Probing protein side chain dynamics via 13C NMR relaxation

Protein side chain dynamics is associated with protein stability, folding, and intermolecular interactions. Detailed dynamics information is crucial for the understanding of protein function and biochemical and biophysical properties, which can be obtained using NMR relaxation techniques. In this review, (13)C relaxation of methine, methylene and methyl groups with and without (1)H decoupling are described briefly for a better understanding of how spin relaxation is associated with motional (dynamics) parameters. Developments in the measurement and interpretation of (13)C auto-relaxation and cross-correlated relaxation data are presented too. Finally, recent progress in the use of (13)C relaxation to probe the dynamics of protein side chains is detailed mainly for the dynamics of non-deuterated proteins on picoseconds-nanosecond timescales.     

Rigidifying Acyl Carrier Protein Domain in Iterative Type I PKS CalE8 Does Not Affect Its Function

Acyl carrier protein (ACP) domains shuttle acyl intermediates among the catalytic domains of multidomain type I fatty acid synthase and polyketide synthase (PKS) systems. It is believed that the unique function of ACPs is associated with their dynamic property, but it remains to be fully elucidated what type of protein dynamics is critical for the shuttling domain. Using NMR techniques, we found that the ACP domain of iterative type I PKS CalE8 from Micromonospora echinospora is highly dynamic on the millisecond-second timescale. Introduction of an interhelical disulfide linkage in the ACP domain suppresses the dynamics on the millisecond-second timescale and reduces the mobility on the picosecond-nanosecond timescale. We demonstrate that the full-length PKS is fully functional upon rigidification of the ACP domain, suggesting that although the flexibility of the disordered terminal linkers may be important for the function of the ACP domain, the internal dynamics of the helical regions is not critical for that function.     

The fungal community and its interaction with the concentration of short-chain fatty acids in the faeces of Chenghua,Yorkshire and Tibetan pigs

Despite their important roles in host nutrition and metabolism, and potential to cause disease, our knowledge of the fungal community in the mammalian gut is quite limited. To date, diversity and composition of fungi in swine gut still remains unknown. Therefore, the first internal transcribed spacer of fungi in faecal samples from three breeds of pigs (10 pigs for each breed) was sequenced based on an Illumina HiSeq 2500 platform, and the relationship between the fungal community and the concentrations of main short-chain fatty acids (SCFAs) was also analysed. Results indicated that Chenghua (local, higher body fat rate), Yorkshire (foreign, higher lean meat and growth rate) and Tibetan (plateau, stronger disease resistance) pigs harboured distinct fungal community. The Basidiomycota and Ascomycota presented as the two predominant phyla, with Loreleia, Russula and Candida as the top three genera in all samples. Network analysis revealed a total of 35 correlations among different fungal genera, with 27 (77.14%) positive and 8 (22.86%) negative pairwise interactions. Canonical correspondence analysis suggested that fungi in the faeces of pigs were more correlated to the concentration of acetate and butyrate rather than propionate. Spearman’s correlation further showed that Tomentella was positively correlated to both acetate and butyrate, and Loreleia was positively correlated to propionate (P < 0.05), while Nephroma and Taiwanofungus were negatively correlated to acetate and propionate (P < 0.05). These findings expanded our knowledge on the intestinal fungi in pigs with different genotypes and phenotypes, indicating that fungi may play an indispensable role during the metabolism of host and the maintenance of intestinal health. The cross-feeding between fungi and other microorganisms may be crucial during the digestion of dietary carbohydrates and the associated physiological processes, which is worthy to be further studied.     

MicroRNA-27a promotes myoblast proliferation by targeting myostatin

MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3'UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.     

Improved lineshape and sensitivity in the HNCO-family of triple resonance experiments

A simple scheme is presented for the suppression of dispersive contributions to cross peaks in HNCO-type spectra where the ¹⁵N chemical shift is recorded in a constant-time manner immediately prior to the transfer from ¹⁵ N to ¹HN at the end of the sequence. These dispersive contributions arise when the delay for refocusing the ¹⁵N-¹³CO one-bond coupling is set to less than 0.5/¹J_N,CO and when ² J_HN,CO 0. Improvements in sensitivity in ¹HN detected experiments recorded on ¹⁵ N,¹³C-labeled samples can be realized by application of ¹³CO/¹³ decoupling during acquisition. Sensitivity gains on the order of 15% and 5% have been obtained for an SH3 domain (62 residues) and maltose binding protein (370 residues), respectively.     

Chronic Glucocorticoid Exposure-Induced Epididymal Adiposity Is Associated with Mitochondrial Dysfunction in White Adipose Tissue of Male C57BL/6J Mice

Prolonged and excessive glucocorticoids (GC) exposure resulted from Cushing''s syndrome or GC therapy develops central obesity. Moreover, mitochondria are crucial in adipose energy homeostasis. Thus, we tested the hypothesis that mitochondrial dysfunction may contribute to chronic GC exposure-induced epididymal adiposity in the present study. A total of thirty-six 5-week-old male C57BL/6J mice (∼20 g) were administrated with 100 µg/ml corticosterone (CORT) or vehicle through drinking water for 4 weeks. Chronic CORT exposure mildly decreased body weight without altering food and water intake in mice. The epididymal fat accumulation was increased, but adipocyte size was decreased by CORT. CORT also increased plasma CORT, insulin, leptin, and fibroblast growth factor 21 concentrations as measured by RIA or ELISA. Interestingly, CORT increased plasma levels of triacylglycerols and nonesterified fatty acids, and up-regulated the expression of both lipolytic and lipogenic genes as determined by real-time RT-PCR. Furthermore, CORT impaired mitochondrial biogenesis and oxidative function in epididymal WAT. The reactive oxygen species production was increased and the activities of anti-oxidative enzymes were reduced by CORT treatment as well. Taken together, these findings reveal that chronic CORT administration-induced epididymal adiposity is, at least in part, associated with mitochondrial dysfunction in mouse epididymal white adipose tissue.     

Development of a method based on surface enhanced laser desorption and ionization time of flight mass spectrometry for rapid identification of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A method based on surface enhanced laser desorption and ionization time of flight mass spectrometry (SELDI-TOF MS) was developed for the rapid identification of Klebsiella pneumoniae by directly applying bacterial colonies without further protein extraction. A total of 40 K. pneumoniae and 114 other related microorganisms isolated clinically were analyzed by SELDI-TOF MS. An identification model for K. pneumoniae was established by artificial neural networks (ANNs) with classification accuracy of 100%. The model was blindly tested with 43 K. pneumoniae and 53 control bacteria again. The results showed that the model was successful with accuracy of 96.9%, sensitivity of 100% and specificity of 943%. This strategy is potential for rapid identification of K. pneumoniae.     

Different messenger RNA expression for the antimicrobial peptides β-defensins between Meishan and crossbred pigs

β-defensins are cysteine-rich endogenously produced antimicrobial peptides that play an important role in innate immunity. In this study, the expressions of genes porcine β-defensins-1(pBD-1), pBD-2 and pBD-3 were determined using real-time PCR for Chinese Meishan pigs and Crossbred (Duroc × Yorkshire × Landrace) pigs of 7 days old in various tissues. The results showed that expressions of pBD-1, 2 and 3 of Meishan pigs in most tissues were higher than those of crossbred pigs and main expression sites for pBD-1 and pBD-3 were tongue and oral mucosa in two varieties of pigs, whereas pBD-2 of crossbred pig was mainly expressed in kidney and liver, and pBD-2 of Meishan pigs mainly in tongue and oral mucosa. The higher expression of pBDs might be the reason of Meishan pigs has higher immunity and disease resistance. The mechanisms of this need a further research.     

SCAssign: a sparky extension for the NMR resonance assignment of aliphatic side-chains of uniformly 13C,15N-labeled large proteins

SCAssign (side-chain assignment) is a Sparky extension written in Python to assist the NMR resonance assignment of aliphatic side-chains of uniformly (13)C,(15)N-labeled large proteins. It is based on a general strategy recently developed in our laboratory that makes use of 4D (13)C,(15)N-edited NOESY, 3D MQ-(H)CC(m)H(m)-TOCSY, and prior backbone assignments. The program runs on all operating systems for which Sparky is available, and is easy to install, setup and use. Not only can it accelerate the assignment process, it also allows assignments of weak NOEs in 4D NOESY, which used to be very difficult with manual approach. AVAILABILITY: The program, in the form of source code, is provided as free download at http://yangdw.science.nus.edu.sg/SCAssign. The website also contains installation guide, user manual and demonstrations recorded in Flash.