The review discusses differences between the specific protein interactions with single- and double-stranded RNA molecules using the data on the structure of RNA–protein complexes. Proteins ...interacting with the single-stranded RNAs form contacts with RNA bases, which ensures recognition of specific nucleotide sequences. Formation of such contacts with the double-stranded RNAs is hindered, so that the proteins recognize unique conformations of the RNA spatial structure and interact mainly with the RNA sugar-phosphate backbone.
A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing β-sandwiches, as ...well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500-1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMT
, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β structure-unlike other amyloidogenic proteins-formed without changes in the SMT
secondary structure. SMT
aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMT
amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.
Members of the Lsm protein family are found in all three domains of life: bacteria, archaea, and eukarya. They are involved in numerous processes associated with RNA processing and gene expression ...regulation. A common structural feature of all Lsm family proteins is the presence of the Sm fold consisting of a five-stranded β-sheet and an α-helix at the N-terminus. Heteroheptameric eukaryotic Sm and Lsm proteins participate in the formation of spliceosomes and mRNA decapping. Homohexameric bacterial Lsm protein, Hfq, is involved in the regulation of transcription of different mRNAs by facilitating their interactions with small regulatory RNAs. Furthermore, recently obtained data indicate a new role of Hfq as a ribosome biogenesis factor, as it mediates formation of the productive structure of the 17S rRNA 3′- and 5′-sequences, facilitating their further processing by RNases. Lsm archaeal proteins (SmAPs) form homoheptamers and likely interact with single-stranded uridine-rich RNA elements, although the role of these proteins in archaea is still poorly understood. In this review, we discuss the structural features of the Lsm family proteins from different life domains and their structure–function relationships.
ExuR and UxuR are paralogous proteins belonging to the GntR family of transcriptional regulators. Both are known to control hexuronic acid metabolism in a variety of Gammaproteobacteria but the ...relative impact of each of them is still unclear. Here, we apply 2D difference electrophoresis followed by mass-spectrometry to characterise the changes in the Escherichia coli proteome in response to a uxuR or exuR deletion. Our data clearly show that the effects are different: deletion of uxuR resulted in strongly enhanced expression of D-mannonate dehydratase UxuA and flagellar protein FliC, and in a reduced amount of outer membrane porin OmpF, while the absence of ExuR did not significantly alter the spectrum of detected proteins. Consequently, the physiological roles of proteins predicted as homologs seem to be far from identical. Effects of uxuR deletion were largely dependent on the cultivation conditions: during growth with glucose, UxuA and FliC were dramatically altered, while during growth with glucuronate, activation of both was not so prominent. During the growth with glucose, maximal activation was detected for FliC. This was further confirmed by expression analysis and physiological tests, thus suggesting the involvement of UxuR in the regulation of bacterial motility and biofilm formation.
This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric ...A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5-10 min) formation of large (>2 μm) aggregates. sMyBP-C oligomers formed both at the initial 5-10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7-10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-β quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S-S bonds providing for general stability.
YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: ...its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions.
The structure and the RNA-binding properties of the Lsm protein from
Halobacterium salinarum
have been determined. A distinctive feature of this protein is the presence of a short L4 loop connecting ...the β3 and β4 strands. Since bacterial Lsm proteins (also called Hfq proteins) have a short L4 loop and form hexamers, whereas archaeal Lsm proteins (SmAP) have a long L4 loop and form heptamers, it has been suggested that the length of the L4 loop may affect the quaternary structure of Lsm proteins. Moreover, the L4 loop covers the region of SmAP corresponding to one of the RNA-binding sites in Hfq, and thus can affect the RNA-binding properties of the protein. Our results show that the SmAP from
H. salinarum
forms heptamers and possesses the same RNA-binding properties as homologous proteins with the long L4 loop. Therefore, the length of the L4 does not govern the number of monomers in the protein particles and does not affect the RNA-binding properties of Lsm proteins.
We performed a comparative study of the process of amyloid formation by short homologous peptides with a substitution of aspartate for glutamate in position 2 – VDSWNVLVAG (AspNB) and VESWNVLVAG ...(GluNB) – with unblocked termini. Peptide AspNB (residues 166–175) corresponded to the predicted amyloidogenic region of the protein glucantransferase Bgl2 from the Saccharomyces cerevisiae cell wall. The process of amyloid formation was monitored by fluorescence spectroscopy (FS), electron microscopy (EM), tandem mass spectrometry (TMS), and X-ray diffraction (XD) methods. The experimental study at pH3.0 revealed formation of amyloid fibrils with similar morphology for both peptides. Moreover, we found that the morphology of fibrils made of untreated ammonia peptide is not mentioned in the literature. This morphology resembles snakes lying side by side in the form of a wave without intertwining. Irrespective of the way of the peptide preparation, the rate of fibril formation is higher for AspNB than for GluNB. However, preliminary treatment with ammonia highly affected fibril morphology especially for AspNB. Such treatment allowed us to obtain a lag period during the process of amyloid formation. It showed that the process was nucleation-dependent. With or without treatment, amyloid fibrils consisted of ring-like oligomers with the diameter of about 6nm packed either directly ring-to-ring or ring-on-ring with a slight shift. We also proposed the molecular structure of amyloid fibrils for two studied peptides.
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•We conclude on the basis of EM, FS and XD data that fibrils formed of AspNB peptide and its analog GluNB are amyloids.•We have found the morphology of fibrils not mentioned in the literature.•The rate of fibril formation is higher for AspNB than for GluNB.•We have found that amyloid fibrils consist of ring-like oligomers.•We propose the molecular structure of amyloid fibrils for two studied peptides.
The aim of this study was to investigate the process of amyloidogenesis of amyloid-β (Aβ)42 peptide, by means of fluorescence spectroscopy, electron microscopy, X-ray diffraction, and mass ...spectrometry. It has been repeatedly reported in the literature that the process of fibril formation by Aβ42 peptide depends considerably not only upon the specific conditions (ionic conditions, pH, temperature, mixing, etc.), as well as the manufacturing route (synthetic or recombinant), but also on the methods of synthesis and purification. We have, for the first time, systematically analyzed samples of Aβ42 peptide supplied by five different companies (Anaspec, Invitrogen, Enzo, Sigma-Aldrich, and SynthAssist) and obtained evidence of significant variability, including lot to lot variations. All studied samples formed amyloid-like fibrils at pH3-6, and the fibrils contained cross-β structures. Samples from Anaspec, Invitrogen, and Enzo formed one particular type of amyloid-like fibrils, while the samples from Sigma-Aldrich and SynthAssist formed another distinct type of fibrils. The observed polymorphism emphasizes the capacity of the Aβ42 peptide to act as a prion agent with varying structural characteristics. The presented data have allowed us to propose a possible mechanism of formation of amyloid-like fibrils.
Transcription factors play a crucial role in control of life of a bacterial cell, working as switchers to a different life style or pathogenicity. To reconstruct the network of regulatory events ...taking place in changing growth conditions, we need to know regulons of as many transcription factors as possible, and motifs recognized by them. Experimentally this can be attained via ChIP-seq in vivo, SELEX and DNAse I footprinting in vitro. All these approaches require large amounts of purified proteins. However, overproduction of transcription factors leading to their extensive binding to the regulatory elements on the DNA make them toxic to a bacterial cell thus significantly complicating production of a soluble protein. Here, on the example of three regulators from Escherichia coli, UxuR, ExuR, and LeuO, we show that stable production of toxic transcription factors in a soluble fraction can be significantly enhanced by holding the expression of a recombinant protein back at the early stages of bacterial growth. This can be achieved by cloning genes together with their regulatory regions containing repressor sites, with subsequent growth in a very rich media where activity of excessive regulators is not crucial, followed by induction with a very low concentration of an inducer. Schemes of further purification of these proteins were developed, and functional activity was confirmed.
•Vectors were constructed and conditions were found to improve the yield of proteins, potentially toxic to a bacterial cell.•Using this approach, three transcription regulators from E. coli, ExuR, LeuO, and UxuR, were successfully overproduced.•Purification scheme to obtain significant amounts of electrophoretically homogenous UxuR and ExuR was developed.•The functionality of the purified proteins was confirmed by EMSA and ChIP experiments.
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