Biophysical studies of membrane proteins are often impeded by the requirement for a membrane mimicking environment. Detergent micelles are the most common choice, but the denaturing properties make them unsatisfactory for studies of many membrane proteins and their interactions. In the present work, we explore phospholipid bilayer nanodiscs as membrane mimics and employ electron microscopy and solution NMR spectroscopy to characterize the structure and function of the human voltage dependent anion channel (VDAC-1) as an example of a polytopic integral membrane protein. Electron microscopy reveals the formation of VDAC-1 multimers, an observation that is consistent with results obtained in native mitochondrial outer membranes. High-resolution NMR spectroscopy demonstrates a well folded VDAC-1 protein and native NADH binding functionality. The observed chemical shift changes upon addition of the native ligand NADH to nanodisc-embedded VDAC-1 resemble those of micelle-embedded VDAC-1, indicating a similar structure and function in the two membrane-mimicking environments. Overall, the ability to study integral membrane proteins at atomic resolution with solution NMR in phospholipid bilayers, rather than in detergent micelles, offers exciting novel possibilities to approach the biophysical properties of membrane proteins under non-denaturing conditions, which makes this technology particular suitable for protein-protein interactions and other functional studies.Membrane proteins are responsible for a wide range of essential physiological processes and are involved in various diseases. Most biophysical methods for studying membrane proteins in vitro require a membrane mimicking environment to stabilize the protein. 1 The most commonly used membrane mimics are detergents, but since they often have deteriorating effects on the structure and activity of a protein, the search for a suitable detergent is an empirical and thus time-consuming process. A better mimic are phospholipid bilayers, since their biophysical properties resemble more closely those of the native membrane and they are thus more likely to maintain membrane proteins in a stable and active state. Phospholipid bilayers in the form of bicelles have been used to study membrane proteins by high resolution biophysical methods such as NMR spectroscopy 2 and x-ray crystallography. 3 However, the morphology of bicelles is complex, depending on various sample conditions, such as gerhard_wagner@hms.harvard.edu. Supporting Information Available: Experimental procedures and data on the purification, refolding and reconstitution of VDAC-1 into nanodiscs. This material is available free of charge via the Internet at http://pubs.acs.org. A promising new approach for studies of membrane proteins in a phospholipid bilayer uses high-density lipoprotein nanodiscs consisting of a central disk-shaped core of lipids, confined by two copies of an α-helical amphipathic protein. [5][6][7] These nanoscale lipid bilayers with reported diameters ranging from 9.5 to 12 nm prov...

Many key aspects of control of quantum systems involve manipulating a large quantum ensemble exhibiting variation in the value of parameters characterizing the system dynamics. Developing electromagnetic pulses to produce a desired evolution in the presence of such variation is a fundamental and challenging problem in this research area. We present such robust pulse designs as an optimal control problem of a continuum of bilinear systems with a common control function. We map this control problem of infinite dimension to a problem of polynomial approximation employing tools from geometric control theory. We then adopt this new notion and develop a unified computational method for optimal pulse design using ideas from pseudospectral approximations, by which a continuous-time optimal control problem of pulse design can be discretized to a constrained optimization problem with spectral accuracy. Furthermore, this is a highly flexible and efficient numerical method that requires low order of discretization and yields inherently smooth solutions. We demonstrate this method by designing effective broadband π∕2 and π pulses with reduced rf energy and pulse duration, which show significant sensitivity enhancement at the edge of the spectrum over conventional pulses in 1D and 2D NMR spectroscopy experiments.pseudospectral methods | ensemble control | Lie algebra | broadband excitation C ompelling applications for quantum control have received particular attention and have motivated seminal studies in wide-ranging areas from coherent spectroscopy and MRI to quantum optics. Designing and implementing time-varying excitations (rf pulses) to manipulate complex dynamics of a large quantum ensemble on the order of Avogadro's number is a long-standing problem and an indispensable step that enables every application of quantum control (1). For example, magnetic resonance applications often suffer from imperfections such as inhomogeneity in the static magnetic field (B 0 inhomogeneity) and in the applied rf field (rf inhomogeneity). In addition, there is dispersion in the Larmor frequency of spins due to chemical shifts. A good pulse design strategy must be robust to these effects, and such variations need to be considered in the modeling and pulse design stages in order to match theoretical predictions to experimental outcomes. As difficult experiments with more demanding performance specifications have emerged, the complexity of finding optimal pulse sequences has drastically increased. For example, as high-field NMR spectrometers are increasingly more accessible and required, broadband excitation pulses are needed to cover a wide 13 C chemical-shift range (e.g., up to 40 kHz). In addition, to design excitation and inversion pulses that are practical for a typical NMR spectrometer, methods must accommodate realistic maximum rf power and pulse duration while accomplishing the desired spin evolution. In the majority of cases, the length of an rf pulse is constrained by the fixed delays that dictate a certain coherence transfer. S...

The increasing frequency of Enterococcus faecium isolates with multidrug resistance is a serious clinical problem given the severely limited number of therapeutic options available to treat these infections. Oritavancin is a promising new alternative in clinical development that has potent antimicrobial activity against both staphylococcal and enterococcal vancomycin-resistant pathogens. Using solid-state NMR to detect changes in the cell-wall structure and peptidoglycan precursors of whole cells after antibiotic-induced stress, we report that vancomycin and oritavancin have different modes of action in E. faecium. Our results show the accumulation of peptidoglycan precursors after vancomycin treatment, consistent with transglycosylase inhibition, but no measurable difference in cross-linking. In contrast, after oritavancin exposure, we do not observe the accumulation of peptidoglycan precursors. Instead, the number of cross-links is significantly reduced, showing that oritavancin primarily inhibits transpeptidation. We propose that the activity of oritavancin is the result of a secondary-binding interaction with the E. faecium peptidoglycan. The hypothesis is supported by results from 13 C{ 19 F} REDOR experiments on whole cells enriched with L-[1-13 C] lysine and complexed with desleucyl [ 19 F]oritavancin. These experiments establish that an oritavancin derivative with a damaged D-Ala-D-Ala binding pocket still binds to E. faecium peptidoglycan. The 13 C{ 19 F} REDOR dephasing maximum indicates that the secondary-binding site of oritavancin is specific to nascent and template peptidoglycan. We conclude that the inhibition of transpeptidation by oritavancin in E. faecium is the result of the large number of secondary-binding sites relative to the number of primary-binding sites.

Lipid nanodiscs are widely used platforms for studying membrane proteins in a near-native environment. Lipid nanodiscs made with membrane scaffold proteins (MSPs) in the linear form have been well studied. Recently, a new kind of nanodisc made with MSPs in the circular form, referred to as covalently circularized nanodiscs (cNDs), has been reported to have some possible advantages in various applications. Given the potential of nanodisc technology, researchers in the field are very interested in learning more about this new kind of nanodisc, such as its reproducibility, production yield, and the possible pros and cons of using it. However, research on these issues is lacking. Here, we report a new study on nanodiscs made with circular MSPs, which are produced from a method different from the previously reported method. We show that our novel production method, detergent-assisted sortase-mediated ligation, can effectively avoid high-molecular-weight byproducts and also significantly improve the yield of the target proteins up to around 80% for larger circular MSP constructs. In terms of the application of circular MSPs, we demonstrate that they can be used to assemble nanodiscs using both synthetic lipids and native lipid extract as the source of lipids. We also show that bacteriorhodopsin can be successfully incorporated into this new kind of cND. Moreover, we found that cNDs have improved stability against both heat and high-concentration-induced aggregations, making them more beneficial for related applications.

Bacteriophage T4 is a large-tailed E. coli virus whose capsid is 120 × 86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 minutes during viral infection. We have isolated 50-mg quantities of uniform 15 N and [ε-15 N]lysine-labeled bacteriophage T4. We have also introduced 15 NH 4 + into filled, unlabeled capsids from synthetic medium by exchange. We have examined lyo-and cryoprotected lyophilized T4 using 15 N{ 31 P} and 31 P{ 15 N} rotational-echo double resonance. The results of these experiments have shown that: (i) packaged DNA is in an unperturbed duplex B-form conformation; (ii) the DNA phosphate negative charge is balanced by lysyl amines (3.2%), polyamines (5.8%), and monovalent cations (40%); and (iii) 11% of lysyl amines, 40% of -NH 2 groups of polyamines, and 80% of monovalent cations within the lyophilized T4 capsid, are involved in the DNA charge balance. The NMR evidence suggests that DNA enters the T4 capsid in a charge-unbalanced state. We propose that electrostatic interactions may provide free energy to supplement the nanomotor-driven T4 DNA packaging.

Real-time tracking of membrane proteins is essential to gain an in-depth understanding of their dynamics on the cell surface. However, conventional fluorescence imaging with molecular probes like organic dyes and fluorescent proteins often suffers from photobleaching of the fluorophores, thus hindering their use for continuous long-term observations. With the availability of fluorescent nanodiamonds (FNDs), which have superb biocompatibility and excellent photostability, it is now possible to conduct the imaging in both short and long terms with high temporal and spatial resolution. To realize the concept, we have developed a facile method (e.g., one-pot preparation) to produce alkyne-functionalized hyperbranched-polyglycerol-coated FNDs for bioorthogonal labeling of azide-modified membrane proteins and azide-modified antibodies of membrane proteins. The high specificity of this labeling method has allowed us to continuously monitor the movements of the proteins of interest (such as integrin α5) on/in living cells over 2 h. The results open a new horizon for live cell imaging with functional nanoparticles and fluorescence microscopy.

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Plusbacin-A3 (pb-A3) is a cyclic lipodepsipeptide which exhibits antibacterial activity against multidrug-resistant Gram-positive pathogens. Plusbacin-A3 is thought not to enter the cell cytoplasm and its lipophilic isotridecanyl side chain is presumed to insert into the membrane bilayer thereby facilitating either lipid II binding or some form of membrane disruption. Analogues of pb-A3, [2H]pb-A3 and deslipo-pb-A3, were synthesized to test membrane insertion as key to the mode of action. [2H]pb-A3 has a 2H-isotopically labeled isopropyl subunit of the lipid side chain, and deslipo-pb-A3 is missing the isotridecanyl side chain. Both analogues have the pb-A3 core structure. The loss of antimicrobial activity in deslipo-pb-A3 showed that the isotridecanyl side chain is crucial for the drug mode of action. However, rotational-echo double resonance NMR characterization of [2H]pb-A3 bound to [1-13C]glycine labeled whole-cells of Staphylococcus aureus showed that the isotridecanyl side chain does not insert into the lipid membrane, but instead is found in the staphylococcal cell wall, positioned near the pentaglycyl cross-bridge of the cell-wall peptidoglycan. Addition of [2H]pb-A3 during S. aureus growth resulted in an accumulation of Park’s nucleotide, consistent with the inhibition of the transglycosylation step of peptidoglycan biosynthesis.