Global conformations, hydrodynamics, and x-ray scattering properties of Taq and Escherichia coli DNA polymerases in solution

Escherichia coli polymerase 1 (Pol 1) and Thermus aquaticus Taq polymerase are homologous Type I DNA polymerases, each comprised of a polymerase domain, a proofreading domain (inactive in Taq), and a 5′ nuclease domain. Klenow and Klentaq are the large fragments of Pol 1 and Taq and are functional polymerases lacking the 5′ nuclease domain. In the available crystal structures of full-length Taq, the 5′ nuclease domain is positioned in two different orientations: in one structure, it is extended out into solution, whereas in the other, it is folded up against the polymerase domain in a more compact structure. Analytical ultracentrifugation experiments report s20,w values of 5.05 for Taq, 4.1 for Klentaq, 5.3 for E. coli Pol 1, and 4.6 for Klenow. Measured partial specific volumes are all quite similar, indicating no significant differences in packing density between the mesophilic and thermophilic proteins. Small angle x-ray scattering studies report radii of gyration of 38.3 Å for Taq, 30.7 Å for Klentaq, and 30.5 Å for Klenow. The hydrodynamic and x-ray scattering properties of the polymerases were also calculated directly from the different crystal structures using the programs HYDROPRO (Garcia De La Torre, J., Huertas, M. L., and Carrasco, B. (2000) Biophys J. 78, 719-730) and CRYSOL (Svergun, D. I., Barberato, C., and Koch, M. H. J. (1995) J. Appl. Crystalogr. 28, 768-773), respectively. The combined experimental and computational characterizations indicate that the full-length polymerases in solution are in a conformation where the 5′ nuclease domain is extended into solution. Further, the radius of gyration, and hence the global conformation of Taq polymerase, is not altered by the binding of either matched primer template DNA or ddATP

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding protein

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its ΔG of unfolding by 3-5 kcal/mol. At 37 °C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilino-naphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP

Extreme Free Energy Stabilization of Taq DNA Polymerase

We have examined the chemical denaturations of the Klentaq and Klenow large-fragment domains of the Type 1 DNA polymerases from Thermus aquaticus (Klentaq) and Escherichia coli (Klenow) under identical solution conditions in order to directly compare the stabilization energetics of the two proteins. The high temperature stability of Taq DNA polymerase is common knowledge, and is the basis of its use in the polymerase chain reaction. This study, however, is aimed at understanding the thermodynamic basis for this high-temperature stability. Chemical denaturations with guanidine hydrochloride report a folding free energy (ΔG) for Klentaq that is over 20 kcal/mol more favorable than that for Klenow under the conditions examined. This difference between the stabilization free energies of a homologous mesophilic-thermophilic protein pair is significantly larger than generally observed. This is due in part to the fact that the stabilization free energy for Klentaq polymerase, at 27.5 kcal/ol, is one of the largest ever determined for a monomeric protein. Large differences in the chemical midpoints of the unfolding (Cm) and the dependences of the unfolding free energy on denaturant concentration in the transition region (m-value) between the two proteins are also observed. Measurements of the sedimentation coefficients of the two proteins in the native and denatured states report that both proteins approximately double in hydrodynamic size upon denaturation, but that Klentaq expands somewhat more than Klenow. © 2004 Wiley-Liss, Inc

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Mapping the human genetic architecture of COVID-19

The genetic make-up of an individual contributes to the susceptibility and response to viral infection. Although environmental, clinical and social factors have a role in the chance of exposure to SARS-CoV-2 and the severity of COVID-191,2, host genetics may also be important. Identifying host-specific genetic factors may reveal biological mechanisms of therapeutic relevance and clarify causal relationships of modifiable environmental risk factors for SARS-CoV-2 infection and outcomes. We formed a global network of researchers to investigate the role of human genetics in SARS-CoV-2 infection and COVID-19 severity. Here we describe the results of three genome-wide association meta-analyses that consist of up to 49,562 patients with COVID-19 from 46 studies across 19 countries. We report 13 genome-wide significant loci that are associated with SARS-CoV-2 infection or severe manifestations of COVID-19. Several of these loci correspond to previously documented associations to lung or autoimmune and inflammatory diseases3–7. They also represent potentially actionable mechanisms in response to infection. Mendelian randomization analyses support a causal role for smoking and body-mass index for severe COVID-19 although not for type II diabetes. The identification of novel host genetic factors associated with COVID-19 was made possible by the community of human genetics researchers coming together to prioritize the sharing of data, results, resources and analytical frameworks. This working model of international collaboration underscores what is possible for future genetic discoveries in emerging pandemics, or indeed for any complex human disease