OneG: A Computational Tool for Predicting Cryptic Intermediates in the Unfolding Kinetics of Proteins under Native Conditions

Understanding the relationships between conformations of proteins and their stabilities is one key to address the protein folding paradigm. The free energy change (ΔG) of unfolding reactions of proteins is measured by traditional denaturation methods and native hydrogen-deuterium (H/D) exchange methods. However, the free energy of unfolding (ΔGU) and the free energy of exchange (ΔGHX) of proteins are not in good agreement, though the experimental conditions of both methods are well matching to each other. The anomaly is due to any one or combinations of the following reasons: (i) effects of cis-trans proline isomerisation under equilibrium unfolding reactions of proteins (ii) inappropriateness in accounting the baselines of melting curves (iii) presence of cryptic intermediates, which may elude the melting curve analysis and (iv) existence of higher energy metastable states in the H/D exchange reactions of proteins. Herein, we have developed a novel computational tool, OneG, which accounts the discrepancy between ΔGU and ΔGHX of proteins by systematically accounting all the four factors mentioned above. The program is fully automated and requires four inputs: three-dimensional structures of proteins, ΔGU, ΔGU* and residue-specific ΔGHX determined under EX2-exchange conditions in the absence of denaturants. The robustness of the program has been validated using experimental data available for proteins such as cytochrome c and apocytochrome b562 and the data analyses revealed that cryptic intermediates of the proteins detected by the experimental methods and the cryptic intermediates predicted by the OneG for those proteins were in good agreement. Furthermore, using OneG, we have shown possible existence of cryptic intermediates and metastable states in the unfolding pathways of cardiotoxin III and cobrotoxin, respectively, which are homologous proteins. The unique application of the program to map the unfolding pathways of proteins under native conditions have been brought into fore and the program is publicly available at http://sblab.sastra.edu/oneg.htm

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

Calculation of k_rc of NHs in proteins from their 3D structures.

<p>Correlation between k_rc values estimated by manual calculation and the OneG program for NHs in proteins (A) Ubiquitin (1UBQ) and (B) Cardiotoxin III (2CRT) at pH 7.0, 298 K.</p

Possible existence of cryptic intermediates of CTX III.

<p>The five β-strands (S1–S5), three loops and a globular head in the structure of CTX III (2CRT) are shown by ribbon diagram. The blue and red sticks represent residues in the cryptic intermediates I & II, respectively, as predicted by OneG program.</p

Figurative representation of cryptic intermediates of apocytochrome b₅₆₂.

<p>Three cryptic intermediates of the protein detected by experimental methods and predicted by OneG are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032465#pone-0032465-g005" target="_blank">Figure 5A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032465#pone-0032465-g005" target="_blank">Figure 5B</a>, respectively. The intermediates are denoted by blue, green and red colour codes in both cases. The backbone structures of the protein and residues representing each intermediate are shown in ribbon and stick models, respectively. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032465#pone-0032465-g005" target="_blank">Figure 5A</a> shows residues for which exchange kinetic data were reported in the literature.</p

Comparison of the actual and the predicted conformations (by OneG program) of Xaa-Pro peptide bonds in Ubiquitin, Rnase A and Cardiotoxin III.

<p>*The manual distance measurements for determining the conformations of the Xaa-pro peptide bonds in proteins were carried-out using PyMol molecular visualization tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032465#pone.0032465-DeLano1" target="_blank">[23]</a>.</p

The values of ΔG_U, ΔG_HX and ΔG_HX* (free energy of exchange corrected to effect of <i>cis-trans</i> proline isomerisation) of sixteen different proteins are herein listed.

@<p>Parentheses contain references from which the values of the free energies of the proteins have been referred.</p><p>The values of ΔG_HX* of the proteins have been calculated using the OneG program. Free energy values of the proteins were represented in kcal/mol.</p

Comparison of the actual and the predicted (by OneG program) cysteine and cystine residues in Cardiotoxin III and Cytochrome C.

#<p>Cys denotes Cysteine residue.</p><p>*The manual distance measurements for determining the cysteine and cystine residues in proteins were carried-out using PyMol molecular visualization tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032465#pone.0032465-DeLano1" target="_blank">[23]</a>.</p

Figurative representation of cryptic intermediates of Cytochrome C.

<p>The cryptic intermediates detected by experimental methods and predicted by OneG are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032465#pone-0032465-g004" target="_blank">Figure 4A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032465#pone-0032465-g004" target="_blank">Figure 4B</a>, respectively. The backbone structures of the protein and residues representing each intermediate are shown in ribbon and stick models, respectively. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032465#pone-0032465-g004" target="_blank">Figure 4A</a> shows cryptic intermediates, proposed on the basis of experimental methods, in blue, green, yellow and red colours. The residues (for which exchange kinetics were observed by experiments) representing each intermediate are shown in sticks. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032465#pone-0032465-g004" target="_blank">Figure 4B</a> shows residues constituting three distinct intermediates as predicted by OneG program, in blue, magenta and yellow colours.</p