Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments

摘要

class="head no_bottom_margin" id="sec1title">IntroductionCellular organization relies on compartmentalization by lipid membranes, around which cells install protein networks that establish further function. These membrane proteins are challenging to characterize, as their native environment is complex and heterogeneous. Despite significant work with membrane proteins, the rate at which new structural information is being produced has decreased since 2005 (). Most structural techniques rely on purification of membrane proteins using detergents, followed in some cases by reconstitution into synthetic lipid bilayers. Neither system can fully mimic the complex nature of these proteins' natural environment, and the choice of mimetic can have significant impact on both structure and function (). Purification can also disrupt higher-order structure, including oligomerization (), complex formation (), or metabolic organization (). Therefore, it is desirable to be able to study membrane proteins and their structure in their native membranes. Two methods that allow for structural investigations in native membranes are solid-state nuclear magnetic resonance spectroscopy (ssNMR) and electron cryotomography (cryoET).CryoET and ssNMR provide highly complementary information. CryoET involves imaging individual events, with each molecule potentially in a different state, while ssNMR uses bulk measurements. Motion and dynamics are implicitly recorded in ssNMR experiments from nanosecond to millisecond time scales, while cryoET is limited by the speed at which samples can be vitrified, and is therefore useful for snapshots of biological processes on the seconds-to-hours timescale. Nanometer-scale spatial information is intrinsic to cryoET, while ssNMR is much more sensitive to chemical information on the Ångstrom scale. Furthermore, ssNMR exploits isotope labeling to exclude background signals, whereas cryoET records the full environment, providing macromolecular context for measurements. CryoET and ssNMR have both been used successfully to study fibrillar structures (for a review, see and a more recent study []), secretion systems accessible to in vitro assembly (e.g., , , ) and, most recently, microtubule -protein complexes (). Restraints from ssNMR experiments also recently have been proposed to aid in model refinement from electron cryomicroscopy (cryoEM) ().Magic angle spinning (MAS) ssNMR () is well suited to the analysis of large assemblies such as cell membranes, as it uses spinning to minimize anisotropic interactions. Conventionally, MAS with speeds of <20 kHz, in combination with ¹³C detection, have been used to study local and overall protein structure and dynamics at atomic resolution in bilayers formed by native bacterial membranes (see, e.g., , , href="#bib27" rid="bib27" class=" bibr popnode">Herzfeld and Lansing, 2002, href="#bib29" rid="bib29" class=" bibr popnode">Hong et al., 2012, href="#bib31" rid="bib31" class=" bibr popnode">Jacso et al., 2012, href="#bib48" rid="bib48" class=" bibr popnode">Miao et al., 2012, href="#bib54" rid="bib54" class=" bibr popnode">Renault et al., 2010, href="#bib75" rid="bib75" class=" bibr popnode">Ward et al., 2015a, href="#bib82" rid="bib82" class=" bibr popnode">Yamamoto et al., 2015). These approaches have been extended to study entire bacterial cell envelopes (href="#bib34" rid="bib34" class=" bibr popnode">Kaplan et al., 2015, href="#bib55" rid="bib55" class=" bibr popnode">Renault et al., 2012a) or mammalian membrane proteins embedded in their natural plasma membrane (href="#bib35" rid="bib35" class=" bibr popnode">Kaplan et al., 2016a, href="#bib36" rid="bib36" class=" bibr popnode">Kaplan et al., 2016b). Recent methodological advancements in Dynamic Nuclear Polarization have improved spectral sensitivity for such samples (href="#bib31" rid="bib31" class=" bibr popnode">Jacso et al., 2012, href="#bib34" rid="bib34" class=" bibr popnode">Kaplan et al., 2015, href="#bib35" rid="bib35" class=" bibr popnode">Kaplan et al., 2016a, href="#bib36" rid="bib36" class=" bibr popnode">Kaplan et al., 2016b, href="#bib56" rid="bib56" class=" bibr popnode">Renault et al., 2012b, href="#bib82" rid="bib82" class=" bibr popnode">Yamamoto et al., 2015). Another area of development is in ¹H-detected MAS ssNMR experiments, where the higher gyromagnetic ratio of protons can enhance overall spectroscopic sensitivity provided that MAS spinning rates >40 kHz are used (href="#bib1" rid="bib1" class=" bibr popnode">Andreas et al., 2010, href="#bib3" rid="bib3" class=" bibr popnode">Asami and Reif, 2013, href="#bib46" rid="bib46" class=" bibr popnode">Medeiros-Silva et al., 2016, href="#bib66" rid="bib66" class=" bibr popnode">Sinnige et al., 2014, href="#bib74" rid="bib74" class=" bibr popnode">Ward et al., 2011). With faster spinning, line widths are generally narrower; sample preparation and choice of labels can improve spectral resolution (href="#bib1" rid="bib1" class=" bibr popnode">Andreas et al., 2010, href="#bib3" rid="bib3" class=" bibr popnode">Asami and Reif, 2013, href="#bib18" rid="bib18" class=" bibr popnode">Fricke et al., 2017, href="#bib46" rid="bib46" class=" bibr popnode">Medeiros-Silva et al., 2016, href="#bib66" rid="bib66" class=" bibr popnode">Sinnige et al., 2014, href="#bib74" rid="bib74" class=" bibr popnode">Ward et al., 2011).CryoET has been used to study a wide range of samples, from purified protein complexes to intact viruses, bacteria, and eukaryotic cells, preserved in a frozen, hydrated state that mimics physiological conditions. Briefly, a series of projection images of the same specimen is collected with different orientations relative to the electron beam, followed by computational processing to recover three-dimensional structural information without averaging (for a recent review, see href="#bib8" rid="bib8" class=" bibr popnode">Beck and Baumeister, 2016). As the sample and stage thickness prevent tilting to 90°, there is a “missing wedge” of information in Fourier space. This missing information can be compensated for by averaging together three-dimensional subvolumes extracted from tomograms, which are differentially oriented relative to the missing wedge. CryoET (and other forms of cryoEM) also recently benefited from technological advancements. In particular, direct electron detectors have significantly increased the signal in images (href="#bib45" rid="bib45" class=" bibr popnode">McMullan et al., 2014). Some recent examples of bacterial systems studied by cryoET include work investigating the organization of the pilus in Myxococcus xanthus (href="#bib11" rid="bib11" class=" bibr popnode">Chang et al., 2016), the injection of pathogenic factors into host cells by Chlamydia trachomatis (href="#bib49" rid="bib49" class=" bibr popnode">Nans et al., 2015), and the formation of cellular structures organizing DNA replication during phage infection (href="#bib10" rid="bib10" class=" bibr popnode">Chaikeeratisak et al., 2017).To take full advantage of the complementarity between ssNMR and cryoET, and recent technological improvements in ¹H detection and direct detectors, respectively, we set out to create a sample preparation method for the structural and functional study of membrane proteins in their native environment, where the same specimens could be used for both techniques. To maintain the native membrane environment, we avoided altogether the use of detergents or other extraction strategies. These samples also needed to balance the sensitivity of ¹H-detected ssNMR experiments with reasonable protein expression levels to avoid excess disruption to the membrane environment. As structure is tightly linked to function, accessibility to the membrane surfaces for functional or binding assays was also an important consideration. Similarly, membrane morphologies needed to be reflective of, e.g., native cell envelope ultrastructure. Furthermore, a range of orientations is desirable to compensate for the missing wedge in cryoET.Here, we present a combined ¹H-detected ssNMR and cryoET investigation of the structure, function, and native environment of YidC in Escherichia coli. YidC is an inner membrane protein that helps fold and insert other inner membrane proteins (href="#bib64" rid="bib64" class=" bibr popnode">Scotti et al., 2000). YidC has also been shown to insert some substrates, such as subunit c of ATP synthase, independently of the Sec translocon system (href="#bib42" rid="bib42" class=" bibr popnode">van der Laan et al., 2004). E. coli ribosomes with substrate membrane protein nascent chains (RNCs) bind and insert substrate via purified and reconstituted YidC (href="#bib38" rid="bib38" class=" bibr popnode">Kedrov et al., 2013). The structure of purified YidC was determined in the lipidic cubic phase by X-ray crystallography (href="#bib41" rid="bib41" class=" bibr popnode">Kumazaki et al., 2014) and also, at lower resolution, in nanodiscs bound to RNCs by single-particle cryoEM (href="#bib39" rid="bib39" class=" bibr popnode">Kedrov et al., 2016). YidC can be produced with high yield after overexpression in E. coli, allowing for purified and reconstituted control samples (href="#bib7" rid="bib7" class=" bibr popnode">Baker et al., 2015), and binding of ribosomes to YidC has been shown to differ depending on the membrane mimetic used (href="#bib38" rid="bib38" class=" bibr popnode">Kedrov et al., 2013). We use our hybrid method to show that the conformation and likely dynamics of YidC in native membranes differs from purified and reconstituted YidC, and observe corresponding RNC binding differences.