An efficient method for detecting membrane protein oligomerization and complex using 05SAR-PAGE.

Oligomerization is an important feature of proteins, which gives a defined quaternary structure to complete the biological functions. Although frequently observed in membrane proteins, characterizing the oligomerization state remains complicated and time-consuming. In this study, 0.05% (w/v) sarkosyl-polyacrylamide gel electrophoresis (05SAR-PAGE) was used to identify the oligomer states of the membrane proteins CpxA, EnvZ, and Ma-Mscl with high sensitivity. Furthermore, two-dimensional electrophoresis (05SAR/sodium dodecyl sulfate-PAGE) combined with western blotting and liquid chromatography-tandem mass spectrometry was successfully applied to study the complex of CpxA/OmpA in cell lysate. The results indicated that 05SAR-PAGE is an efficient, economical, and practical gel method that can be widely used for the identification of membrane protein oligomerization and the analysis of weak protein interactions.

Stochastic simulations of self-organized elastogenesis in the developing lung.

In the normal lung, the dominant cable is an elastic "line element" composed of elastin fibers bound to a protein scaffold. The cable line element maintains alveolar geometry by balancing surface forces within the alveolus and changes in lung volume with exercise. Recent work in the postnatal rat lung has suggested that the process of cable development is self-organized in the extracellular matrix. Early in postnatal development, a blanket of tropoelastin (TE) spheres appear in the primitive lung. Within 7 to 10 days, the TE spheres are incorporated into a distributed protein scaffold creating the mature cable line element. To study the process of extracellular assembly, we used cellular automata (CA) simulations. CA simulations demonstrated that the intermediate step of tropoelastin self-aggregation into TE spheres enhanced the efficiency of cable formation more than 5-fold. Similarly, the rate of tropoelastin production had a direct impact on the efficiency of scaffold binding. The binding affinity of the tropoelastin to the protein scaffold, potentially reflecting heritable traits, also had a significant impact on cable development. In contrast, the spatial distribution of TE monomer production, increased Brownian motion and variations in scaffold geometry did not significantly impact simulations of cable development. We conclude that CA simulations are useful in exploring the impact of concentration, geometry, and movement on the fundamental process of elastogenesis.

Improvement of average magnetic field measurement based on magnetic-field-sensitive Ramsey fringes.

Accurate magnetic field measurement is the key to evaluating the second-order Zeeman effect. The conventional method is to deduce the magnetic field by determining the center frequency of the magnetic-field-sensitive Ramsey fringes. In this Letter, we present a more rigorous theory for this method and demonstrate that the current peak-searching method has a non-negligible sub-Hz or even larger deviation. We introduce an improved method that considers more parameters and a strict formula that can correct the deviation and suppress it to below 0.1 Hz. Corresponding experiments on the 85Rb atomic fountain demonstrate that this improved method is expected to enhance the precision of magnetic field measurement and improve the atomic fountain clock.

Serum IL-17A concentration and a IL17RA single nucleotide polymorphism contribute to the risk of autoimmune type 1 diabetes.

AIMS: Interleukin (IL)-17 is associated with autoimmunity. This study aimed to affirm the role of IL-17A, IL-17F and single nucleotide polymorphisms (SNPs) in genes related to them and their receptors in autoimmune type 1 diabetes (T1D) for Chinese population. METHODS: In this study, 130 patients with autoimmune T1D and 140 non-T1D controls were included for analysis. Clinical and biochemical data were collected, and serum levels of IL-17A, IL-17F, IL-6, and high-sensitivity C reactive protein were measured using ELISA. The SNPs rs2275913, rs8193036, rs3819025, rs763780, rs879577, rs4819554, and rs708567 were genotyped using the SNaPshot assay. RESULTS: IL-17A levels were higher in patients with autoimmune T1D than in controls (median [IQR] 28.83[37.38] vs. 16.68[8.10], p < 0.001) and high IL-17A was a risk factor for autoimmune T1D (odds ratio (OR), 1.013; 95% CI, 1.003-1.023; p = 0.013) after adjusting for confounding factors. Linear regression analysis revealed that log10 IL-17A levels were independently associated with fasting C-peptide, IL-6, body mass index, and IL-17F. However, no independent association was found between IL-17F and autoimmune T1D. The GG genotype of SNP rs4819554 in the interleukin 17 receptor A (IL17RA) gene was associated with a decreased risk of autoimmune T1D (OR, 0.458; 95% CI, 0.246-0.852; p = 0.014) after adjusting for other confounders. The IL17RA rs4819554 GG genotype was negatively correlated with serum glutamic acid decarboxylase antibody appearance (p < 0.05). CONCLUSIONS: Increased serum IL-17A, but not IL-17F, is a risk factor for autoimmune T1D. The GG genotype of IL17RA rs4819554 might decrease the risk for autoimmune T1D.

Amyloid-like aggregation and fibril core determination of TDP-43 C-terminal domain.

Cytoplasmic inclusion of TAR DNA-binding protein 43 (TDP-43) is a hallmark of most ALS (amyotrophic lateral sclerosis) and FTLD (Frontotemporal dementia), yet the aggregation of TDP-43 remains unclear. In this study, we proved the existence of amyloid-like structures of C-terminal domain of TDP-43 (TDP-C) in bacterial inclusion bodies (IBs), and obtained a homogenous fibril sample by seeding from the components of aggregated TDP-C in Escherichiacoli IBs. The results from solid-state NMR spectroscopy suggest that the homogenous fibrils were seeded from a tiny amount of aggregated TDP-C compositions in IBs; the structure characteristics of the rigid fibril core are identified of ß-rich structures, and show subtle relativity with the hydrophobicity of residues. Our study here provides a further understanding of TDP-43 protein aggregation and fibrillation.

Development of Multimarker Diagnostic Models from Metabolomics Analysis for Gestational Diabetes Mellitus (GDM).

Although metabolomics are desirable to understand the pathophysiology of gestational diabetes mellitus (GDM), comprehensive metabolomic studies of GDM are rare. We aimed to offer a holistic view of metabolites alteration in GDM patients and investigate the possible multimarker models for GDM diagnosis. Biochemical parameters and perinatal data of 131 GDM cases and 138 controls were collected. Fasting serum samples at 75 g oral glucose tolerance test were used for metabolites by ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry, ultra performance liquid chromatography-triple triple-quadrupole-mass spectrometry and gas chromatography- time-of- flight mass spectrometry platforms. Significant changes were observed in free fatty acids, bile acids, branched chain amino acids, organic acids, lipids and organooxygen compounds between two groups. In receiver operating characteristic (ROC) analysis, different combinations of candidate biomarkers and metabolites in multimarker models achieved satisfactory discriminative abilities for GDM, with the values of area under the curve (AUC) ranging from 0.721 to 0.751. Model consisting of body mass index (BMI), retinol binding protein 4 (RBP4), n-acetylaspartic acid and C16:1 (cis-7) manifested the best discrimination [AUC 0.751 (95% CI: 0.693-0.809), p < 0.001], followed by model consisting of BMI, Cystatin C, acetylaspartic acid and 6,7-diketoLCA [AUC 0.749 (95% CI: 0.691-0.808), p < 0.001]. Metabolites alteration reflected disorders of glucose metabolism, lipid metabolism, amino acid metabolism, bile acid metabolism as well as intestinal flora metabolism in GDM state. Multivariate models combining clinical markers and metabolites have the potential to differentiate GDM subjects from healthy controls.

Structural Mechanism of Barriers to Interspecies Seeding Transmissibility of Full-Length Prion Protein Amyloid.

A puzzling feature of prion diseases is the cross-species barriers. The detailed molecular mechanisms underlying these interspecies barriers remain poorly understood because of a lack of high-resolution structural information on the scrapie isoform of the prion protein (PrPSc ). In this study we identified the critical role of the residues 165/167 in the barrier to seeding mouse PrP (mPrP) fibril seeds to human cellular prion protein (PrPC ). Solid-state NMR revealed a C-terminal ß-sheet core spanning residues 165-230 and the packing arrangement of mPrP fibrils. Residues 165/167 are located on one end of the fibril core. Molecular dynamics simulations demonstrated that the stabilities of the seeding-induced ß-strand structures are significantly impacted by hydrogen bonds involving the side chain of residue 167 and steric resistance involving residue 165. These findings suggest that the α2-ß2 loop containing residues 165/167 could be the initial site of seed-template conformational conversion.

Heterogeneity of insulin resistance and beta cell dysfunction in gestational diabetes mellitus: a prospective cohort study of perinatal outcomes.

BACKGROUND: Insulin resistance and beta cell dysfunction were reported to be responsible for gestational diabetes mellitus (GDM). However, little is known about the heterogeneity of these factors and its influences on perinatal outcomes. We investigated whether subtypes of insulin resistance and beta cell dysfunction in gestational diabetes mellitus have different impacts on perinatal outcomes. METHODS: In this prospective cohort study, we followed 554 pregnant women and glucose challenge test was performed at 24-28th weeks of their gestation. Women with plasma glucose ≥ 7.8 mmol/L would be included and advised to undergo the diagnostic 75-g, 3-h oral glucose tolerance test. According to indices of measuring insulin resistance or beta cell function were below the 25th percentile of women with normal glucose tolerance (NGT), women with GDM were defined as three subtypes: GDM with the beta cell dysfunction, GDM with the insulin resistance defect or GDM with both traits mentioned above (GDM-mixed). Perinatal outcomes were documented. RESULTS: The levels of prepregnancy and maternal BMI in the GDM-mix group were higher compared to women in the NGT group (23.2 ± 4.0 vs 20.8 ± 3.7 kg/m2, P < 0.001; 24.5 ± 4.3 vs 21.8 ± 3.4 kg/m2, P < 0.001, respectively). Furthermore, women in GDM-mix group more likely to be subjected to LGA (P = 0.008) adverse perinatal outcomes (P = 0.005), although these differences were normalized after adjusting age, prepregnancy and maternal BMI (GDM-mix vs. NGT: P = 0.141 for LGA and P = 0.186 for adverse outcomes). On the other hand, all perinatal outcomes were similar between other two GDM subgroups and NGT group. CONCLUSIONS: Women with GDM display respective characteristics on metabolism disorders and confer discriminating risks of adverse perinatal outcomes because of this heterogeneity.

Arg913Gln of SLC12A3 gene promotes development and progression of end-stage renal disease in Chinese type 2 diabetes mellitus.

Whether the Arg913Gln variation (rs11643718, G/A) of SLC12A3 contributes to diabetic nephropathy (DN) remains controversial. We undertook a case-control study to evaluate the association of the SLC12A3-Arg913Gln variation with the risk of end-stage renal disease (ESRD) in Chinese type 2 diabetes mellitus (T2DM) patients undergoing hemodialysis, and analyzed the genotype-phenotype interaction. Unrelated Chinese T2DM patients (n = 372) with diabetic retinopathy were classified into the non-DN (control) group (n = 151; duration of T2DM >15 years, no signs of renal involvement) and the DN-ESRD group (n = 221; ESRD due to T2DM, receiving hemodialysis). Polymerase chain reaction-direct sequencing was used to genotype the SLC12A3-Arg913Gln variation for all participants. The frequency of the GA+AA genotype in the DN-ESRD group was significantly higher than that of the non-DN group (23.1 vs. 9.9%; adjusted OR 2.2 (95% CI 1.3-4.5), P = 0.019). In the non-DN group, GA+AA carriers had a significantly higher urinary albumin excretion rate (UAER) and diastolic blood pressure compared with GG carriers (both P < 0.05). The SLC12A3-Arg913Gln variation may be associated with increased blood pressure and UAER and, therefore, could be used to predict the development and progression of DN-ESRD in Chinese T2DM patients undergoing hemodialysis.

The angiotensin-I converting enzyme gene I/D variation contributes to end-stage renal disease risk in Chinese patients with type 2 diabetes receiving hemodialysis.

Whether the DD genotype of the angiotensin-I converting enzyme (ACE) I/D variation contributes to end-stage renal disease (ESRD) risk in type 2 diabetes mellitus (T2DM) remains controversial. Differences in study design, case and control definition, sample size and ethnicity may contribute to the discrepancies reported in association studies. We performed a case-control study to evaluate the association of the ACE I/D variation with ESRD risk in Chinese patients with T2DM receiving hemodialysis and analyzed the genotype-phenotype interaction. Unrelated Chinese patients (n = 432) were classified into the non-diabetic nephropathy (DN) control group (n = 222, duration of diabetes >10 years, no signs of renal involvement) and the DN-ESRD group (n = 210; ESRD due to T2DM, receiving hemodialysis). Polymerase chain reaction was used to genotype ACE I/D for all 432 subjects. The frequencies of the ID + DD genotypes were higher in the DN-ESRD group than non-DN control group (65.2 vs. 50.9 %; adjusted OR 1.98 (95 % CI, 1.31-3.00; P = 0.001). In the DN-ESRD group, the DD genotypic subgroup had significantly elevated HbA1c and diastolic blood pressure (DBP) compared to the II subgroup (both P < 0.05). The DD genotype of the ACE I/D variation may be associated with more elevated blood pressure and HbA1c, and therefore may predict the development, progression and severity of DN-ESRD in Chinese patients with T2DM undergoing hemodialysis.

Heating and temperature gradients of lipid bilayer samples induced by RF irradiation in MAS solid-state NMR experiments.

The MAS solid-state NMR has been a powerful technique for studying membrane proteins within the native-like lipid bilayer environment. In general, RF irradiation in MAS NMR experiments can heat and potentially destroy expensive membrane protein samples. However, under practical MAS NMR experimental conditions, detailed characterization of RF heating effect of lipid bilayer samples is still lacking. Herein, using 1 H chemical shift of water for temperature calibration, we systematically study the dependence of RF heating on hydration levels and salt concentrations of three lipids in MAS NMR experiments. Under practical 1 H decoupling conditions used in biological MAS NMR experiments, three lipids show different dependence of RF heating on hydration levels as well as salt concentrations, which are closely associated with the properties of lipids. The maximum temperature elevation of about 10 °C is similar for the three lipids containing 200% hydration, which is much lower than that in static solid-state NMR experiments. The RF heating due to salt is observed to be less than that due to hydration, with a maximum temperature elevation of less than 4 °C in the hydrated samples containing 120 mmol l-1 of salt. Upon RF irradiation, the temperature gradient across the sample is observed to be greatly increased up to 20 °C, as demonstrated by the remarkable broadening of 1 H signal of water. Based on detailed characterization of RF heating effect, we demonstrate that RF heating and temperature gradient can be significantly reduced by decreasing the hydration levels of lipid bilayer samples from 200% to 30%. Copyright © 2016 John Wiley & Sons, Ltd.

The E23K and A190A variations of the KCNJ11 gene are associated with early-onset type 2 diabetes and blood pressure in the Chinese population.

Conflicting associations between define (KCNJ11) variations and susceptibility to late-onset (>40 years old) type 2 diabetes mellitus (T2DM) have been reported in different ethnic groups. We investigated whether the E23K (G→A, rs5219) or A190A (C→T, rs5218) variations in KCNJ11 are associated with early-onset T2DM and blood pressure in the Chinese population. Case-control study of 175 unrelated Chinese patients with early-onset T2DM (age of onset <40 years old) who receive (ins+, n = 57) or do not receive insulin (ins-, n = 118), and 182 non-diabetic control subjects. PCR-direct sequencing was performed to genotype E23K and A190A; the genotypic frequencies and associations with clinical characteristics were analyzed. The genotypic frequencies of E23K-GA+AA were higher and A190A-TT was lower in the early-onset T2DM group, especially the T2D-ins+ group, compared to the non-diabetic control group (p < 0.01 or 0.05, respectively). In non-diabetic subjects, E23K-AA carriers had significantly higher 2 h plasma glucose and lower 2 h insulin than E23K-GG carriers (both p < 0.05). A190A-TT or E23K-GG carriers had higher systolic blood pressure (SBP) than CC or AA carriers in the non-diabetic control and T2DM groups (both p < 0.05). In the T2DM ins+ group, E23K-AA carriers had lower onset age and duration of diabetes and higher BMI than GG carriers, and A190A-TT carriers had higher SBP than CC carriers (all p < 0.05). The E23K-GA or AA genotypes may increase the susceptibility to early-onset T2DM, while A190A-TT may protect against early-onset T2DM. On the other hand the A190A-TT or E23K-GG genotypes may increase the risk of hypertension in the Chinese population.

The Leu72Met polymorphism of the GHRL gene prevents the development of diabetic nephropathy in Chinese patients with type 2 diabetes mellitus.

The preproghrelin (GHRL) Leu72Met polymorphism (rs 696217) is associated with obesity, reduced glucose-induced insulin secretion in healthy or diabetic subjects, and reduced serum creatinine (Scr) levels in type 2 diabetes. We evaluated the association of the Leu72Met polymorphism with measures of insulin sensitivity in non-diabetic control individuals and type 2 diabetics, and whether this variation contributes to the development of diabetic nephropathy (DN) in type 2 diabetes. A case-control study was performed of 291 non-diabetic control subjects and 466 patients with type 2 diabetes, of whom 238 had DN with overt albuminuria (DN group; albuminuric excretion rate [AER] ≥ 300 mg/24 h) and 228 did not have DN, but had diabetes for more than 10 years (non-DN group). Genotyping was performed using a TaqMan PCR assay. The Leu/Leu, Leu/Met, and Met/Met genotype frequencies were significantly different between the non-DN and DN groups (p = 0.011). The frequency of the variant genotypes (Leu/Met, Met/Met) was significantly lower in the DN group than the non-DN group (23.5 vs. 36.0 %, p = 0.003). Met/Met non-diabetic control subjects had lower BMI and Scr levels and higher eGFR level than Leu/Leu or Leu/Met individuals (p < 0.05). Leu/Met and Met/Met type 2 diabetics had significantly lower AER and Scr levels and higher eGFR level than Leu/Leu type 2 diabetics (all p < 0.001). The GHRL Leu72Met polymorphism may help to maintain normal renal function and may protect against the development of DN by reducing albuminuria and improving renal function in Chinese patients with type 2 diabetes.

The molecular structure of Alzheimer ß-amyloid fibrils formed in the presence of phospholipid vesicles.

ß-amyloid (Aß) fibrils are the major species involved in Alzheimer's disease (AD). An atomic-resolution molecular structure of Aß40 fibrils formed in the presence of lipid vesicles was obtained by using magic angle spinning (MAS) solid-state NMR spectroscopy. The fibril structures formed in the presence of the lipid vesicles are remarkably different from those formed in solution. These results provide insights into the molecular mechanism of Aß aggregation in the presence of lipid vesicles.

Exploring the economic toxicity experiences and coping styles of patients with colorectal cancer fistula in Jiangsu province, China: a qualitative study.

OBJECTIVES: In recent years, economic toxicity has significantly affected the physical and mental health as well as the quality of life of patients with colorectal cancer. However, this issue has not garnered adequate attention from healthcare professionals. This study aims to investigate the experiences of economic toxicity and coping strategies among patients with colorectal cancer fistula. The findings are intended to inform the development of suitable and effective intervention programmes to address economic toxicity within this patient population. DESIGN: A descriptive phenomenological approach was employed in this qualitative research, using a semistructured method for data collection and analysis of interview data. Traditional content analysis methods were applied, encompassing coding, categorisation and theme distillation. Data analysis continued until thematic saturation was achieved, with no new themes emerging. SETTING: Nanjing Medical University Lianyungang Clinical Medical College. PARTICIPANTS: A total of 21 patients with colorectal cancer fistula were selected as interview subjects through purposive sampling. The selection took place from May 2022 to May 2023, involving patients during their stay at a tertiary hospital in Lianyungang city, Jiangsu province, China. RESULTS: In total, three pieces and eight subthemes were distilled: subjective feelings (worries about treatment costs, concerns about uncertainty about the future, worries about daily life), coping styles (coping alone, unwillingness to help, prepurchased insurance, dealing with illness, giving up treatment, inability to afford costs) and needs and aspirations (need for health policies, need for social support). CONCLUSIONS: Patients with colorectal cancer fistulae experience economic toxicity, leading to significant impairment in both physical and mental health. Despite employing various coping strategies, healthcare professionals must prioritise addressing the economic toxicity issue in patients. Implementing rational and effective interventions can greatly assist patients in effectively managing economic toxicity.

Selective correlations between aliphatic 13C nuclei in protein solid-state NMR.

Solid-state nuclear magnetic resonance (NMR) is a potent tool for studying the structures and dynamics of insoluble proteins. It starts with signal assignment through multi-dimensional correlation experiments, where the aliphatic 13Cα-13Cß correlation is indispensable for identifying specific residues. However, developing efficient methods for achieving this correlation is a challenge in solid-state NMR. We present a simple band-selective zero-quantum (ZQ) recoupling method, named POST-C4161 (PC4), which enhances 13Cα-13Cß correlations under moderate magic-angle spinning (MAS) conditions. PC4 requires minimal 13C radio-frequency (RF) field and proton decoupling, exhibits high stability against RF variations, and achieves superior efficiency. Comparative tests on various samples, including the formyl-Met-Leu-Phe (fMLF) tripeptide, microcrystalline ß1 immunoglobulin binding domain of protein G (GB1), and membrane protein of mechanosensitive channel of large conductance from Methanosarcina acetivorans (MaMscL), demonstrate that PC4 selectively enhances 13Cα-13Cß correlations by up to 50 % while suppressing unwanted correlations, as compared to the popular dipolar-assisted rotational resonance (DARR). It has addressed the long-standing need for selective 13C-13C correlation methods. We anticipate that this simple but efficient PC4 method will have immediate applications in structural biology by solid-state NMR.

Near-Infrared Stimuli-Responsive Hydrogel Promotes Cell Migration for Accelerated Diabetic Wound Healing.

Diabetic wound healing including diabetic foot ulcers is a major clinical challenge, which could bring an increased level of mortality and morbidity. However, conventional wound dressings exhibit limited healing efficacy due to their lack of active modulation for the healing process. Here, a near-infrared (NIR) stimuli-responsive composite hydrogel dressing with the synergistic effect of both mechanical contraction and epithelial-mesenchymal transition (EMT) was developed to facilitate cell migration and vascularization for diabetic wound healing. In the methacrylated gelatin-based composite hydrogel, N-isopropylacrylamide and polydopamine nanoparticles were incorporated to endow the composite hydrogel with thermosensitive and photothermal properties. Linagliptin (LIN) was loaded into the composite hydrogel, and the drug release rate could be controlled by NIR laser irradiation. NIR-triggered on-demand active contraction of wound area and LIN release for biological stimulation were potentially realized in this responsive system due to the thermally induced sol-gel transition of the composite hydrogel. The release of loaded LIN could effectively promote cell migration by activating EMT and enhancing angiogenesis. In the full-thickness skin defect model, the LIN-loaded composite hydrogel with NIR laser irradiation had the highest wound closure rate as compared with the pure hydrogel and LIN-loaded hydrogel groups. Therefore, this composite hydrogel can serve as an excellent platform for promoting wound healing and will find more practical value in clinical treatment.

Association of sudomotor dysfunction with risk of diabetic retinopathy in patients with type 2 diabetes.

PURPOSE: Sudomotor dysfunction is considered as one of the earliest manifestations of diabetic peripheral neuropathy. We aimed to investigate the association between sudomotor dysfunction non-invasively detected by the SUDOSCAN device and diabetic retinopathy (DR) in patients with type 2 diabetes. METHODS: A total of 2010 patients admitted to a tertiary hospital located in Shanghai were included from March 2020 to September 2023. Sudomotor function was assessed by the SUDOSCAN device, and sudomotor dysfunction was defined as feet electrochemical skin conductance (FESC) <60 µs. Fundus radiography was used for DR assessment, which was graded according to the severity, specifically: (1) non-DR; (2) mild nonproliferative DR (NPDR); (3) moderate NPDR/vision-threatening DR (VTDR). RESULTS: Among the enrolled 2010 patients, 525 patients had sudomotor dysfunction; 648 were diagnosed with DR, which was equivalent to 32.2% of all patients. Patients with sudomotor dysfunction had a significantly higher prevalence of DR, compared to those with normal sudomotor function (40.8% vs. 29.2%, P < 0.05). After controlling for confounding factors including HbA1c, sudomotor dysfunction was significantly associated with any DR (odd ratio [OR] = 1.57, 95% CI 1.26-1.96). When FESC was considered as a continuous variable, the multivariable-adjusted OR of DR was 1.29 (95% CI 1.17-1.42) for per 1-SD decrease in FESC. Furthermore, multinomial logistic regression revealed significant associations between sudomotor dysfunction and all stages of DR (mild NPDR: OR = 1.40, 95% CI 1.11-1.78; moderate NPDR/VTDR: OR = 2.35, 95% CI 1.60-3.46). CONCLUSIONS: Sudomotor dysfunction was significantly associated with DR in patients with type 2 diabetes.

Crystal structure of elongator subcomplex Elp4-6.

Elongator is a multiprotein complex composed of two subcomplexes, Elp1-3 and Elp4-6. Elongator is highly conserved between yeast and humans and plays an important role in RNA polymerase II-mediated transcriptional elongation and many other processes, including cytoskeleton organization, exocytosis, and tRNA modification. Here, we determined the crystal structure of the Elp4-6 subcomplex of yeast. The overall structure of Elp4-6 revealed that Elp6 acts as a bridge to assemble Elp4 and Elp5. Detailed structural and sequence analyses revealed that each subunit in the Elp4-6 subcomplex forms a RecA-ATPase-like fold, although it lacks the key sequence signature of ATPases. Site-directed mutagenesis and biochemical analyses indicated that the Elp4-6 subcomplex can assemble into a hexameric ring-shaped structure in vitro and in vivo. Furthermore, GST pulldown assays showed that the ring-shaped assembly of the Elp4-6 subcomplex is important for its specific histone H3 binding. Our results may shed light on the substrate recognition and assembly of the holo-Elongator complex.

Solid-state NMR structure determination of a membrane protein in E. coli cellular inner membrane.

Structure determination of membrane proteins in native cellular membranes is critical to precisely reveal their structures in physiological conditions. However, it remains challenging for solid-state nuclear magnetic resonance (ssNMR) due to the low sensitivity and high complexity of ssNMR spectra of cellular membranes. Here, we present the structure determination of aquaporin Z (AqpZ) by ssNMR in Escherichia coli inner membranes. To enhance the signal sensitivity of AqpZ, we optimized protein overexpression and removed outer membrane components. To suppress the interference of background proteins, we used a "dual-media" expression approach and antibiotic treatment. Using 1017 distance restraints obtained from two-dimensional 13C-13C spectra based on the complete chemical shift assignments, the 1.7-Å ssNMR structure of AqpZ is determined in E. coli inner membranes. This cellular ssNMR structure determination paves the way for analyzing the atomic structural details for membrane proteins in native cellular membranes.