Value of turbo spin echo-based diffusion-weighted imaging in the differential diagnosis of benign and malignant solitary pulmonary lesions.

To quantitatively assess the diagnostic efficacy of multiple parameters derived from multi-b-value diffusion-weighted imaging (DWI) using turbo spin echo (TSE)-based acquisition techniques in patients with solitary pulmonary lesions (SPLs). A total of 105 patients with SPLs underwent lung DWI using single-shot TSE-based acquisition techniques and multiple b values. The apparent diffusion coefficient (ADC), intravoxel incoherent motion (IVIM) parameters, and lesion-to-spinal cord signal intensity ratio (LSR), were analyzed to compare the benign and malignant groups using the Mann-Whitney U test and receiver operating characteristic analysis. The Dstar values observed in lung cancer were slightly lower than those observed in pulmonary benign lesions (28.164 ± 31.950 versus 32.917 ± 34.184; Z = -2.239, p = 0.025). The LSR values were significantly higher in lung cancer than in benign lesions (1.137 ± 0.581 versus 0.614 ± 0.442; Z = - 4.522, p < 0.001). Additionally, the ADC800, ADCtotal, and D values were all significantly lower in lung cancer than in the benign lesions (Z = - 5.054, -5.370, and -6.047, respectively, all p < 0.001), whereas the f values did not exhibit any statistically significant difference between the two groups. D had the highest area under the curve (AUC = 0.887), followed by ADCtotal (AUC = 0.844), ADC800 (AUC = 0.824), and LSR (AUC = 0.789). The LSR, ADC800, ADCtotal, and D values did not differ statistically significantly in diagnostic effectiveness. Lung DWI using TSE is feasible for differentiating SPLs. The LSR method, conventional DWI, and IVIM have comparable diagnostic efficacy for assessing SPLs.

Primary Structural Units "D+A-" Ion Pairs Dominating Near-Infrared Photothermal Conversion of Organic Ionic Cocrystals.

The specific stacking mode of D/A blocks is often considered to largely determine the physicochemical properties of cocrystals. However, this rule may fail when encountering a large degree of (integer or near-integer) charge transfer situations. Herein, we explore the extensive correlations between the possible smallest structural units, stacking modes, and near-infrared photothermal conversion (NIR-PTC) properties of F4TCNQ-based cocrystals with typical features of integer-charge-transfer. Surprisingly, these cocrystals with distinct stacking modes display analogous D-A interactions, broad red-shift absorption, ultrafast (1-3 ps) relaxation dynamics of excited states, and excellent NIR-PTC properties. This supports that the resulting "D+A-" ion pairs from integer-charge-transfer may serve as the primary structural units beneath the secondary stacking modes to dominate the property of cocrystals. The stacking modes play an important but only secondary role. This work provides new insights into the structure-dynamics-property correlations and modular design of organic cocrystals for PTC and other applications.

Long-Acting Nanohybrid Hydrogel Induces Persistent Immunogenic Chemotherapy for Suppressing Postoperative Tumor Recurrence and Metastasis.

Despite the continuous advancement of surgical resection techniques, postoperative tumor recurrence and metastasis remain a huge challenge. Here, we constructed an injectable curcumin/doxorubicin-loaded nanoparticle (NanoCD) hydrogel, which could effectively inhibit tumor regrowth and metastasis via reshaping the tumor immune microenvironment (TIME) for highly effective postsurgical cancer treatment. NanoCD was prepared by the controlled assembly of curcumin (CUR) and doxorubicin (DOX) via π-π stacking and hydrogen bonding in the presence of human serum albumin. To facilitate prolonged treatment of postsurgical tumors, NanoCD was further incorporated into the temperature-sensitive Poloxamer 407 gel (NanoCD@Gel) for intracavity administration. Mechanistically, DOX induced the generation of intracellular reactive oxygen species (ROS) and CUR reduced the ROS metabolism by inhibiting thioredoxin reductase (TrxR). The synergy of DOX and CUR amplified intracellular ROS levels and thus resulted in enhanced immunogenic cell death (ICD) of tumor cells. Upon being injected into the tumor cavity after resection, the in situ-generated NanoCD@Gel allowed the local release of CUR and DOX in a controlled manner to induce local chemotherapy and persistently activate the antitumor immune response, thereby achieving enhanced immunogenic chemotherapy with reduced systemic toxicity. Our work provides an elegant strategy for persistently stimulating effective antitumor immunity to prevent postsurgical tumor recurrence and metastasis.

Dietary oregano essential oil supplementation alters meat quality, oxidative stability, and fatty acid profiles of beef cattle.

This study was conducted to elucidate the effects of oregano essential oil (OEO) supplementation on the meat quality, antioxidant capacity, and nutritional value of the longissimus thoracis muscle in steers. Steers were divided into three groups (n = 9) and fed either a basal diet, or a basal diet supplemented with 130 mg/d OEO, or 230 mg/d OEO for 390 days. The results demonstrated that dietary OEO supplementation increased the total antioxidant capacity and activity of catalase, glutathione peroxidase, and superoxide dismutase, and decreased pH30min, pH24h, cooking loss, and malondialdehyde content. OEO increased the concentrations of polyunsaturated fatty acids and conjugated linoleic acid. In contrast, saturated fatty acids decreased, accompanied by increased essential amino acids, flavor amino acids, and total amino acids in the longissimus thoracis muscle. In summary, dietary OEO supplementation promotes the nutritional and meat quality of beef by maintaining its water-holding capacity and meat color, enhancing its antioxidative capacity, and preventing lipid oxidation.

1,2-Dicarbofunctionalization of Trifluoromethyl Alkenes with Pyridinium Salts via a Cycloaddition/Visible-Light-Enabled Fragmentation Cascade.

Although trifluoromethyl alkenes have great synthetic potential, their 1,2-difunctionalization has been a challenge. In this Letter, we disclose the first 1,2-dicarbofunctionalization of trifluoromethyl alkenes with pyridinium salts via a cascade process involving a base-promoted [3 + 2] cycloaddition followed by a visible-light-mediated Norrish-type-II fragmentation. This protocol allows for the formation of pyridines bearing a trifluoromethyl-substituted quaternary center in moderate to excellent yields under mild conditions.

Convergence or divergence? The effects of economic openness on low-carbon innovation in Chinese manufacturing industry.

Low-carbon innovation can address both economic and environmental concerns; patterns of low-carbon innovation convergence can determine the effectiveness of mitigating the adverse consequences of climate change. Considering that economic openness has a huge impact on the development of innovation capability, this paper uses a conditional ß convergence model to examine the convergence of low-carbon innovation in Chinese manufacturing industry and its relationship with economic openness. We incorporate the spatial spillover effect into the convergence function by constructing spatial error model, spatial lag model, and spatial Durbin model. Based on a panel data set of 30 Chinese provinces over the period 2004-2016, the results show that low-carbon innovation in Chinese manufacturing industry has a strong feature of conditional ß convergence. The convergence rate of low-carbon innovation is slightly slowed down by economic openness, and the main reason is that the spillover effect is weak and the convergence rate is slow in lower open areas, so the convergence rate of the whole country is slowed down by that of the lower open areas. Although the economic openness in adjacent areas can contribute to the development of local innovation ability, but generally speaking, economic openness in local areas takes a stronger effect in promoting the convergence of low-carbon innovation than that in adjacent areas. The findings have important policy implications as they suggest the need for a more equal degree of economic openness among Chinese provinces to speed up the convergence of low-carbon innovation.

Values of Apparent Diffusion Coefficient and Lesion-to-Spinal Cord Signal Intensity in Diagnosing Solitary Pulmonary Lesions: Turbo Spin-Echo versus Echo-Planar Imaging Diffusion-Weighted Imaging.

OBJECTIVE: This study is aimed at comparing the image quality and diagnostic performance of mean apparent diffusion coefficient (ADC) and lesion-to-spinal cord signal intensity ratio (LSR) derived from turbo spin-echo diffusion-weighted imaging (TSE-DWI) and echo-planar imaging- (EPI-) DWI in patients with a solitary pulmonary lesion (SPL). METHODS: 33 patients with SPL underwent chest imaging using EPI-DWI and TSE-DWI with b = 600 s/mm2 in free breathing. A comparison of the distortion ratio (DR), signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) was drawn between the two techniques using a Wilcoxon signed-rank test. The interprotocol reproducibility between quantitative parameters of EPI-DWI and TSE-DWI was evaluated using a Bland-Altman plot. ADCs and LSRs derived from EPI-DWI and TSE-DWI were calculated and compared between malignant and benign groups using the Mann-Whitney test. RESULTS: TSE-DWI had similar SNR and CNR compared with EPI-DWI. DR was significantly lower on TSE-DWI than EPI-DWI. ADC and LSR showed slightly higher values with TSE-DWI, while the Bland-Altman analysis showed unacceptable limits of agreement between the two sequences. ADC and LSR of both DWI techniques differed significantly between lung cancer and benign lesions (P < 0.05). The LSR(EPI-DWI) showed the highest area under the curve (AUC = 0.818), followed by ADC(EPI-DWI) (AUC = 0.789), ADC(TSE-DWI) (AUC = 0.781), and LSR(TSE-DWI) (AUC = 0.748), respectively. Among these parameters, the difference in diagnostic accuracy was not statistically significant. CONCLUSIONS: TSE-DWI provides significantly improved image quality in patients with SPL as compared with EPI-DWI. However, there was no difference in diagnostic efficacy between these two techniques, according to ADC and LSR.

Comparative transcriptome and histological analyses provide insights into the skin pigmentation in Minxian black fur sheep (Ovis aries).

BACKGROUND: Minxian black fur (MBF) sheep are found in the northwestern parts of China. These sheep have developed several special traits. Skin color is a phenotype subject to strong natural selection and diverse skin colors are likely a consequence of differences in gene regulation. METHODS: Skin structure, color differences, and gene expression (determined by RNA sequencing) were evaluated the Minxian black fur and Small-tail Han sheep (n = 3 each group), which are both native Chinese sheep breeds. RESULTS: Small-tail Han sheep have a thicker skin and dermis than the Minxian black fur sheep (P < 0.01); however, the quantity of melanin granules is greater (P < 0.01) in Minxian black fur sheep with a more extensive distribution in skin tissue and hair follicles. One hundred thirty-three differentially expressed genes were significantly associated with 37 ontological terms and two critical KEGG pathways for pigmentation ("tyrosine metabolism" and "melanogenesis" pathways). Important genes from those pathways with known involvement in pigmentation included OCA2 melanosomal transmembrane protein (OCA2), dopachrome tautomerase (DCT), tyrosinase (TYR) and tyrosinase related protein (TYRP1), melanocortin 1 receptor (MC1R), and premelanosome protein (PMEL). The results from our histological and transcriptome analyses will form a foundation for additional investigation into the genetic basis and regulation of pigmentation in these sheep breeds.

Effects of oregano essential oil on in vitro ruminal fermentation, methane production, and ruminal microbial community.

Different inclusion rates of oregano essential oil (OEO) were investigated for their effects on ruminal in vitro fermentation parameters, total gas, methane production, and bacterial communities. Treatments were (1) control, 0 mg/L of OEO (CON); 13 mg/L (OEO1); 52 mg/L (OEO2); 91 mg/L (OEO3); and 130 mg/L (OEO4), each incubated with 150 mL of buffered rumen fluid and 1,200 mg of substrate for 24 h using the Ankom in vitro gas production system (Ankom Technology Corp., Fairport, NY). Treatment responses were statistically analyzed using polynomial contrasts. Digestibility of DM, NDF, and ADF increased quadratically with increasing OEO inclusion rates. Digestibility of DM and NDF were highest for OEO2, whereas ADF digestibility was highest for OEO3, compared with CON, with the remaining treatments being intermediate and similar. Ammonia nitrogen concentrations decreased from CON at a quadratic rate with increasing OEO inclusion rates, and OEO2 had the lowest concentration compared with the other groups. Total VFA, acetate, propionate, butyrate, valerate, and isovalerate concentrations linearly decreased with increasing OEO inclusion rates. Total gas production levels by CON and OEO4 were greater than those of OEO1, OEO2, and OEO3 in a quadratic response, and methane production linearly decreased from CON, compared with OEO4, at a decreasing rate with OEO inclusion rates. As determined by 16S rRNA sequencing, the α biodiversity of ruminal bacteria was similar among OEO inclusion rates. Increasing OEO inclusion rates linearly increased the relative abundance of Prevotella and Dialister bacteria. Several bacteria demonstrated different polynomial responses, whereas several bacteria were similar among increasing OEO inclusion rates. These results suggested that OEO supplementation can modify ruminal fermentation to alter VFA concentrations and reduce methane emissions by extensively altering the ruminal bacterial community, suggesting an optimal feeding rate for future animal studies of approximately 52 mg/L for mature ruminants.

Effects of oregano essential oil on the ruminal pH and microbial population of sheep.

Oregano essential oil (OEO), which has antimicrobial properties, may be used for altering the ruminal pH and microbial populations of sheep, as observed by the altered volatile fatty acid patterns. To further elucidate the effects of OEO on ruminal pH and microbial populations of sheep, 3 German merino sheep × local sheep crossbred rams with permanent ruminal fistulas were randomly assigned to a 3 × 3 Latin square design with 12-d periods. The treatments were as follows: control (CON); OEO4: OEO supplied at 4 g•d-1; and OEO7: OEO supplied at 7 g•d-1. Starting on day 11, rumen fluid was collected at 0 h, and at 4, 8, 12, 24 and 48 h after supplying OEO, and then pH values of rumen fluid were immediately measured. The abundance of microbial populations was determined by using qPCR. The ruminal pH values were similar among the sheep from all treatments. The abundance of ruminal fungi was higher for the sheep supplied OEO7 compared with the sheep supplied CON and OEO4, especially at 4 and 12 h. The abundance of ruminal protozoa decreased with supplied OEO, indicating that OEO could inhibit the protozoa. The abundance of the total ruminal bacteria was similar for the sheep from all treatments, but R. flavefaciens, R. albus and F. succinogenes increased in the sheep supplied OEO4 compared with those in the sheep supplied CON, however, the sheep supplied OEO7 had higher abundances of R. flavefaciens than the sheep supplied CON. These results demonstrated that supplying OEO to sheep did not affect the ruminal pH but could shift the rumen microbial population to one with less protozoa. Supplying OEO can preferentially enhance the growth of certain rumen microbial populations, but the shifts were influenced by the supply rate. Therefore, supplying low amount (i.e. 4 g•d-1) of OEO could have positive effects on ruminal microbial populations, whereas supplying elevated doses of OEO could be detrimental to those same ruminal microbial populations.

The effect of periodic energy restriction on growth performance, serum biochemical indices, and meat quality in sheep.

Energy is a key factor regulated by the neuroendocrine system. The objective of this study was to compare growth performance, serum biochemical indices, carcass traits, meat quality, and nutrient composition of lambs fed restricted metabolizable energy (ME) intakes to mimic the seasonal changes of the natural grasslands of northwest China. Nineteen male Dorper × Small Tailed Han lambs were assigned to treatments, control (CON) fed at 1.0 MJ/W0.75 × d-1 throughout the 180 d study, and restricted ME (RES) fed by sequentially restricting ME every 30 d (0.56, 0.84, 1.0, 0.84, 0.56, and 0.28 MJ/W0.75 × d-1). Lambs were harvested at the end of the study. Lambs fed the CON diet demonstrated higher (P < 0.05) average daily gain (ADG) compared with RES-fed lambs (158.79 and 57.01 g/d, for CON and RES, respectively), although ADG were lower in each period with the last period being a negative ADG for RES-fed lambs resulting in a loss of body weight. Both treatments demonstrated lower ADG in the last 3 study periods compared with the first 2 periods, which may be explained by cold stress (<-3 °C) increasing the lamb's energy requirement. Feeding lambs a restricted ME diet resulted in lower (P < 0.05) blood serum glucose, triglycerides, total protein, calcium, phosphorus, immunoglobulin G, immunoglobulin M, and immunoglobulin A concentrations compared with CON-fed lambs, but both treatments were similar (P > 0.05) for cholesterol and high-density lipoprotein-cholesterol. Feeding lambs a restricted ME diet increased (P < 0.05) meat pH, while reducing the cooked meat rate compared with CON-fed lambs. Thus, the carcass yield, rib eye muscle area, and shear force were lower (P < 0.05) for RES-fed lambs compared with CON-fed lambs. The meat water concentration was higher (P < 0.05), whereas the concentrations of dry matter, crude fat, crude protein, and carbohydrate were lower (P < 0.05) for lambs fed a RES ME diet compared to CON-fed lambs. These results demonstrate that sequentially restricting ME intake, as natural grasslands might experience during seasonal forage quality and quantity changes, resulted in a reduction of body weight because lambs were mobilizing body reserves as a source of ME. The reduction in concentrations of key serum nutrients, as well as carcass traits and meat composition, demonstrate the body is responding to the meet the lambs ME requirements, which has implications for both meat quality and the animal's physiological functions.

Investigating the Secondary Structure of Membrane Peptides Utilizing Multiple 2H-Labeled Hydrophobic Amino Acids via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

An electron spin echo envelope modulation (ESEEM) approach was used to probe local secondary structures of membrane proteins and peptides. This ESEEM method detects dipolar couplings between 2H-labeled nuclei on the side chains of an amino acid (Leu or Val) and a strategically placed nitroxide spin-label in the proximity up to 8 Å. ESEEM spectra patterns for different samples correlate directly to the periodic structural feature of different secondary structures. Since this pattern can be affected by the side chain length and flexibility of the 2H-labeled amino acid used in the experiment, it is important to examine several different hydrophobic amino acids (d3 Ala, d8 Val, d8 Phe) utilizing this ESEEM approach. In this work, a series of ESEEM data were collected on the AChR M2δ membrane peptide to build a reference for the future application of this approach for various biological systems. The results indicate that, despite the relative intensity and signal-to-noise level, all amino acids share a similar ESEEM modulation pattern for α-helical structures. Thus, all commercially available 2H-labeled hydrophobic amino acids can be utilized as probes for the further application of this ESEEM approach. Also, the ESEEM signal intensities increase as the side chain length gets longer or less rigid. In addition, longer side chain amino acids had a larger 2H ESEEM FT peak centered at the 2H Larmor frequency for the i ± 4 sample when compared to the corresponding i ± 3 sample. For shorter side chain amino acids, the 2H ESEEM FT peak intensity ratio between i ± 4 and i ± 3 was not well-defined.

A Dynamic Protein-Protein Coupling between the TonB-Dependent Transporter FhuA and TonB.

Bacterial outer membrane TonB-dependent transporters function by executing cycles of binding and unbinding to the inner membrane protein TonB. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, substrate binding increases the periplasmic exposure of the Ton box, an energy-coupling segment. This increased exposure appears to enhance the affinity of the transporter for TonB. Here, continuous wave and pulse EPR spectroscopy were used to examine the state of the Ton box in the Escherichia coli ferrichrome transporter FhuA. In its apo state, the Ton box of FhuA samples a broad range of positions and multiple conformational substates. When bound to ferrichrome, the Ton box does not extend further into the periplasm, although the structural states sampled by the FhuA Ton box are altered. When bound to a soluble fragment of TonB, the TonB-FhuA complex remains heterogeneous and dynamic, indicating that TonB does not make strong, specific contacts with either the FhuA barrel or the core region of the transporter. This result differs from that seen in the crystal structure of the TonB-FhuA complex. These data indicate that unlike BtuB and FecA, the periplasmic exposure of the Ton box in FhuA does not change significantly in the presence of substrate and that allosteric control of transporter-TonB interactions functions by a different mechanism than that seen in either BtuB or FecA. Moreover, the data indicate that models involving a rotation of TonB relative to the transporter are unlikely to underlie the mechanism that drives TonB-dependent transport.

Probing the Local Secondary Structure of Human Vimentin with Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Previously, an electron spin echo envelope modulation (ESEEM) spectroscopic approach was established to probe the local secondary structure of membrane proteins and peptides utilizing site-directed spin-labeling (SDSL). In this method, the side chain of one amino acid residue is selectively 2H-labeled and a nitroxide spin label is strategically placed 1, 2, 3, or 4 amino acids away from the 2H-labeled amino acid (denoted as i ± 1 to i ± 4, i represents the 2H-labeled amino acid). ESEEM can detect the dipolar coupling between the nitroxide spin label and 2H atoms on the amino acid side chain. Due to the periodicity of different secondary structures, different ESEEM patterns can be revealed to probe the structure. For an α-helical structural component, a 2H ESEEM signal can be detected for i ± 3 and i ± 4 samples, but not for i ± 1 or i ± 2 samples. Several 2H-labeled hydrophobic amino acids have been demonstrated in model system that can be utilized to identify local secondary structures via this ESEEM approach in an extremely efficient fashion. In this study, the ESEEM approach was used to investigate the rod 2B region of the full-length intermediate filament protein human vimentin. Consistent with previous EPR and X-ray crystallography results, our ESEEM results indicated helical structural components within this region. Thus, this ESEEM approach is able to identify α-helical structural components despite the coiled-coil nature of the vimentin structure. The data show that the human vimentin rod 2B adapted a typical α-helical structure around residue Leu309. This result is consistent with the X-ray data from fragmented protein segments and continuous wave EPR data on the full-length vimentin. Finally, the ESEEM data suggested that a local secondary structure slightly different from a typical α-helix was adopted around residue 340.

Probing the Secondary Structure of Membrane Peptides Using (2)H-Labeled d(10)-Leucine via Site-Directed Spin-Labeling and Electron Spin Echo Envelope Modulation Spectroscopy.

Previously, we reported an electron spin echo envelope modulation (ESEEM) spectroscopic approach for probing the local secondary structure of membrane proteins and peptides utilizing (2)H isotopic labeling and site-directed spin-labeling (SDSL). In order to probe the secondary structure of a peptide sequence, an amino acid residue (i) side chain was (2)H-labeled, such as (2)H-labeled d10-Leucine, and a cysteine residue was strategically placed at a subsequent nearby position (denoted as i + 1 to i + 4) to which a nitroxide spin label was attached. In order to fully access and demonstrate the feasibility of this new ESEEM approach with (2)H-labeled d10-Leu, four Leu residues within the AChR M2δ peptide were fully mapped out using this ESEEM method. Unique (2)H-ESEEM patterns were observed with the (2)H-labeled d10-Leu for the AChR M2δ α-helical model peptide. For proteins and peptides with an α-helical secondary structure, deuterium modulation can be clearly observed for i ± 3 and i ± 4 samples, but not for i ± 2 samples. Also, a deuterium peak centered at the (2)H Larmor frequency of each i ± 4 sample always had a significantly higher intensity than the corresponding i + 3 sample. This unique feature can be potentially used to distinguish an α-helix from a π-helix or 310-helix. Moreover, (2)H modulation depth for ESEEM samples on Leu10 were significantly enhanced which was consistent with a kinked or curved structural model of the AChR M2δ peptide as suggested by previous MD simulations and NMR experiments.

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Revealing detailed structural and dynamic information of membrane embedded or associated proteins is challenging due to their hydrophobic nature which makes NMR and X-ray crystallographic studies challenging or impossible. Electron paramagnetic resonance (EPR) has emerged as a powerful technique to provide essential structural and dynamic information for membrane proteins with no size limitations in membrane systems which mimic their natural lipid bilayer environment. Therefore, tremendous efforts have been devoted toward the development and application of EPR spectroscopic techniques to study the structure of biological systems such as membrane proteins and peptides. This chapter introduces a novel approach established and developed in the Lorigan lab to investigate membrane protein and peptide local secondary structures utilizing the pulsed EPR technique electron spin echo envelope modulation (ESEEM) spectroscopy. Detailed sample preparation strategies in model membrane protein systems and the experimental setup are described. Also, the ability of this approach to identify local secondary structure of membrane proteins and peptides with unprecedented efficiency is demonstrated in model systems. Finally, applications and further developments of this ESEEM approach for probing larger size membrane proteins produced by overexpression systems are discussed.

Development of electron spin echo envelope modulation spectroscopy to probe the secondary structure of recombinant membrane proteins in a lipid bilayer.

Membrane proteins conduct many important biological functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is very difficult to obtain structural information on membrane-bound proteins using traditional biophysical techniques. We are developing a new approach to probe the secondary structure of membrane proteins using the pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. This method has been successfully applied to model peptides made synthetically. However, in order for this ESEEM technique to be widely applicable to larger membrane protein systems with no size limitations, protein samples with deuterated residues need to be prepared via protein expression methods. For the first time, this study shows that the ESEEM approach can be used to probe the local secondary structure of a (2) H-labeled d8 -Val overexpressed membrane protein in a membrane mimetic environment. The membrane-bound human KCNE1 protein was used with a known solution NMR structure to demonstrate the applicability of this methodology. Three different α-helical regions of KCNE1 were probed: the extracellular domain (Val21), transmembrane domain (Val50), and cytoplasmic domain (Val95). These results indicated α-helical structures in all three segments, consistent with the micelle structure of KCNE1. Furthermore, KCNE1 was incorporated into a lipid bilayer and the secondary structure of the transmembrane domain (Val50) was shown to be α-helical in a more native-like environment. This study extends the application of this ESEEM approach to much larger membrane protein systems that are difficult to study with X-ray crystallography and/or NMR spectroscopy.

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies.

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30 nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment.

Solid-state NMR (31)P paramagnetic relaxation enhancement membrane protein immersion depth measurements.

Paramagnetic relaxation enhancement (PRE) is a widely used approach for measuring long-range distance constraints in biomolecular solution NMR spectroscopy. In this paper, we show that (31)P PRE solid-state NMR spectroscopy can be utilized to determine the immersion depth of spin-labeled membrane peptides and proteins. Changes in the (31)P NMR PRE times coupled with modeling studies can be used to describe the spin-label position/amino acid within the lipid bilayer and the corresponding helical tilt. This method provides valuable insight on protein-lipid interactions and membrane protein structural topology. Solid-state (31)P NMR data on the 23 amino acid α-helical nicotinic acetylcholine receptor nAChR M2δ transmembrane domain model peptide followed predicted behavior of (31)P PRE rates of the phospholipid headgroup as the spin-label moves from the membrane surface toward the center of the membrane. Residue 11 showed the smallest changes in (31)P PRE (center of the membrane), while residue 22 shows the largest (31)P PRE change (near the membrane surface), when compared to the diamagnetic control M2δ sample. This PRE SS-NMR technique can be used as a molecular ruler to measure membrane immersion depth.

Investigating the interaction between peptides of the amphipathic helix of Hcf106 and the phospholipid bilayer by solid-state NMR spectroscopy.

The chloroplast twin arginine translocation (cpTat) system transports highly folded precursor proteins into the thylakoid lumen using the protonmotive force as its only energy source. Hcf106, as one of the core components of the cpTat system, is part of the precursor receptor complex and functions in the initial precursor-binding step. Hcf106 is predicted to contain a single amino terminal transmembrane domain followed by a Pro-Gly hinge, a predicted amphipathic α-helix (APH), and a loosely structured carboxy terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes. To better understand the membrane active capabilities of Hcf106, we used solid-state NMR spectroscopy to investigate those properties of the APH. In this study, synthesized peptides of the predicted Hcf106 APH (amino acids 28-65) were incorporated at increasing mol.% into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and POPC/MGDG (monogalactosyldiacylglycerol; mole ratio 85:15) multilamellar vesicles (MLVs) to probe the peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic experiments revealed that the peptide perturbs the headgroup and the acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy (CSA) line width, and (2)H order SCD parameters. In addition, the comparison between POPC MLVs and POPC/MGDG MLVs indicated that the lipid bilayer composition affected peptide perturbation of the lipids, and such perturbation appeared to be more intense in a system more closely mimicking a thylakoid membrane.