Manuel Etzkorn

Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins.

Structural studies of membrane proteins are still hampered by difficulties of finding appropriate membrane-mimicking media that maintain protein structure and function. Phospholipid nanodiscs seem promising to overcome the intrinsic problems of detergent-containing environments. While nanodiscs can offer a near-native environment, the large particle size complicates their routine use in the structural analysis of membrane proteins by solution NMR. Here, we introduce nanodiscs assembled from shorter ApoA-I protein variants that are of markedly smaller diameter and show that the resulting discs provide critical improvements for the structure determination of membrane proteins by NMR. Using the bacterial outer-membrane protein OmpX as an example, we demonstrate that the combination of small nanodisc size, high deuteration levels of protein and lipids, and the use of advanced non-uniform NMR sampling methods enable the NMR resonance assignment as well as the high-resolution structure determination of polytopic membrane proteins in a detergent-free, near-native lipid bilayer setting. By applying this method to bacteriorhodopsin, we show that our smaller nanodiscs can also be beneficial for the structural characterization of the important class of seven-transmembrane helical proteins. Our set of engineered nanodiscs of subsequently smaller diameters can be used to screen for optimal NMR spectral quality for small to medium-sized membrane proteins while still providing a functional environment. In addition to their key improvements for de novo structure determination, due to their smaller size these nanodiscs enable the investigation of interactions between membrane proteins and their (soluble) partner proteins, unbiased by the presence of detergents that might disrupt biologically relevant interactions.

Nonmicellar systems for solution NMR spectroscopy of membrane proteins.

Integral membrane proteins play essential roles in many biological processes, such as energy transduction, transport of molecules, and signaling. The correct function of membrane proteins is likely to depend strongly on the chemical and physical properties of the membrane. However, membrane proteins are not accessible to many biophysical methods in their native cellular membrane. A major limitation for their functional and structural characterization is thus the requirement for an artificial environment that mimics the native membrane to preserve the integrity and stability of the membrane protein. Most commonly employed are detergent micelles, which can however be detrimental to membrane protein activity and stability. Here, we review recent developments for alternative, nonmicellar solubilization techniques, with a particular focus on their application to solution NMR studies. We discuss the use of amphipols and lipid bilayer systems, such as bicelles and nanolipoprotein particles (NLPs). The latter show great promise for structural studies in near native membranes.

Cell-Free Expressed Bacteriorhodopsin in Different Soluble Membrane Mimetics: Biophysical Properties and NMR Accessibility

Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Nonconventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here, we present a comparative study of the heptahelical membrane protein bacteriorhodopsin in detergent micelles, amphipols, and nanodiscs. Our results confirm that nonconventional environments can increase stability of functional bacteriorhodopsin, and demonstrate that well-folded heptahelical membrane proteins are, in principle, accessible by solution-NMR methods in amphipols and phospholipid nanodiscs. Our data distinguish regions of bacteriorhodopsin that mediate membrane/solvent contacts in the tested environments, whereas the proteins functional inner core remains almost unperturbed. The presented data allow comparing the investigated membrane mimetics in terms of NMR spectral quality and thermal stability required for structural studies.

Structural rearrangements of membrane proteins probed by water-edited solid-state NMR spectroscopy

We show that water-edited solid-state NMR spectroscopy allows for probing global protein conformation and residue-specific solvent accessibility in a lipid bilayer environment. The transfer dynamics can be well described by a general time constant, irrespective of protein topology and lipid environment. This approach was used to follow structural changes in response to protein function in the chimeric potassium channel KcsA-Kv1.3. Data obtained as a function of pH link earlier biochemical data to changes in protein structure in a functional bilayer setting.

Plasticity of the PAS domain and a potential role for signal transduction in the histidine kinase DcuS.

The mechanistic understanding of how membrane-embedded sensor kinases recognize signals and regulate kinase activity is currently limited. Here we report structure-function relationships of the multidomain membrane sensor kinase DcuS using solid-state NMR, structural modeling and mutagenesis. Experimental data of an individual cytoplasmic Per-Arnt-Sim (PAS) domain were compared to structural models generated in silico. These studies, together with previous NMR work on the periplasmic PAS domain, enabled structural investigations of a membrane-embedded 40-kDa construct by solid-state NMR, comprising both PAS segments and the membrane domain. Structural alterations are largely limited to protein regions close to the transmembrane segment. Data from isolated and multidomain constructs favor a disordered N-terminal helix in the cytoplasmic domain. Mutations of residues in this region strongly influence function, suggesting that protein flexibility is related to signal transduction toward the kinase domain and regulation of kinase activity.

High‐Resolution Solid‐State NMR Studies on Uniformly [13C,15N]‐Labeled Ubiquitin

Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high‐resolution magic‐angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]‐labeled sample of ubiquitin, we discuss solid‐state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid‐phase protein preparation on solid‐state NMR spectra. A comparative analysis of 13C′, 13Cα, and 13Cβ resonance frequencies suggests that 13C chemical‐shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid‐state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high‐resolution solid‐state NMR.

Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells

Significance Tumor metastasis is the major cause of cancer lethality, whereas the underlying mechanisms are obscure. Breast cancer stem cells (CSCs) are essential for breast cancer relapse and metastasis and stromal cell-derived factor 1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) is a key regulator of tumor dissemination. We report a large-scale quantification of SDF-1/CXCR4–induced phosphoproteome events and identify several previously unidentified phosphoproteins and signaling pathways in breast CSCs. This study provides insights into the understanding of the mechanisms of breast cancer metastasis. Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4–mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4–mediated phosphoproteome, including construction of kinase–substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, δ-catenin, and PPP1Cα in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.

The native conformation of the human VDAC1 N terminus.

The voltage-dependent anion channel (VDAC) is located in the mitochondrial outer membrane and constitutes the major pathway for the transport of ADP, ATP, and other metabolites. It is also considered a key player in mitochondrial apoptosis. 2] Recently, the three-dimensional structure of the VDAC1 isoform was elucidated independently by three different experimental approaches. All structures reveal a novel 19-stranded b-barrel architecture with an N-terminal a helix positioned horizontally inside the pore. Although all three structures are highly similar in terms of the b barrel, they exhibit clear differences in the functionally important Nterminal region (Figure 1a). There is a general consensus that the N terminus is involved in the voltage-dependent gating process of the channel and that it may adopt different conformations depending on external factors. Based on these structural data, different models for voltage gating have been proposed. Ujwal et al. suggested that the whole helix may move towards the middle of the pore and thereby close the channel. On the other hand, as residues 11–20 are difficult to observe in solution-state NMR, Hiller and Wagner argued that they may be subject to conformational exchange and that movements in this part of the N terminus alone may explain the gating behavior. Alternatively, larger conformational rearrangements upon voltage gating that also involve the b barrel have been suggested based on electron microscopy and electrophysiological data. Additionally, some previous biophysical findings are at odds with all three published structures, and the question has been raised as to whether they really represent the native conformation in a natural membrane environment. In particular, N-terminal truncation mutants of VDAC1 were found to exhibit lower conductance than the full-length channel, which has been taken as an indication that the N-terminal helix may not lie inside the pore, but form part of the barrel wall. Solid-state NMR spectroscopy has proven to be a very useful method for structural investigations of membrane proteins in a natural lipid environment (see, for example, reference [13] for a recent review). We therefore investigated functional human VDAC1 in lipid bilayers using solid-state NMR spectroscopy, with a focus on the conformation of its N terminus. Functionality of our hVDAC1 preparation was confirmed by electrophysiological measurements in lipid bilayers (see the Supporting Information, Figure S1). Figure 1b shows a C–C proton-driven spin diffusion (PDSD) correlation spectrum of uniformly [C, N] isotope-labeled hVDAC1 reconstituted into dimyristoylphosphocholine (DMPC) liposomes. The spectra exhibit excellent sensitivity and resolution. A prediction of the Ca–Cb region of the spectrum based on the crystal structure of mouse VDAC1 (Supporting Information, Figure S2) agrees very well with the spectrum and indicates that the overall 19-stranded b-barrel fold is conserved in liposomes. Comprising 283 amino acids, VDAC1 is a challenging protein for solid-state NMR spectroscopy. To identify the N-terminal residues, we made use of different isotope labeling schemes, involving a (Lys, Trp, Tyr, Val) reverse-labeled and an (Ala, Asp, Leu, Val) forward-labeled protein variant along with the uniformly isotope-labeled sample. Furthermore, we studied an N-terminally truncated hVDAC1 variant (D(1-20)-hVDAC1). Figure 1c illustrates how a combination of results from the different samples was used in the assignment process. With a large set of homoand heteronuclear correlation spectra of the different protein variants (see, for example, the Supporting Information, Figure S3) we were able to obtain unambiguous de novo sequential resonance assignments for residues Ala2–Val17 (Supporting Information, Figure S4 and Table S1). All of these residues give rise to distinct narrow cross-peaks in experiments based on dipolar transfer schemes at sample temperatures between + 5 and + 25 8C (Supporting Information, Figure S5 and S6). This result demonstrates that the hVDAC1 N terminus assumes a well-defined conformation in liposomes and does not exhibit sizable dynamics on the sub-millisecond timescale. No peak broadening or doubling owing to chemical exchange on slower timescales was [*] Dr. R. Schneider, K. Giller, V. Daebel, Prof. Dr. M. Zweckstetter, Prof. Dr. C. Griesinger, Dr. S. Becker, Dr. A. Lange Department for NMR-based Structural Biology Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 G ttingen (Germany) Fax: (+ 49)551-201-2202 E-mail: adla@nmr.mpibpc.mpg.de Homepage: http://www.mpibpc.mpg.de/english/research/ags/ lange/

Complex Formation and Light Activation in Membrane-Embedded Sensory Rhodopsin II as Seen by Solid-State NMR Spectroscopy

Microbial rhodopsins execute diverse biological functions in the cellular membrane. A mechanistic understanding of their functional profile is, however, still limited. We used solid-state NMR (ssNMR) spectroscopy to study structure and dynamics of a 2 x 400 amino acid sensory rhodopsin/transducer (SRII/HtrII) complex from Natronomonas pharaonis in a natural membrane environment. We found a receptor-transducer binding interface in the ground state that significantly extends beyond the available X-ray structure. This binding domain involves the EF loop of the receptor and stabilizes the functionally relevant, directly adjacent HAMP domain of the transducer. Using 2D ssNMR difference spectroscopy, we identified protein residues that may act as a functional module around the retinal binding site during the early events of protein activation. These latter protein segments, the inherent plasticity of the HAMP domain, and the observation of an extended SRII/HtrII membrane-embedded interface may be crucial components for optimal signal relay efficiency across the cell membrane.

How Amphipols Embed Membrane Proteins: Global Solvent Accessibility and Interaction with a Flexible Protein Terminus

AbstractAmphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.