Tsyr-Yan Yu

Structural and functional characterization of the integral membrane protein VDAC-1 in lipid bilayer nanodiscs

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 nondenaturing conditions, which makes this technology particular suitable for protein-protein interactions and other functional studies.

Optimal pulse design in quantum control: A unified computational method

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.

Solution NMR Spectroscopic Characterization of Human VDAC-2 in Detergent Micelles and Lipid Bilayer Nanodiscs

Three isoforms of the human voltage-dependent anion channel (VDAC), located in the outer mitochondrial membrane, are crucial regulators of mitochondrial function. Numerous studies have been carried out to elucidate biochemical properties, as well as the three-dimensional structure of VDAC-1. However, functional and structural studies of VDAC-2 and VDAC-3 at atomic resolution are still scarce. VDAC-2 is highly similar to VDAC-1 in amino acid sequence, but has substantially different biochemical functions and expression profiles. Here, we report the reconstitution of functional VDAC-2 in lauryldimethylamine-oxide (LDAO) detergent micelles and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer nanodiscs. We find that VDAC-2 is properly folded in both membrane-mimicking systems and that structural and functional characterization by solution NMR spectroscopy is feasible. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

REDOR NMR characterization of DNA packaging in bacteriophage T4.

Bacteriophage T4 is a large-tailed Escherichia coli virus whose capsid is 120x86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 min during viral infection. We have isolated 50-mg quantities of uniform (15)N- and [epsilon-(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.

The Isotridecanyl Side Chain of Plusbacin-A3 Is Essential for the Transglycosylase Inhibition of Peptidoglycan Biosynthesis

Plusbacin-A3 (pb-A3) is a cyclic lipodepsipeptide that 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, [(2)H]pb-A3 and deslipo-pb-A3, were synthesized to test membrane insertion as a key to the mode of action. [(2)H]pb-A3 has an isotopically (2)H-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 mode of action of the drug. However, rotational-echo double-resonance nuclear magnetic resonance characterization of [(2)H]pb-A3 bound to [1-(13)C]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 [(2)H]pb-A3 during the growth of S. aureus resulted in the accumulation of Parks nucleotide, consistent with the inhibition of the transglycosylation step of peptidoglycan biosynthesis.

Tuning the Photocycle Kinetics of Bacteriorhodopsin in Lipid Nanodiscs

Monodisperse lipid nanodiscs are particularly suitable for characterizing membrane protein in near-native environment. To study the lipid-composition dependence of photocycle kinetics of bacteriorhodopsin (bR), transient absorption spectroscopy was utilized to monitor the evolution of the photocycle intermediates of bR reconstituted in nanodiscs composed of different ratios of the zwitterionic lipid (DMPC, dimyristoyl phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine) to the negatively charged lipid (DOPG, dioleoyl phosphatidylglycerol; DMPG, dimyristoyl phosphatidylglycerol). The characterization of ion-exchange chromatography showed that the negative surface charge of nanodiscs increased as the content of DOPG or DMPG was increased. The steady-state absorption contours of the light-adapted monomeric bR in nanodiscs composed of different lipid ratios exhibited highly similar absorption features of the retinal moiety at 560 nm, referring to the conservation of the tertiary structure of bR in nanodiscs of different lipid compositions. In addition, transient absorption contours showed that the photocycle kinetics of bR was significantly retarded and the transient populations of intermediates N and O were decreased as the content of DMPG or DOPG was reduced. This observation could be attributed to the negatively charged lipid heads of DMPG and DOPG, exhibiting similar proton relay capability as the native phosphatidylglycerol (PG) analog lipids in the purple membrane. In this work, we not only demonstrated the usefulness of nanodiscs as a membrane-mimicking system, but also showed that the surrounding lipids play a crucial role in altering the biological functions, e.g., the ion translocation kinetics of the transmembrane proteins.

Variability in C3-Plant Cell-Wall Biosynthesis in a High-CO2 Atmosphere by Solid-State NMR Spectroscopy

We have used a frequency-selective rotational-echo double-resonance (REDOR) solid-state NMR experiment to measure the concentrations of glycine-glycine pairs in proteins (and protein precursors) of intact leaves of plants exposed to both high- and low-CO(2) atomospheres. The results are interpreted in terms of differences in cell-wall biosynthesis between plant species. We illustrate this variability by comparing the assimilation of label in cheatgrass and soybean leaves labeled using (15)N-fertilizer and (13)CO(2) atmospheres. Cheatgrass and soybean are both C(3) plants but differ in their response to a high-CO(2) environment. Based on REDOR results, we determined that cheatgrass (a plant that seems likely to flourish in future low-water, high-CO(2) environments) routes 2% of the assimilated carbon label that remains in the leaf after 1 h in a 600-ppm (13)CO(2) atmosphere to glycine-rich protein (or its precursors), a structural component of cell walls cross-linked to lignins. In contrast, soybean under the same conditions routes none of its assimilated carbon to glycine-rich protein.

Oxygen-17 Appears Only in Protein in Water-Stressed Soybean Leaves Labeled by 17O2

We have used a rotational-echo adiabatic-passage double-resonance (13)C{(17)O} solid-state NMR experiment to prove that the glycine produced in the oxygenase reaction of ribulose bisphosphate carboxylase-oxygenase is incorporated exclusively into protein (or protein precursors) of intact, water-stressed soybean leaves exposed to (13)CO(2) and (17)O(2). The water stress increased stomatal resistance and decreased gas exchange so that the Calvin cycle in the leaf chloroplasts was no more than 35% (13)C isotopically enriched. Labeled O(2) levels were sufficient, however, to increase the (17)O isotopic concentration of oxygenase products 20-fold over the natural-abundance level of 0.04%. The observed direct incorporation of glycine into protein shows that water stress suppresses photorespiration in soybean leaves.

Optimization of the production of covalently circularized nanodiscs and their characterization in physiological conditions

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.

Lipids influence the proton pump activity of photosynthetic protein embedded in nanodiscs

We report the lipid-composition dependent photocycle kinetics and proton pump activity of bacteriorhodopsin (bR) embedded in nanodiscs composed of different lipids. Using time-resolved spectroscopy and electrochemical methods, we were able to comprehensively understand the kinetics of the photocycle and the corresponding proton pumping activity as the composition of the charged lipids were systematically adjusted. We found that positively-charged lipids assist in repulsing protons from bR, thus increasing the concentration of the non-bounded protons in the bulk. In contrast, the negatively-charged lipids assist in entrapping the protons in the proximity of bR during the photocycle, preserving the electromotive force across the lipid bilayers which is essential for the vitality of the lateral proton transport and the bioenergetics.