Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents

G-protein coupled receptors (GPCRs) constitute the largest family of intercellular signaling molecules and are estimated to be the target of more than 50% of all modern drugs. As with most integral membrane proteins (IMPs), a major bottleneck in the structural and biochemical analysis of GPCRs is their expression by conventional expression systems. Cell-free (CF) expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs. However, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. Here, we report that using CF expression with the help of a fructose-based polymer, NV10 polymer (NVoy), we obtained preparative amounts of homogeneous GPCRs from the three GPCR families. We demonstrate that two GPCR B family members, corticotrophin-releasing factor receptors 1 and 2β are not only solubilized in NVoy but also have functional ligand-binding characteristics with different agonists and antagonists in a detergent-free environment as well. Our findings open new possibilities for functional and structural studies of GPCRs and IMPs in general.     

New data on birds and mammals population of the Sette-Daban Mountain Ridge

Original materials on the fauna of the northern macroslope of the Sette-Daban Mountain Ridge are presented. Birds and mammals population in different habitats in the basin of the Allakh-Yun River are characterized.     

Genome‐wide SNP analysis unveils genetic structure and phylogeographic history of snow sheep (Ovis nivicola) populations inhabiting the Verkhoyansk Mountains and Momsky Ridge (northeastern Siberia)

Insights into the genetic characteristics of a species provide important information for wildlife conservation programs. Here, we used the OvineSNP50 BeadChip developed for domestic sheep to examine population structure and evaluate genetic diversity of snow sheep (Ovis nivicola) inhabiting Verkhoyansk Range and Momsky Ridge. A total of 1,121 polymorphic SNPs were used to test 80 specimens representing five populations, including four populations of the Verkhoyansk Mountain chain: Kharaulakh Ridge–Tiksi Bay (TIK, n = 22), Orulgan Ridge (ORU, n = 22), the central part of Verkhoyansk Range (VER, n = 15), Suntar‐Khayata Ridge (SKH, n = 13), and Momsky Ridge (MOM, n = 8). We showed that the studied populations were genetically structured according to a geographic pattern. Pairwise F_ST values ranged from 0.044 to 0.205. Admixture analysis identified K = 2 as the most likely number of ancestral populations. A Neighbor‐Net tree showed that TIK was an isolated group related to the main network through ORU. TreeMix analysis revealed that TIK and MOM originated from two different ancestral populations and detected gene flow from MOM to ORU. This was supported by the f3 statistic, which showed that ORU is an admixed population with TIK and MOM/SKH heritage. Genetic diversity in the studied groups was increasing southward. Minimum values of observed (Ho) and expected (He) heterozygosity and allelic richness (Ar) were observed in the most northern population—TIK, and maximum values were observed in the most southern population—SKH. Thus, our results revealed clear genetic structure in the studied populations of snow sheep and showed that TIK has a different origin from MOM, SKH, and VER even though they are conventionally considered a single subspecies known as Yakut snow sheep (Ovis nivicola lydekkeri). Most likely, TIK was an isolated group during the Late Pleistocene glaciations of Verkhoyansk Range.     

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation

Abstract  Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.     

NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes

Background  

Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization.     

Drug screening strategy for human membrane proteins: From NMR protein backbone structure to in silica- and NMR-screened hits

About 8000 genes encode membrane proteins in the human genome. The information about their druggability will be very useful to facilitate drug discovery and development. The main problem, however, consists of limited structural and functional information about these proteins because they are difficult to produce biochemically and to study. In this paper we describe the strategy that combines Cell-free protein expression, NMR spectroscopy, and molecular DYnamics simulation (CNDY) techniques. Results of a pilot CNDY experiment provide us with a guiding light towards expedited identification of the hit compounds against a new uncharacterized membrane protein as a potentially druggable target. These hits can then be further characterized and optimized to develop the initial lead compound quicker. We illustrate such “omics” approach for drug discovery with the CNDY strategy applied to two example proteins: hypoxia-induced genes HIGD1A and HIGD1B.     

Genetic characteristics of Kodar snow sheep using SNP markers

For the first time nuclear DNA polymorphisms were studied in Kodar snow sheep (Ovis nivicola kodarensis, KOD). KOD is a representative of a small isolated population of Asian snow sheep (Ovis nivicola Eschscholtz, 1829) inhabiting the Kodar Ridge (Irkutsk oblast, Transbaikal krai). We compared KOD with the geographically closest Yakut subspecies (Ovis nivicola lydekkeri). Genome-wide study of single-nucleotide polymorphisms (SNPs) was performed using the Illumina OvineSNP50 BeadChip (Illumina, United States). The final set of markers for analysis included 1030 SNPs. We found that Kodar snow sheep had almost 10 times lower level of genetic diversity evaluated by multilocus heterozygosity—MLH (0.027 for KOD vs 0.215–0.270 for individuals of Yakut subspecies) and standardized MLH—stMLH (0.116 against 0.910–1.147). The results of multidimensional scaling (MDS), Nei distances calculations (D_N) and STRUCTURE analysis showed a clear genetic differentiation of Kodar snow sheep from Yakut subspecies. Our data is the first step to understanding the demographic history of the original Kodar population of snow sheep.     

Genomic patterns in the widespread Eurasian lynx shaped by Late Quaternary climatic fluctuations and anthropogenic impacts

Disentangling the contribution of long‐term evolutionary processes and recent anthropogenic impacts to current genetic patterns of wildlife species is key to assessing genetic risks and designing conservation strategies. Here, we used 80 whole nuclear genomes and 96 mitogenomes from populations of the Eurasian lynx covering a range of conservation statuses, climatic zones and subspecies across Eurasia to infer the demographic history, reconstruct genetic patterns, and discuss the influence of long‐term isolation and/or more recent human‐driven changes. Our results show that Eurasian lynx populations shared a common history until 100,000 years ago, when Asian and European populations started to diverge and both entered a period of continuous and widespread decline, with western populations, except Kirov, maintaining lower effective sizes than eastern populations. Population declines and increased isolation in more recent times probably drove the genetic differentiation between geographically and ecologically close westernmost European populations. By contrast, and despite the wide range of habitats covered, populations are quite homogeneous genetically across the Asian range, showing a pattern of isolation by distance and providing little genetic support for the several proposed subspecies. Mitogenomic and nuclear divergences and population declines starting during the Late Pleistocene can be mostly attributed to climatic fluctuations and early human influence, but the widespread and sustained decline since the Holocene is more probably the consequence of anthropogenic impacts which intensified in recent centuries, especially in western Europe. Genetic erosion in isolated European populations and lack of evidence for long‐term isolation argue for the restoration of lost population connectivity.     

Whole genome SNP scanning of snow sheep (<Emphasis Type="Italic">Ovis nivicola</Emphasis>)

This is the first report performing the whole genome SNP scanning of snow sheep (Ovis nivicola). Samples of snow sheep (n = 18) collected in six different regions of the Republic of Sakha (Yakutia) from 64° to 71° N. For SNP genotyping, we applied Ovine 50K SNP BeadChip (Illumina, United States), designed for domestic sheep. The total number of genotyped SNPs (call rate 90%) was 47796 (88.1% of total SNPs), wherein 1006 SNPs were polymorphic (2.1%). Principal component analysis (PCA) showed the clear differentiation within the species O. nivicola: studied individuals were distributed among five distinct arrays corresponding to the geographical locations of sampling points. Our results demonstrate that the DNA chip designed for domestic sheep can be successfully used to study the allele pool and the genetic structure of snow sheep populations.