while other alkylation reagents can convert cysteines to methyl-lysine analogs (D) [33, 38]. Desulfurization reactions can transform a cysteine residue into other amino acids such as alanine (E) or useful functional groups such as dehydrolanine (F) [45, 47].

Figure 2.

Strategy to produce segmentally labeled proteins for NMR applications.

Figure 3.

Intein splicing mechanism. (A) A contiguous intein-extein construct denoting the position of residues that are important in the splicing mechanism. C₁ is the first cysteine residue of the intein sequence, while N_n denotes the last asparagine amino acid. C₊₁ is the first cysteine residue of the N-extein. (B) Splicing mechanism for contiguous inteins. (C) Splicing mechanism for split inteins.

Figure 4.

Strategy for tagless protein purification based on contiguous inteins.

Figure 5.

Native chemical ligation (NCL) mechanism and applications. (A) NCL begins with a trans-thioseterificaiton step where the thiol group on the cysteine residue of peptide 2 attacks the C-terminal thioester on peptide 1. After trans-thioesterification, the peptide backbone rearranges to form a native peptide bond. The reactive cysteine residue can be subsequently converted to alanine through an optional desulfurization step. (B) Possible applications for NCL, including segmental labeling, residue specific labeling, and site-specific installation of post-translational modifications.

Figure 6.

Overview of unnatural amino acid incorporation by genetic means. (A) Unnatural amino acid (UAA) incorporation starts with an engineered aminoacyl-tRNA synthetase (aaRS) and tRNA pair. The aaRS recognizes the UAA and attaches it to the tRNA. At the same time, the amber codon UAG is incorporated at the desired position in the protein of interest. The ribosome uses the matching tRNA to install the UAA into the protein. (B) Incorporation of spectroscopic probes through bio-orthogonal norborne-tetrazine chemistry.