Calculation of protein extinction coefficients from amino acid sequence data

Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.     

Early Cenozoic increases in mammal diversity cannot be explained solely by expansion into larger body sizes

A prominent hypothesis in the diversification of placental mammals after the Cretaceous–Palaeogene (K/Pg) boundary suggests that the extinction of non-avian dinosaurs resulted in the ecological release of mammals, which were previously constrained to small body sizes and limited species richness. This ‘dinosaur incumbency hypothesis’ may therefore explain increases in mammalian diversity via expansion into larger body size niches, that were previously occupied by dinosaurs, but does not directly predict increases in other body size classes. To evaluate this, we estimate sampling-standardized diversity patterns of terrestrial North American fossil mammals within body size classes, during the Cretaceous and Palaeogene. We find strong evidence for post-extinction diversity increases in all size classes. Increases in the diversity of small-bodied species (less than 100 g, the common body size class of Cretaceous mammals, and much smaller than the smallest non-avialan dinosaurs (c. 400 g)) were similar to those of larger species. We propose that small-bodied mammals had access to greater energetic resources or were able to partition resources more finely after the K/Pg mass extinction. This is likely to be the result of a combination of widespread niche clearing due to the K/Pg mass extinctions, alongside a suite of biotic and abiotic changes that occurred during the Late Cretaceous and across the K/Pg boundary, such as shifting floral composition, and novel key innovations among eutherian mammals.     

Multiple comparison of entropies with application to dinosaur biodiversity

Summary. Did the biodiversity of dinosaurs decline, or did it remain more or less constant before their mass extinction 65 million years ago? Sheehan et al. (1991, Science, 835–839) reported that the biodiversity of families of dinosaur species remained more or less constant preceding their extinction, suggesting extinction due to a cataclysmic event such as an asteroid strike. But that claim was based on the incorrect interpretation that a large p value associated with a test of null hypothesis of equality supports that null hypothesis. To assess whether there is a basis for such a claim, we formulate the problem as one of practical equivalence, in analogy to bioequivalence. We then develop reliable practical equivalence confidence intervals for differences of entropies by applying the bootstrap-t technique to a nearly pivotal quantity. Confidence intervals for changes in the biodiversity of dinosaurs are then computed, allowing the reader to assess whether there is evidence of near constancy of dinosaur biodiversity before extinction.     

The extinction of the dinosaurs

Non‐avian dinosaurs went extinct 66 million years ago, geologically coincident with the impact of a large bolide (comet or asteroid) during an interval of massive volcanic eruptions and changes in temperature and sea level. There has long been fervent debate about how these events affected dinosaurs. We review a wealth of new data accumulated over the past two decades, provide updated and novel analyses of long‐term dinosaur diversity trends during the latest Cretaceous, and discuss an emerging consensus on the extinction's tempo and causes. Little support exists for a global, long‐term decline across non‐avian dinosaur diversity prior to their extinction at the end of the Cretaceous. However, restructuring of latest Cretaceous dinosaur faunas in North America led to reduced diversity of large‐bodied herbivores, perhaps making communities more susceptible to cascading extinctions. The abruptness of the dinosaur extinction suggests a key role for the bolide impact, although the coarseness of the fossil record makes testing the effects of Deccan volcanism difficult.     

Footprints pull origin and diversification of dinosaur stem lineage deep into Early Triassic

The ascent of dinosaurs in the Triassic is an exemplary evolutionary radiation, but the earliest phase of dinosaur history remains poorly understood. Body fossils of close dinosaur relatives are rare, but indicate that the dinosaur stem lineage (Dinosauromorpha) originated by the latest Anisian (ca 242-244 Ma). Here, we report footprints from the Early-Middle Triassic of Poland, stratigraphically well constrained and identified using a conservative synapomorphy-based approach, which shifts the origin of the dinosaur stem lineage back to the Early Olenekian (ca 249-251 Ma), approximately 5-9 Myr earlier than indicated by body fossils, earlier than demonstrated by previous footprint records, and just a few million years after the Permian/Triassic mass extinction (252.3 Ma). Dinosauromorph tracks are rare in all Polish assemblages, suggesting that these animals were minor faunal components. The oldest tracks are quadrupedal, a morphology uncommon among the earliest dinosauromorph body fossils, but bipedality and moderately large body size had arisen by the Early Anisian (ca 246 Ma). Integrating trace fossils and body fossils demonstrates that the rise of dinosaurs was a drawn-out affair, perhaps initiated during recovery from the Permo-Triassic extinction.     

An analysis of dinosaurian biogeography: evidence for the existence of vicariance and dispersal patterns caused by geological events

As the supercontinent Pangaea fragmented during the Mesozoic era, dinosaur faunas were divided into isolated populations living on separate continents. It has been predicted, therefore, that dinosaur distributions should display a branching ('vicariance') pattern that corresponds with the sequence and timing of continental break-up. Several recent studies, however, minimize the importance of plate tectonics and instead suggest that dispersal and regional extinction were the main controls on dinosaur biogeography. Here, in order to test the vicariance hypothesis, we apply a cladistic biogeographical method to a large dataset on dinosaur relationships and distributions. We also introduce a methodological refinement termed 'time-slicing', which is shown to be a key step in the detection of ancient biogeographical patterns. These analyses reveal biogeographical patterns that closely correlate with palaeogeography. The results provide the first statistically robust evidence that, from Middle Jurassic to mid-Cretaceous times, tectonic events had a major role in determining where and when particular dinosaur groups flourished. The fact that evolutionary trees for extinct organisms preserve such distribution patterns opens up a new and fruitful direction for palaeobiogeographical research.     

Reproductive response of generalist and specialist aphid morphs with the same genotype to plant secondary compounds and amino acids

Many studies have paid particular attention to the role of either secondary plant compounds or amino acids as determinants of host-plant range in phytophagous insects. Here we examine the relative importance of both of these classes of compound in host acceptance by generalist and specialist morphs of the black bean aphid, Aphis fabae, that are morphologically similar and genetically identical. Eleven secondary plant compounds and six amino acids with known biological activity in aphids are presented to insects within an artificial membrane system as single compounds, mixtures of same-class compounds and combined mixtures of the two classes of compound. It is found that 1) when specific single secondary compounds and amino acids are presented to generalist and specialist morphs of A. fabae, differential responses are exclusively consistent with plant-use strategy for amino acids but not for secondary compounds, 2) neither secondary compound nor amino acid mixtures give reproductive responses entirely consistent with plant range, but the response to secondary compounds is broadly consistent with plant range whereas the response to amino acids is not, 3) when secondary compounds and amino acid mixtures are combined, the response to secondary compounds generally dominates that to amino acids. Some scenarios of plant-range determination by secondary plant compounds and amino acids, suggested by results, are discussed.     

A possible molecular metric for biological evolvability

Proteins manifest themselves as phenotypic traits, retained or lost in living systems via evolutionary pressures. Simply put, survival is essentially the ability of a living system to synthesize a functional protein that allows for a response to environmental perturbations (adaptation). Loss of functional proteins leads to extinction. Currently there are no universally applicable quantitative metrics at the molecular level for either measuring 'evolvability' of life or for assessing the conditions under which a living system would go extinct and why. In this work, we show emergence of the first such metric by utilizing the recently discovered stoichiometric margin of life for all known naturally occurring (and functional) proteins. The constraint of having well-defined stoichiometries of the 20 amino acids in naturally occurring protein sequences requires utilization of the full scope of degeneracy in the genetic code, i.e. usage of all codons coding for an amino acid, by only 11 of the 20 amino acids. This shows that the non-availability of individual codons for these 11 amino acids would disturb the fine stoichiometric balance resulting in non-functional proteins and hence extinction. Remarkably, these amino acids are found in close proximity of any given amino acid in the backbones of thousands of known crystal structures of folded proteins. On the other hand, stoichiometry of the remaining 9 amino acids, found to be farther/distal from any given amino acid in backbones of folded proteins, is maintained independent of the number of codons available to synthesize them, thereby providing some robustness and hence survivability.     

A palaeoequatorial ornithischian and new constraints on early dinosaur diversification

Current characterizations of early dinosaur evolution are incomplete: existing palaeobiological and phylogenetic scenarios are based on a fossil record dominated by saurischians and the implications of the early ornithischian record are often overlooked. Moreover, the timings of deep phylogenetic divergences within Dinosauria are poorly constrained owing to the absence of a rigorous chronostratigraphical framework for key Late Triassic–Early Jurassic localities. A new dinosaur from the earliest Jurassic of the Venezuelan Andes is the first basal ornithischian recovered from terrestrial deposits directly associated with a precise radioisotopic date and the first-named dinosaur from northern South America. It expands the early palaeogeographical range of Ornithischia to palaeoequatorial regions, an area sometimes thought to be devoid of early dinosaur taxa, and offers insights into early dinosaur growth rates, the evolution of sociality and the rapid tempo of the global dinosaur radiation following the end-Triassic mass extinction, helping to underscore the importance of the ornithischian record in broad-scale discussions of early dinosaur history.     

Crystal structure of the ribosomal protein S6 from Thermus thermophilus.

The amino acid sequence and crystal structure of the ribosomal protein S6 from the small ribosomal subunit of Thermus thermophilus have been determined. S6 is a small protein with 101 amino acid residues. The 3D structure, which was determined to 2.0 A resolution, consists of a four-stranded anti-parallel beta-sheet with two alpha-helices packed on one side. Similar folding patterns have been observed for other ribosomal proteins and may suggest an original RNA-interacting motif. Related topologies are also found in several other nucleic acid-interacting proteins and based on the assumption that the structure of the ribosome was established early in the molecular evolution, the possibility that an ancestral RNA-interacting motif in ribosomal proteins is the evolutionary origin for the nucleic acid-interacting domain in large classes of ribonucleic acid binding proteins should be considered.     

谷子叶绿体乙酰辅酶A羧化酶cDNA的克隆和禾本科除草剂靶位点的序列分析

乙酰辅酶A羧化酶是一个生物素羧化酶,它所催化的反应是脂肪酸生物合成中的第一个植物叶绿体中的乙酰辅酶A羧化酶是两类禾本科除草剂的靶蛋白.从抗除草剂拿捕净和感拿捕净的谷子(SetariaitalicaBeauv.)中克隆了两个乙酰辅酶A羧化酶的全长cDNA,分别命名为foxACC-R和foxACC-S,它们推导的蛋白质均编码2 321个氨基酸,然而在第1 780个氨基酸处,foxACC-R编码亮氨酸,而foxACC-S编码异亮氨酸.采用生物信息学方法,我们推断这个cDNA编码的是叶绿体中的乙酰辅酶A羧化酶,并预测了它的功能域和保守区.通过这两个cDNA编码的氨基酸序列与其他乙酰辅酶A羧化酶的序列比较得出结论,亮氨酸/异亮氨酸位点可能是APPs和CHDs两类除草剂作用的关键位点.Southern杂交分析的结果显示,该基因在谷子基因组中只有一个拷贝.     

谷子叶绿体乙酰辅酶A羧化酶cDNA的克隆和禾本科除草剂靶位点的序列分析

乙酰辅酶A羧化酶是一个生物素羧化酶,它所催化的反应是脂肪酸生物合成中的第一个关键步骤。禾本科植物叶绿体中的乙酰辅酶A羧化酶是两类禾本科除草剂的靶蛋白。从抗除草剂拿捕净和感拿捕净的谷子(Setaria italicaBeauv.)中克隆了两个乙酰辅酶A羧化酶的全长cDNA,分别命名为foxACC-R和foxACC-S,它们推导的蛋白质均编码2 321个氨基酸,然而在第1 780个氨基酸处,foxACC-R编码亮氨酸,而foxACC-S编码异亮氨酸。采用生物信息学方法,我们推断这个cDNA编码的是叶绿体中的乙酰辅酶A羧化酶,并预测了它的功能域和保守区。通过这两个cDNA编码的氨基酸序列与其他乙酰辅酶A羧化酶的序列比较得出结论,亮氨酸/异亮氨酸位点可能是APPs和CHDs两类除草剂作用的关键位点。Southern 杂交分析的结果显示,该基因在谷子基因组中只有一个拷贝。     

Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii

A serine protease, named as "Milin" was purified to homogeneity from the latex of Euphorbia milii, a medicinal plant of Euphorbiaceae family. The molecular mass (SDS-PAGE), optimum pH and temperature of the enzyme were 51kDa, pH 8.0 and 60 degrees C, respectively. Milin retains full proteolytic activity over a wide range of pH (5.5-12) and temperature (up to 65 degrees C) with casein and azoalbumin as substrates. The activity of milin is inhibited by serine proteases inhibitors like PMSF, APMSF and DFP, but not by any other protease inhibitors such as E-64 and PCMB. Like the other serine proteases from the genus Euphorbia, the activity of milin was not inhibited by the proteinaceous inhibitor soyabean trypsin inhibitor (SBTI) even at very high concentrations that is naturally present in plants. The specific extinction coefficient (epsilon(280 nm)(1%)), molar extinction coefficient (a(m)) and isoelectric point of the enzyme were found to be 29, 152,500 M(-1) cm(-1) and pH 7.2, respectively. The enzyme is a glycoprotein with detectable carbohydrate moiety (7-8%) in its constitution, which is essential for the activity. The numbers of tryptophan, tyrosine and cysteine residues in the sequence of milin were estimated chemically and are 23, 14 and 14, respectively. Of the 14-cysteine residues, 12 constituted 6-disulfide linkages while two are free cysteines. The N-terminal sequence (first 12 amino acid residues) was determined and does not match with any sequence of known plant serine proteases. Perturbation studies by temperature, pH and chaotropes of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Thus, this serine protease may have potential applications in food industry.     

Dinosaur peptides suggest mechanisms of protein survival

Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.     

A method for the determination of the molar extinction coefficient of structure-linked chromophores

Synopsis This paper describes a general method for the determination of the molar extinction ceefficient of a chromophore covalently bound to structure-linked groups, without isolating the compound formed. The method is illustrated by the determination of the molar extinction coefficient of the reaction product of 2,4-dinitro-1-fluorobenzene (DNFB) with films of aminoethyl-cellulose (AE-cellulose). The method is based on the relation between the decrease in extinction of a DNFB staining solution and the increase in extinction of the AE-cellulose after staining as measured in a film-spectrophotometer. In addition, the value of the molar extinction coefficient was used in establishing reaction conditions for a quantitative staining procedure for determining amino groups with DNFB and picric acid. Conditions of optimum DNFB staining were determined and the measured extinction was converted into concentration of amino groups using the molar extinction coefficient. The amino group concentration of the same batch of AE-cellulose was also determined, after finding the optimum reaction conditions, by staining with picric acid. The results, when compared, showed a linear relationship with a slope of unity for batches of AE-cellulose of varying amino group concentrations. This is consistent with the same stoichiometry in both cases and indicates that in both procedures one chromophore molecule has reacted with one amino group and that this reaction has proceeded to completion. The general applicability of the method is discussed.     

Genome-wide prediction of G protein-coupled receptors in Verticillium spp

G protein-coupled receptors (GPCRs) are critical factors in regulating morphogenesis, mating, infection and virulence in fungi. In this study, various computational strategies were applied to identify GPCR-like proteins from the genomes of both Verticillium dahliae and Verticillium albo-atrum. The putative GPCRs were distributed over 13 classes, and significantly, three of those represented novel classes of GPCR-like proteins in fungi. The three novel GPCRs had high levels of identity to their counterparts in higher eukaryotes, including Homo sapiens. The numbers of GPCR-like proteins in the two Verticillium spp. were similar to those seen in other filamentous fungi, such as Magnaporthe grisea, Neurospora crassa and Fusarium graminearum. Additionally, the carbon/amino acid receptors were divided into three different subclasses, indicating that differences among the GPCRs existed not only among different classes but also within classes. In conclusion, the identification and classification of GPCRs and their homology to some well-studied fungi will be an important starting point for future research in Verticillium spp.     

The first 50Myr of dinosaur evolution: macroevolutionary pattern and morphological disparity

The evolutionary radiation of dinosaurs in the Late Triassic and Early Jurassic was a pivotal event in the Earth's history but is poorly understood, as previous studies have focused on vague driving mechanisms and have not untangled different macroevolutionary components (origination, diversity, abundance and disparity). We calculate the morphological disparity (morphospace occupation) of dinosaurs throughout the Late Triassic and Early Jurassic and present new measures of taxonomic diversity. Crurotarsan archosaurs, the primary dinosaur 'competitors', were significantly more disparate than dinosaurs throughout the Triassic, but underwent a devastating extinction at the Triassic-Jurassic boundary. However, dinosaur disparity showed only a slight non-significant increase after this event, arguing against the hypothesis of ecological release-driven morphospace expansion in the Early Jurassic. Instead, the main jump in dinosaur disparity occurred between the Carnian and Norian stages of the Triassic. Conversely, dinosaur diversity shows a steady increase over this time, and measures of diversification and faunal abundance indicate that the Early Jurassic was a key episode in dinosaur evolution. Thus, different aspects of the dinosaur radiation (diversity, disparity and abundance) were decoupled, and the overall macroevolutionary pattern of the first 50Myr of dinosaur evolution is more complex than often considered.     

Properties of common wheat ferredoxin, and a comparison with ferredoxins from related species of triticum and aegilops.

Wheat ferredoxin was purified from the leaves of common wheat (Triticum aestivum). The absorption spectrum showed maxima at 465, 425, 332, and 278 nm. The absorbance ratio, A425 nm/A278 nm was 0.49, and the millimolar extinction coefficient at 425 nm was 10.8 mM-1. cm-1. The amino acid composition was determined to be Lys5, His2, Arg1, Asp11, Thr5, Ser7, Glu18, Pro5, Gly6, Ala7, Cys5, Val7, Met1, Ile4, Leu7, Tyr4, Phe1, and Trp1. The total number of amino acid residues was 97. The molecular weight was calculated from the amino acid composition to be 10,829, including iron and sulfur atoms. This value was confirmed by other methods, which were based on the contents of non-heme iron and of terminal amino acid. The N-terminal amino acid was alanine, and the C-terminal amino acid sequence was -Glu-Leu-Thr-AlaCOOH. Comparative studies were performed between T. aestivum ferredoxin and ferredoxins isolated from closely related species; these were T. aegilopoides, T. durum, Ae. squarrosa, and Ae. ovata. No significant differences in the properties of these ferredoxins were detected. It was also shown that these ferredoxins are immunologically homologous. It is, therefore, likely that one molecular species of ferredoxin is distributed through two genera of Triticum and Aegilops.     

Downsizing a giant: re-evaluating Dreadnoughtus body mass

Estimates of body mass often represent the founding assumption on which biomechanical and macroevolutionary hypotheses are based. Recently, a scaling equation was applied to a newly discovered titanosaurian sauropod dinosaur (Dreadnoughtus), yielding a 59 300 kg body mass estimate for this animal. Herein, we use a modelling approach to examine the plausibility of this mass estimate for Dreadnoughtus. We find that 59 300 kg for Dreadnoughtus is highly implausible and demonstrate that masses above 40 000 kg require high body densities and expansions of soft tissue volume outside the skeleton several times greater than found in living quadrupedal mammals. Similar results from a small sample of other archosaurs suggests that lower-end mass estimates derived from scaling equations are most plausible for Dreadnoughtus, based on existing volumetric and density data from extant animals. Although volumetric models appear to more tightly constrain dinosaur body mass, there remains a clear need to further support these models with more exhaustive data from living animals. The relative and absolute discrepancies in mass predictions between volumetric models and scaling equations also indicate a need to systematically compare predictions across a wide size and taxonomic range to better inform studies of dinosaur body size.