From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins.
ABSTRACT: Nanodiscs and isotropic bicelles are promising membrane mimetics in the field of solution nuclear magnetic resonance (NMR) spectroscopy of integral membrane proteins (IMPs). Despite varied challenges to solution NMR studies of IMPs, we attribute the paucity of solution NMR structures in these environments to the inability of diverse IMPs to withstand detergent treatment during standard nanodisc and bicelle preparations. Here, we present a strategy that creates small isotropic bicelles from IMPs co-translationally embedded in large nanodiscs using cell-free expression. Our results demonstrate appreciable gains in NMR spectral quality while preserving lipid-IMP contacts. We validate the approach on the detergent-sensitive LspA, which finally allowed us to perform high-quality triple-resonance NMR experiments for structural studies. Our strategy of producing bicelles from nanodiscs comprehensively avoids detergent during expression and preparation and is suitable for solution NMR spectroscopy of lipid-IMP complexes.
Project description:Solution NMR spectroscopy has become a robust method to determine structures and explore the dynamics of integral membrane proteins. The vast majority of previous studies on membrane proteins by solution NMR have been conducted in lipid micelles. Contrary to the lipids that form a lipid bilayer in biological membranes, micellar lipids typically contain only a single hydrocarbon chain or two chains that are too short to form a bilayer. Therefore, there is a need to explore alternative more bilayer-like media to mimic the natural environment of membrane proteins. Lipid bicelles and lipid nanodiscs have emerged as two alternative membrane mimetics that are compatible with solution NMR spectroscopy. Here, we have conducted a comprehensive comparison of the physical and spectroscopic behavior of two outer membrane proteins from Pseudomonas aeruginosa, OprG and OprH, in lipid micelles, bicelles, and nanodiscs of five different sizes. Bicelles stabilized with a fraction of negatively charged lipids yielded spectra of almost comparable quality as in the best micellar solutions and the secondary structures were found to be almost indistinguishable in the two environments. Of the five nanodiscs tested, nanodiscs assembled from MSP1D1?H5 performed the best with both proteins in terms of sample stability and spectral resolution. Even in these optimal nanodiscs some broad signals from the membrane embedded barrel were severely overlapped with sharp signals from the flexible loops making their assignments difficult. A mutant OprH that had two of the flexible loops truncated yielded very promising spectra for further structural and dynamical analysis in MSP1D1?H5 nanodiscs.
Project description:Bicelles are model membranes generally made of long-chain dimyristoylphosphatidylcholine (DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensively used in the study of membrane interactions and structure determination of membrane-associated peptides, since their composition and morphology mimic the widespread PC-rich natural eukaryotic membranes. At low DMPC/DHPC (q) molar ratios, fast-tumbling bicelles are formed in which the DMPC bilayer is stabilized by DHPC molecules in the high-curvature rim region. Experimental constraints imposed by techniques such as circular dichroism, dynamic light scattering, or microscopy may require the use of bicelles at high dilutions. Studies have shown that such conditions induce the formation of small aggregates and alter the lipid-to-detergent ratio of the bicelle assemblies. The objectives of this work were to determine the exact composition of those DMPC/DHPC isotropic bicelles and study the lipid miscibility. This was done using (31)P nuclear magnetic resonance (NMR) and exploring a wide range of lipid concentrations (2-400 mM) and q ratios (0.15-2). Our data demonstrate how dilution modifies the actual DMPC/DHPC molar ratio in the bicelles. Care must be taken for samples with a total lipid concentration ?250 mM and especially at q ? 1.5-2, since moderate dilutions could lead to the formation of large and slow-tumbling lipid structures that could hinder the use of solution NMR methods, circular dichroism or dynamic light scattering studies. Our results, supported by infrared spectroscopy and molecular dynamics simulations, also show that phospholipids in bicelles are largely segregated only when q > 1. Boundaries are presented within which control of the bicelles' q ratio is possible. This work, thus, intends to guide the choice of q ratio and total phospholipid concentration when using isotropic bicelles.
Project description:Bicelles are used in many membrane protein studies because they are thought to be more bilayer-like than micelles. We investigated the properties of "isotropic" bicelles by small-angle neutron scattering, small-angle X-ray scattering, fluorescence anisotropy, and molecular dynamics. All data suggest that bicelles with a q value below 1 deviate from the classic bicelle that contains lipids in the core and detergent in the rim. Thus not all isotropic bicelles are bilayer-like.
Project description:It is challenging to find membrane mimics that stabilize the native structures, dynamics, and functions of membrane proteins. In a recent advance, nanodiscs have been shown to provide a bilayer environment compatible with solution NMR. We show that increasing the lipid to "belt" peptide ratio expands their diameter, slows their reorientation rate, and allows the protein-containing discs to be aligned in a magnetic field for oriented sample solid-state NMR. The spectroscopic properties of membrane proteins with one to seven transmembrane helices in q = 0.1 isotropic bicelles, ~10 nm diameter isotropic nanodiscs, ~30 nm diameter magnetically aligned macrodiscs, and q = 5 magnetically aligned bicelles are compared.
Project description:Bicelles are a major medium form to produce weak alignment of soluble proteins for residual dipolar coupling (RDC) measurements. The obstacle to using the same type of bicelles for transmembrane proteins with solution-state NMR spectroscopy is the loss of signals due to the adhesion or penetration of the proteins into large bicelles, resulting in slow protein tumbling. In this study, weak alignment of the second and third transmembrane domains (TM23) of the human glycine receptor (GlyR) was achieved in low-q bicelles (q = DMPC/DHPC). Although protein-free bicelles with such low q would likely show isotropic properties, the insertion of TM23 induced weakly preferred orientations so that the RDC of the embedded protein can be measured. The extent of the alignment increased but the TM23 signal intensity decreased when q was varied from 0.19 to 0.60. A q of 0.50 was found to be an optimal compromise between alignment and the signal-to-noise ratio. In each pair of NMR experiments for RDC measurements, the same sample and pulse sequence were used, with one being performed at high-resolution magic-angle spinning to obtain pure J-couplings without RDC. A meaningful structure refinement in bicelles was possible by iteratively fitting the experimental RDCs to the back-calculated RDCs using the high-resolution NMR structure of GlyR TM23 in trifluoroethanol as the starting template. Combination of this method with the conventional high-resolution NMR in membrane mimicking mixtures of water and organic solvents offers an attractive way to derive structural information for membrane proteins in their native environment.
Project description:In spite of recent technological advances in NMR spectroscopy, its low sensitivity continues to be a major limitation particularly for the structural studies of membrane proteins. The need for a large quantity of a membrane protein and acquisition of NMR data for a long duration are not desirable. Therefore, there is considerable interest in the development of methods to speed up the NMR data acquisition from model membrane samples. In this study, we demonstrate the feasibility of acquiring two-dimensional spectra of an antimicrobial peptide (MSI-78; also known as pexiganan) embedded in isotropic bicelles using natural-abundance (15)N nuclei. A copper-chelated lipid embedded in bicelles is used to speed-up the spin-lattice relaxation of protons without affecting the spectral resolution and thus enabling fast data acquisition. Our results suggest that even a 2D SOFAST-HMQC spectrum can be obtained four times faster using a very small amount (?3 mM) of a copper-chelated lipid. These results demonstrate that this approach will be useful in the structural studies of membrane-associated peptides and proteins without the need for isotopic enrichment for solution NMR studies.
Project description:Structural characterization of transmembrane proteins in isotropic bicelles has become an increasingly popular application of solution NMR spectroscopy, as the fast-tumbling bicelles are membrane-like, yet can often yield spectral quality comparable to those of detergent micelles. While larger bicelles are closer to the true lipid bilayer, it remains unclear how large the bicelles need to be to allow accurate assessment of the protein transmembrane partition in the lipid bilayer. Here, we address the above question from the perspective of the protein residing in the bicelles, through systematic measurement of the protein chemical shift and transmembrane partition at different lipid/detergent ratios (q), ranging from 0.3 to 0.7, using the transmembrane domain of the human Fas receptor as model system. We found that the lipid environment of the bicelles, as reflected by the protein chemical shift, begins to be perturbed when q is reduced to below 0.6. We also implemented a solvent paramagnetic relaxation enhancement (PRE) approach for bicelles to show that the protein transmembrane partition in bicelles with q=0.5 and 0.7 are very similar, but at q=0.3 the solvent PRE profile is significantly different. Our data indicate that q values between 0.5 and 0.6 are a good compromise between high resolution NMR and closeness to the membrane environment, and allow accurate characterization of the protein position in the lipid bilayer.
Project description:We designed ?-strand peptides that stabilize integral membrane proteins (IMPs). ?-strand peptides self-assemble in solution as filaments and become restructured upon association with IMPs; resulting IMP-?-strand peptide complexes resisted aggregation when diluted in detergent-free buffer and were visible as stable, single particles with low detergent background in electron micrographs. ?-strand peptides enabled clear visualization of flexible conformations in the highly dynamic ATP-binding cassette (ABC) transporter MsbA.
Project description:Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per-residue resolution that while OmpX forms a stable β-barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico- to nanosecond and micro- to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β-strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane-mimetic-dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility.
Project description:Integral membrane proteins (IMPs) play a central role in cell communication with the environment. Their structures are essential for our understanding of the molecular mechanisms of signaling and for drug design, yet they remain badly underrepresented in the protein structure databank. Solution NMR is, aside from X-ray crystallography, the major tool in structural biology. Here we review recently reported solution NMR structures of polytopic IMPs and discuss the new approaches, which were developed in the course of these studies to overcome barriers in the field. Advances in cell-free protein expression, combinatorial isotope labeling, resonance assignment, and collection of structural data greatly accelerated IMP structure determination by solution NMR. In addition, novel membrane-mimicking media made possible determination of solution NMR structures of IMPs in a native-like lipid environment.