Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

Abstract

13C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

“…Lastly, we formed and isolated a high-affinity complex between purified NECA-bound A 2A R and purified G s heterotrimer in the absence of GDP or GTP (Figure 2-figure supplement 1, Materials and methods). Like the apo spectrum, the G protein complex spectrum had fewer well-dispersed Ile peaks in a deuterated background as previously described (Clark et al, 2015). Crystal structures of A 2A R are shown complexed with agonist NECA (red, 2YDV (Lebon et al, 2011)) and inverse agonist ZM241385 (gray, 4EIY (Liu et al, 2012b), with isoleucine residues displayed as spheres.…”

“…was 0.42 nM in NaCl-containing buffer, while the K i for NECA was 210 nM in low ionic strength and 420 nM in KCl-containing buffer, in agreement with previous binding studies (Xu et al, 2011;Carpenter et al, 2016;Bertheleme et al, 2013) We developed a purification protocol for A 2A R from Pichia-derived membranes, in which we can solubilize and purify~0.5 mg of monodisperse biochemically pure wild-type A 2A R in dodecylmaltoside (DDM) detergent from 1 L of culture (Figure 1-figure supplement 1). Coupling this protocol with our previously-described labeling method (Clark et al, 2015) Figure 1B). These data show that the perdeuterated A 2A R is capable of activating G s at wild-type levels in vitro, and can serve as a valid model for GPCR dynamics.…”

“…We have addressed many of these practical issues with our generation (Clark et al, 2015) of highly-deuterated 1 H/ 13 C Ile d1-methyl labeled proteins in Pichia pastoris, a methylotrophic yeast (Cereghino and Cregg, 2000) that is adaptable to D 2 O-based minimal media, will utilize the Ile precursor a-ketobutyrate, and enables expression of GPCRs at levels sufficient for crystallography (Shimamura et al, 2011;Hino et al, 2012;Yurugi-Kobayashi et al, 2009). Here we apply this labeling method to the wild-type human A 2A R, an important regulator of vascular function with a well-developed pharmacology including the natural hormone adenosine and high-affinity synthetic agonists and antagonists (Preti et al, 2015;de Lera Ruiz et al, 2014).…”

Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization

“…This host has also been used to obtain isotopically labeled protein for NMR studies [27][28][29]. In this case, the protein should be enriched in active nuclei, for example 15 N and/or 13 C. Although E. coli is usually employed to express heavy isotope-labeled recombinant protein, the product of interest is often expressed as inclusion bodies and has to be refolded.…”

Isotope-labeling strategies for solution NMR studies of macromolecular assemblies

“…Recently, selective [ 1 H, 13 C]-labeling of Ile-d1 methyl groups in perdeuterated proteins has been described in glucose-controlled Kluyveromyces lactis [68] and methanol-controlled Pichia pastoris [68,69]. The 42 kDa maltose-binding protein was perdeuterated to high levels (90%) with Ile-d1 labeling efficiency of 45% and 67% for P. pastoris and K. lactis, respectively.…”