Recognition of errors in three-dimensional structures of proteins

A major problem in the determination of the three-dimensional structure of proteins concerns the quality of the structural models obtained from the interpretation of experimental data. New developments in X-ray crystallography and nuclear magnetic resonance spectroscopy have acceleratedd the process of structure determination and the biological community is confronted with a steadily increasing number of experimentally determined protein folds. However, in the recent past several experimentally determined protein structures have been proven to contain major errors, indicating that in some cases the interpretation of experimental data is difficult and may yield incorrect models. Such problems can be avoided when computational methods are employed which complement experimental structure determinations. A prerequisite of such computational tools is that they are independent of the parameters obtained from a particular experiment. In addition such techniques are able to support and accelerate experimental structure determinations. Here we present techniques based on knowledge based mean fields which can be used to judge the quality of protein folds. The methods can be used to identify misfolded structures as well as faulty parts of structural models. The techniques are even applicable in cases where only the C_α trace of a protein conformation is available. The capabilities of the technique are demonstrated using correct and incorrect protein folds. © 1993 Wiley-Liss, Inc.     

Synthesis of (+)-(R)- and (−)-(S)-5-hydroxy-2-methyl-2-dipropylaminotetralin: Effects on rat hippocampal output of 5-HT, 5-HIAA,and DOPAC as determined by in vivo microdialysis

Racemic 5-methoxy-2-methyl-2-dipropylaminotetralin ( 3 ) has been prepared by a short synthetic route, in which the N,N-dipropyliminium perchlorate of 5-methoxy-2-tetralone ( 4 ) is a key intermediate. Racemic 3 was resolved by crystallization of the corresponding diastereomeric di-p-toluoyltartrates. The enantiomeric excess (%ee) of the phenolic derivatives of (+)-(R)- and (?)-(S)-3 [(+)-(R)- and (?)-(S)-2] was determined by ¹HNMR spectroscopic analysis of the corresponding diastereomeric (?)-(R)-1,1′-binaphthyl-2,2′-diylphosphoric acid salts utilizing ¹³C satellites. X-ray crystallography established the absolute configuration of (?)-(S)-2 · HCl. The enantiomers of 2 were tested for hippocampal output of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and dihydroxyphenylacetic acid in rats by use of in vivo microdialysis. The (?)-(S)-enantiomer appeared to affect 5-HT-turnover, whereas (+)-(R)- 2 was inactive. Results obtained provide support for the previously reported hypothesis that the inactivity of (?)-(S)- 2 at central DA receptors is caused by the steric bulk of the C(2)-methyl group. This makes it possible to define a “DA D2 receptor essential volume.” © 1993 Wiley-Liss, Inc.     

Identification of a genomic region linked with sex determination of Undaria pinnatifida (Alariaceae) through genomic resequencing and genetic linkage analyses of a segregating gametophyte family

Different from the traditional knowledge about kelp, three sexual phenotypes (female, male, and monoecious) exist in the haploid gametophytes of Undaria pinnatifida. However, the sex-determining mechanisms remain unknown. Genetic linkage mapping is an efficient tool to identify sex-linked regions. In the present study, we resequenced a segregating gametophyte family based on the male genome of U. pinnatifida. A high-density genetic linkage map was constructed using 9887 SNPs, with an average distance of 0.41 cM between adjacent SNPs. On the basis of this genetic map and using the composite interval mapping method, we identified 62 SNPs significantly linked with the sexual phenotype. They were located at a position of 67.67 cM on the linkage group 23, corresponding to a physical range of 14.67 Mbp on the HiC_Scaffold_23 of the genome. Reanalysis of the previous specific length amplified fragment sequencing data according to the reference genome led to the identification of a sex-linked genomic region that encompassed the above-mentioned 14.67 Mbp region. Hence, this overlapped genomic range was likely the sex-determining region. Within this region, 129 genes were retrieved and 39 of them were annotated with explicit function, including the potential male sex-determining gene-encoding high mobility group (HMG) domain protein. Relative expression analysis of the HMG gene showed that its expression was higher in male gametophytes during the vegetative phase and monoecious gametophytes during both the vegetative and gametogenesis phases, but significantly lower in male gametophytes during the gametogenesis phase. These results provide a foundation for deciphering the sex-determining mechanism of U. pinnatifida.     

单链抗体展示系统研究进展

单链抗体(single chain antibody fragment,scFv)是由抗体重链可变区(variable region of heavy chain,VH)和轻链可变区(variable region of light chain,VL)通过柔性短肽连接组成的小分子,是具有完整抗原结合活性的最小功能片段,包含抗体识别及抗原结合部位。相比于其他抗体,scFv具有分子量小、穿透性强、免疫原性弱、易构建表达等优点。目前,scFv最常用的展示系统主要有噬菌体展示系统、核糖体展示系统、mRNA展示系统、酵母细胞表面展示系统和哺乳动物细胞展示系统等。近年来,随着scFv在医学、生物学、食品安全学等领域的发展,使得其在生物合成和应用研究方面备受关注。本文对近年来scFv展示系统的研究进展作一综述,以期为scFv的筛选及应用提供理论基础。     

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.     

GENETIC EVIDENCE FOR MULTIPLE ORIGINS OF DIOECY IN THE HAWAIIAN SHRUB WIKSTROEMIA (THYMELAEACEAE)

Dioecy is unusually common in the Hawaiian Islands, yet little is known about the evolutionary biology of this breeding system. A native shrub, Wikstroemia, has an unusually diverse array of breeding systems: two forms of dioecy, cryptic and morphological dioecy, as well as hermaphroditism (perfect flowers). The existence of two forms of dioecy is significant for three reasons: 1) the presence of cryptic unisexuals that are functionally unisexual, but retain the appearance of hermaphroditism in both sexes, is strong evidence for the ancestral status of hermaphroditism; 2) the production of nonfunctional pollen, by female cryptic unisexuals, is a new instance of a phenomenon which has previously been reported for a few other species; 3) the two forms of dioecy are morphological markers which are useful in hybridization studies for tracing the genetic basis of their inheritance. Crosses were made between cryptically unisexual individuals (C), between morphologically unisexual individuals (M), and between the two types of unisexuality. The offspring of crosses between individuals with the same sex type usually resulted in offspring with that sex type, but most of the progeny of between-sex type crosses were, unexpectedly, perfect-flowered hermaphrodites. These results show that genetic control of sex determination is not homologous in all populations, suggesting that dioecy has evolved at least twice in Hawaiian Wikstroemia. The genetic data further suggest that males are the heterozygous sex.     

COMPLEMENTARY SEX DETERMINATION IN HYMENOPTERAN PARASITOIDS AND ITS IMPLICATIONS FOR BIOLOGICAL CONTROL

Abstract In haplodiploid Hymenoptera, unfertilized eggs produce haploid males while fertilized eggs lead to diploid females under most circumstances. Diploid males can also be produced from fertilization under a system of sex determination known as complementary sex determination (CSD). Under single-locus CSD, sex is determined by multiple alleles at a single sex locus. Individuals heterozygous at the sex locus are female while hemizygous and homozygous individuals develop as haploid and diploid males, respectively. In multiple-locus CSD, two or more loci, each with two or more alleles, determine sex. Diploid individuals are female if one or more sex loci are heterozygous, while a diploid is male only if homozygous at all sex loci. Diploid males are known to occur in 43 hymenopteran species and single-locus CSD has been demonstrated in 22 of these species. Diploid males are either developmentally inviable or sterile, so their production constitutes a genetic load. Because diploid male production is more likely under inbreeding, CSD is a form of inbreeding depression. It is crucial to preserve the diversity of sex alleles and reduce the loss of genetic variation in biological control. In the parasitoid species with single-locus CSD, certain precautionary procedures can prevent negative effects of single-locus CSD on biological control.     

Practical applications of time-averaged restrained molecular dynamics to ligand-receptor systems: FK506 bound to the Q50R,A95H,K98I triple mutant of FKBP-13

Summary The ability of time-averaged restrained molecular dynamics (TARMD) to escape local low-energy conformations and explore conformational space is compared with conventional simulated-annealing methods. Practical suggestions are offered for performing TARMD calculations with ligand-receptor systems, and are illustrated for the complex of the immunosuppressant FK506 bound to Q50R,A95H,K98I triple mutant FKBP-13. The structure of ¹³C-labeled FK506 bound to triple-mutant FKBP-13 was determined using a set of 87 NOE distance restraints derived from HSQC-NOESY experiments. TARMD was found to be superior to conventional simulated-annealing methods, and produced structures that were conformationally similar to FK506 bound to wild-type FKBP-12. The individual and combined effects of varying the NOE restraint force constant, using an explicit model for the protein binding pocket, and starting the calculations from different ligand conformations were explored in detail.Abbreviations DG distance geometry - dmFKBP-12 double-mutant (R42K,H87V) FKBP-12 - FKBP-12 FK506-binding protein (12 kDa) - FKBP-13 FK506-binding protein (13 kDa) - HSQC heteronuclear single-quantum coherence - K_NOE force constant (penalty) for NOE-derived distance restraints - MD molecular dynamics - NOE nuclear Overhauser effect - SA simulated annealing - TARMD molecular dynamics with time-averaged restraints - tmFKBP-13 triple-mutant (Q50R,A95H,K98I) FKBP-13 - wtFKBP-12 wild-type FKBP-12     

Treatment of NOE constraints involving equivalent or nonstereoassigned protons in calculations of biomacromolecular structures

Summary Two modifications to the commonly used protocols for calculating NMR structures are developed, relating to the treatment of NOE constraints involving groups of equivalent protons or nonstereoassigned diastereotopic protons. Firstly, a modified method is investigated for correcting for multiplicity, which is applicable whenever all NOE intensities are calibrated as a single set and categorised in broad intensity ranges. Secondly, a new set of values for pseudoatom corrections is proposed for use with calculations employing centre-averaging. The effect of these protocols on structure calculations is demonstrated using two proteins, one of which is well defined by the NOE data, the other less so. It is shown that failure to correct for multiplicity when using r^-6 averaging results in overly precise structures, higher NOE energies and deviations from geometric ideality, while failure to correct for multiplicity when using r^-6 summation can cause an avoidable degradation of precision if the NOE data are sparse. Conversely, when multiplicities are treated correctly, r^-6 averaging, r^-6 summation and centre averaging all give closely comparable results when the structure is well defined by the data. When the NOE data contain less information, r^-6 averaging or r^-6 summation offer a significant advantage over centre averaging, both in terms of precision and in terms of the proportion of calculations that converge on a consisten result.Abbreviations HMG high mobility group - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - rmsd root-mean-square deviation - YASAP yet another simulated-annealing protocol     

A rapid method for determination of Proteus vulgaris with a piezoelectric quartz crystal sensor coated with a thin liquid film

A rapid method for microorganism detection using a piezoelectric quartz crystal sensor (PQC) coated with a thin liquid culture medium film was developed and applied to detect the cell number of Proteus vulgaris. This method employed the viscosity and density response of PQC and utilized the coagulation of gelatine medium solution in which the microorganisms had grown to determine the microorganism indirectly. Three time points (TT₁, DT, TT₂) were obtained from the coagulation curve and were found to be in good linear relationship with the logarithm of the initial number of P. vulgaris in the range 1·3 × 10²−1·3 × 10⁵ cells/ml. The detection was rapid and accurate because the coagulation of the thin liquid culture medium film was quick and the time points in the response curve were sharp and so were easy to determine accurately. The detection time was less than 4 h and only a micro sample was needed. A 5 h preincubation was needed before detection. Some experimental conditions are discussed in detail.