Biochemistry (Moscow) (v.81, #1)
Structure and noncanonical activities of coat proteins of helical plant viruses by V. V. Makarov; N. O. Kalinina (1-18).
The main function of virus coat protein is formation of the capsid that protects the virus genome against degradation. However, besides the structural function, coat proteins have many additional important activities in the infection cycle of the virus and in the defense response of host plants to viral infection. This review focuses on noncanonical functions of coat proteins of helical RNA-containing plant viruses with positive genome polarity. Analysis of data on the structural organization of coat proteins of helical viruses has demonstrated that the presence of intrinsically disordered regions within the protein structure plays an important role in implementation of nonstructural functions and largely determines the multifunctionality of coat proteins.
Keywords: helical plant viruses; coat protein; structure; intrinsically disordered regions; noncanonical functions
Role of protein L25 and its contact with protein L16 in maintaining the active state of Escherichia coli ribosomes in vivo by A. Y. Anikaev; A. B. Isaev; A. V. Korobeinikova; M. B. Garber; G. M. Gongadze (19-27).
A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.
Keywords: 5S rRNA-binding protein L25; ribosomal protein L16; RNA–protein interactions; ribosome; translation; Escherichia coli
Calponin-like protein from mussel smooth muscle is a competitive inhibitor of actomyosin ATPase by V. V. Sirenko; A. V. Dobrzhanskaya; N. S. Shelud’ko; Y. S. Borovikov (28-33).
The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters V max and K ATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in K ATPase value and to a lesser extent the decrease in V max, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of V max and K ATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.
Keywords: calponin-like protein; mussel; ATPase; V max ; K ATPase ; actomyosin; S1
Proteomic analysis of Escherichia coli protein fractions resistant to solubilization by ionic detergents by K. S. Antonets; K. V. Volkov; A. L. Maltseva; L. M. Arshakian; A. P. Galkin; A. A. Nizhnikov (34-46).
Amyloids are protein fibrils adopting structure of cross-beta spine exhibiting either pathogenic or functionally significant properties. In prokaryotes, there are several groups of functional amyloids; however, all of them were identified by specialized approaches that do not reveal all cellular amyloids. Here, using our previously developed PSIA (Proteomic Screening and Identification of Amyloids) approach, we have conducted a proteomic screening for candidates for novel amyloid-forming proteins in Escherichia coli as one of the most important model organisms and biotechnological objects. As a result, we identified 61 proteins in fractions resistant to treatment with ionic detergents. We found that a fraction of proteins bearing potentially amyloidogenic regions predicted by bioinformatics algorithms was 3-5-fold more abundant among the identified proteins compared to those observed in the entire E. coli proteome. Almost all identified proteins contained potentially amyloidogenic regions, and four of them (BcsC, MukB, YfbK, and YghJ) have asparagineand glutamine-rich regions underlying a crucial feature of many known amyloids. In this study, we demonstrate for the first time that at the proteome level there is a correlation between experimentally demonstrated detergent-resistance of proteins and potentially amyloidogenic regions predicted by bioinformatics approaches. The data obtained enable further comprehensive characterization of entirety of amyloids (or amyloidome) in bacterial cells.
Keywords: amyloid; prion; E. coli ; fimbria; curlin; amyloidomics
Recombinant phospholipase A1 of the outer membrane of psychrotrophic Yersinia pseudotuberculosis: Expression, purification, and characterization by S. I. Bakholdina; N. M. Tischenko; E. V. Sidorin; M. P. Isaeva; G. N. Likhatskaya; P. S. Dmitrenok; N. Yu. Kim; O. V. Chernikov; T. F. Solov’eva (47-57).
The pldA gene encoding membrane-bound phospholipase A1 of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A1 (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A1 was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A1 were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A1 from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.
Keywords: Yersinia pseudotuberculosis ; phospholipase A1 ; recombinant protein; spatial structure modeling
Investigation of stability of photosynthetic reaction center and quantum dot hybrid films by E. P. Lukashev; P. P. Knox; I. P. Oleinikov; N. Kh. Seifullina; N. P. Grishanova (58-63).
The efficiency of interaction (efficiency of energy transfer) between various quantum dots (QDs) and photosynthetic reaction centers (RCs) from the purple bacterium Rhodobacter sphaeroides and conditions of long-term stability of functioning of such hybrid complexes in film preparations were investigated. It was found that dry films containing RCs and QDs and maintained at atmospheric humidity are capable to keep their functional activity for at least some months as judging by results of measurement of their spectral characteristics, efficiency of energy transfer from QDs to RCs, and RC electron-transport activity. Addition of trehalose to the films giving them still greater stability is especially expressed for films maintained at low humidity. These stable hybrid film structures are promising for further biotechnological studies for developing new phototransformation devices.
Keywords: photosynthetic reaction center; quantum dots; energy transfer