Sparse labeling of proteins: Structural characterization from long range constraints

Abstract

Structural characterization of biologically important proteins faces many challenges associated with degradation of resolution as molecular size increases and loss of resolution improving tools such as perdeuteration when non-bacterial hosts must be used for expression. In these cases, sparse isotopic labeling (single or small subsets of amino acids) combined with long range paramagnetic constraints and improved computational modeling offer an alternative. This perspective provides a brief overview of this approach and two discussions of potential applications; one involving a very large system (an Hsp90 homolog) in which perdeuteration is possible and methyl-TROSY sequences can potentially be used to improve resolution, and one involving ligand placement in a glycosylated protein where resolution is achieved by single amino acid labeling (the sialyltransferase, ST6Gal1). This is not intended as a comprehensive review, but as a discussion of future prospects that promise impact on important questions in the structural biology area.

Pseudocontact shifts in biomolecular NMR using paramagnetic metal tags

“…Alternatively, however, chemical exchange saturation transfer (CEST) experiments can be used to measure chemical shifts and PCSs of lowpopulated states. This has recently been demonstrated for the Abp1p SH3 domain tagged with 4MMPyMTA (33). In the presence of 3% Ark1p peptide, 15 N PCSs of five residues measured with Tb 3+ were shown to agree between the excited state of apo-Abp1p SH3 domain and its complex with peptide [175].…”

“…Comprehensive reviews on the physical background of paramagnetic effects in biomolecular NMR have been published by Bertini and co-workers [4,[15][16][17] and summarized in later reviews [18][19][20]. Reviews of applications in structural biology have been published with emphasis on metalloproteins [21][22][23][24][25], carbohydrates [26], solid state NMR [27][28][29], PREs for characterising transient, low-population states [30], protein-protein complexes [31], techniques for measuring PCSs [32], sparse protein labelling [33], paramagnetic tags [34][35][36][37] and paramagnetically induced residual dipolar couplings (RDC) and relaxation enhancements [18]. Additional reviews have focused on the use of PCSs and paramagnetically induced RDCs for structure analysis of flexible proteins [38] and comparisons of protein conformations in solution and in single crystals [39].…”

“…Recently, the 4MMPyMTA tag (33) has been reported, which reacts with a cysteine residue by formation of a disulfide linkage [175]. The resulting tether is the same as for the 4VPyMTA tag, except that a CH2 group is replaced by a sulfur atom.…”

The Structural Role of Antibody N-Glycosylation in Receptor Interactions

“…Using selectively 15 N-amino acid enriched HEK293F cell expression medium we recovered high yields (30–50 mg/L) of appropriately glycosylated and 15 N-labeled IgG1 Fc (see Fig S1). Sparse amino acid labeling provides peak separation and supports measurement of residual dipolar couplings (RDCs) to define the relative orientation of each observable N-H bond vector (Prestegard et al, 2014). If the structure of the individual Cγ2 and Cγ3 domains in solution mirrors the conformation of available models, RDCs can be interpreted to reveal the relative orientations of these two domains with high precision.…”

NMR assignments of sparsely labeled proteins using a genetic algorithm

“…However, sparse labeling with single or small subsets of isotopically labeled amino acids is still possible. Certain subsets of isotopically labeled amino acids are relatively inexpensive, making application to glycoproteins economical, and the reduction in numbers of resonances increases resolution for larger proteins (Goto and Kay 2000; Kainosho et al 2006; Prestegard et al 2014). Even with fewer labeled sites chemical shift perturbation can provide information on ligand binding or protein-protein association (Williamson 2014), and residual dipolar couplings (RDCs) can constrain relative orientation of structural units in multiple domain proteins or protein-protein complexes (Chen and Tjandra 2012; Lipsitz and Tjandra 2004).…”