First crystal structure of a non-canonical amino acid linked to a paramagnetic lanthanide tag facilitates protein structure determination using NMR-derived restraints

Abstract

SummarySite-directed spin labeling of proteins via non-canonical amino acids (ncAAs) is a non-traditional method for the measurement of pseudocontact shifts (PCSs) by nuclear magnetic resonance (NMR) spectroscopy. PCSs provide long-range distance and orientational information between a paramagnetic center and protein nuclei that can be used as restraints for computational structural modeling techniques. Here, we present the first experimental structure of an ncAA chemically linked to a lanthanide tag conjugated to the protein, T4-Lysozyme (T4L). T4L was crystallized with a cyclen-based C3 tag coordinated to the paramagnetic ion terbium (Tb3+). The paramagnetic C3-lanthanide tag generated PCSs measured at four different ncAA sites. We show that the addition of these restraints improves structure prediction protocols for T4L using the RosettaNMR framework. Generated models provide insight into T4L conformational flexibility sampled in solution. This integrative modeling protocol is readily transferable to larger proteins. Methods to predict protein structures are advancing into an exciting arena such that reliable experimental data will play important roles for evaluating the biophysical relevance of predicted structural models. Our contribution here caters to the growing interest in using ncAAs for a range of biophysical studies, and these methods can be readily transferred to larger protein systems of interest.

Pseudocontact shift NMR data obtained from a non-canonical amino acid-linked lanthanide tag improves integral membrane protein structure prediction

“…Expression and purification of 15 N-labeled DsbB-pAzF mutants. DsbB was expressed with a C-terminal His6tag and an amber stop codon to replace residues Phe32, Val72, and Tyr97 with pAzF as described 34,36 . Protein genes containing the amber stop codon (UAG) were cloned in a pet22b vector to produce site-specific pAzF-DsbB mutants.…”

“…An orthogonal strategy to circumvent the need to remove all native cysteines is the use of genetically encoded noncanonical amino acids (ncAAs) that either have a paramagnetic center or a reactive group for the attachment of a spin-label. The site-specific incorporation of the ncAA !-azido-ʟ-phenylalanine (pAzF) and ligation with alkyne-bearing lanthanide tags (C3 and C4) via a copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction was first reported by Loh et al for several soluble proteins including ubiquitin, sortase A, and T4-lysozyme [34][35][36] . The synthesis of the 1,4,7,10-tetraazacyclododecane (cyclen)-based C3 and C4 lanthanide tags are based on the cysteine-compatible C1 and C2 tags 37,38 .…”

“…The site-specific incorporation of the ncAA p -azido-ʟ-phenylalanine (pAzF) and ligation with alkyne-bearing lanthanide tags (C3 and C4) via a copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction was first reported by Loh et al . for several soluble proteins including ubiquitin, sortase A, and T4-lysozyme 34–36 . The synthesis of the 1,4,7,10-tetraazacyclododecane (cyclen)-based C3 and C4 lanthanide tags are based on the cysteine-compatible C1 and C2 tags 37,38 .…”