Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca²⁺ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca²⁺ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein’s dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.     

Proposal of Ensifer psoraleae sp. nov., Ensifer sesbaniae sp. nov., Ensifer morelense comb. nov. and Ensifer americanum comb. nov

In a survey of rhizobia associated with the native legumes in Yunnan Province, China, seven and nine strains isolated from the root nodules of Psoralea corylifolia, Sesbania cannabina and Medicago lupulina were respectively classified into the novel genomic species groups I and II in the genus Ensifer (former Sinorhizobium) based on the sequence analyses of the 16S rRNA gene. Analyses of concatenated housekeeping genes (atpD, recA and glnII) further revealed that they were distinct lineages in the genus, and group I was most similar to Ensifer terangae and Ensifer garamanticus (both with 94.2% similarity), while group II was most similar to Ensifer adhaerens (94.0%). These groups could be distinguished from closely related species by DNA–DNA relatedness, MALID-TOF MS, cellular fatty acid profiles and a series of phenotypic characters. Therefore, two novel species were proposed: Ensifer psoraleae sp. nov. (seven strains, type strain CCBAU 65732^T = LMG 26835^T = HAMBI 3286^T) and Ensifer sesbaniae sp. nov. (nine strains, type strain CCBAU 65729^T = LMG 26833^T = HAMBI 3287^T). They had a DNA G + C mol% (T_m) of 58.9 and 60.4, respectively. Both of the type strains formed effective nodules on common bean (Phaseolus vulgaris) and their hosts of origin. In addition, the previously described species Sinorhizobium morelense and Sinorhizobium americanum were renamed as Ensifer morelense comb. nov. and Ensifer americanum comb. nov. according to the accumulated data from different studies.     

Serum proteomic profiling for the early diagnosis of colorectal cancer

No ideal serum biomarker currently exists for the early diagnosis of colorectal cancer (CRC). Magnetic bead‐based fractionation coupled with MALDI‐TOF MS was used to screen serum samples from CRC patients, healthy controls, and other cancer patients. A diagnostic model with five proteomic features (m/z 1778.97, 1866.16, 1934.65, 2022.46, and 4588.53) was generated using Fisher algorithm with best performance. The Fisher‐based model could discriminate CRC patients from the controls with 100% (46/46) sensitivity and 100% (35/35) specificity in the training set, 95.6% (43/45) sensitivity and 83.3% (35/42) specificity in the test set. We further validated the model with 94.4% (254/269) sensitivity and 75.5% (83/110) specificity in the external independent group. In other cancers group, the Fisher‐based model classified 25 of 46 samples (54.3%) as positive and the other 21 as negative. With FT‐ICR‐MS, the proteomic features of m/z 1778.97, 1866.16, 1934.65, and 2022.46, of which intensities decreased significantly in CRC, were identified as fragments of complement C3f. Therefore, the Fisher‐based model containing five proteomic features was able to effectively differentiate CRC patients from healthy controls and other cancers with a high sensitivity and specificity, and may be CRC‐specific. Serum complement C3f, which was significantly decreased in CRC group, may be relevant to the incidence of CRC. J. Cell. Biochem. 114: 448–455, 2013. © 2012 Wiley Periodicals, Inc.     

Arabidopsis CDPK6 phosphorylates ADF1 at N-terminal serine 6 predominantly

Key message

We found that Arabidopsis AtADF1 was phosphorylated by AtCDPK6 at serine 6 predominantly and the phosphoregulation plays a key role in the regulation of ADF1-mediated depolymerization of actin filaments.

Abstract

Since actin-depolymerizing factor (ADF) is highly conserved among eukaryotes, it is one of the key modulators for actin organization. In plants, ADF is directly involved in the depolymerization of actin filaments, and therefore important for F-actin-dependent cellular activities. The activity of ADF is tightly controlled through a number of molecular mechanisms, including phosphorylation-mediated inactivation of ADF. To investigate Arabidopsis ADF1 phosphoregulation, we generated AtADF1 phosphorylation site-specific mutants. Using transient expression and stable transgenic approaches, we analyzed the ADF1 phosphorylation mutants in the regulation of actin filament organizations in plant cells. By in vitro phosphorylation assay, we showed that AtADF1 is phosphorylated by AtCDPK6 at serine 6 predominantly. Chemically induced expression of AtCDPK6 can negatively regulate the wild-type AtADF1 in depolymerizing actin filaments, but not those of the mutants AtADF1(S6A) and AtADF1(S6D). These results demonstrate a regulatory function of Arabidopsis CDPK6 in the N-terminal phosphorylation of AtADF1.     

食品级惰性粉对三种储藏物害虫生长发育的影响

应用粉剂药膜法,一种食品级惰性粉——4号粉相应剂量处理赤拟谷盗Tribolium castaneum(Herbst)、烟草甲Lasioderma serricorne(Fabricius)、锯谷盗Oryzaephilus surinamensis(Linnaeus)的卵和1龄幼虫,赤拟谷盗、锯谷盗、烟草甲的卵平均孵化率均在97%以上,处理后卵孵化的1龄幼虫平均死亡率均在96%以上;处理3种害虫的1龄幼虫其平均死亡率均在99%以上。结果表明食品级惰性粉对3种试虫卵的孵化率几乎没有影响,但对其1龄幼虫有较好的防治效果。通过混粮法应用正交试验研究发现,惰性粉剂量和小麦含水量对赤拟谷盗F1代防治效果均有显著影响。当小麦处于安全水分时(含水量12%¹4%),10014%),100¹50 mg/kg 4号粉能有效防治赤拟谷盗F1代。     

Molecular cloning and characterization of a Brassica juncea yellow stripe-like gene, BjYSL7, whose overexpression increases heavy metal tolerance of tobacco

Key message

BjYSL7 encodes a plasma-localized metal–NA transporter and has transport Fe(II)–NA complexes activity. BjYSL7 is involved in the transport of Cd and Ni from roots to shoots.

Abstract

Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. In this study, we isolated a novel member of the yellow stripe-like (YSL) gene family BjYSL7 from the hyperaccumulator Brassica juncea. BjYSL7 is composed of 688 amino acids with 12 putative transmembrane domains and is over 90 % identical to TcYSL7 and AtYSL7. Real-time PCR analysis revealed that BjYSL7 mRNA was mainly expressed in the stem under normal condition. The expression of BjYSL7 was found to be up-regulated by 127.1-, 12.7-, and 3.4-fold in roots and 6.5-, 4.3-, and 2.8-fold in shoots under FeSO₄, NiCl₂, and CdCl₂ stresses, respectively. We have demonstrated that BjYSL7 is a Fe(II)–NA influx transporter by yeast functional complementation. Moreover, a BjYSL7::enhanced green fluorescent protein (EGFP) fusion localized to the plasma membrane of onion epidermal cells. The BjYSL7-overexpressing transgenic tobacco plants exhibited longer root lengths, lower relative inhibition rate of lengths and superior root hair development compared to that of wild-type (WT) plants in the presence of CdCl₂ and NiCl₂. Furthermore, the concentrations of Cd and Ni in shoots of BjYSL7-overexpressing plants are significantly higher than that of WT plants. Compared with WT plants, BjYSL7-overexpressing plants exhibited Fe concentrations that were higher in the shoots and seeds and lower in the roots. Taken together, these results suggest that BjYSL7 might be involved in the transport of Fe, Cd and Ni to the shoot and improving heavy metal resistance in plants.