Dataset Information

Combining secondary-structure and protein solvent-accessibility predictions in methionine substitution for anomalous dispersion.

ABSTRACT: In X-ray crystallographic analysis, the single-wavelength and multi-wavelength anomalous diffraction (SAD and MAD) methods have been widely used in order to solve the phase problem. Selenium-labelled methionine has been shown to be very effective for anomalous dispersion phasing, and at least one selenomethionine is required for every 100 amino acids. Some proteins, such as the Arabidopsis thaliana thylakoid lumen protein AtTLP18.3, can be overexpressed in an Escherichia coli system and high-quality protein crystals can be obtained. However, AtTLP18.3 contains no methionine residues, and site-directed mutagenesis was required in order to introduce methionine residues into the protein. A criterion for the mutated residues is that they should avoid affecting the structure and function. In this study, several leucine and isoleucine residues were selected for methionine substitution by combining secondary-structure and solvent-accessibility predictions. From the secondary-structure prediction, mutated residues were first determined in the coil or loop regions at the junction of two secondary structures. Since leucine and isoleucine residues are hydrophobic and are normally buried within the protein core, these residues should have a higher solvent-accessibility prediction so that they would be partially buried or exposed in the protein. In addition, five residues (Leu107, Leu202, Ile133, Leu128 and Ile159) of AtTLP18.3 were mutated to methionine residues. After overexpression and purification, only two single-mutant lines, L128M and I159M, could be crystallized. Finally, a double-mutation line of truncated AtTLP18.3 with L128M and I159M mutations was constructed. The structure of the double mutant AtTLP18.3 protein was resolved using the single-wavelength anomalous diffraction method at 2.6?Å resolution. The results indicated that a combination of secondary-structure and solvent-accessibility prediction for methionine substitution is a useful method in SAD and MAD phasing.

SUBMITTER: Wu HY

PROVIDER: S-EPMC3944707 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Combining secondary-structure and protein solvent-accessibility predictions in methionine substitution for anomalous dispersion.

Wu Hsin-Yi HY Cheng Yi-Sheng YS

Acta crystallographica. Section F, Structural biology communications 20140219 Pt 3

In X-ray crystallographic analysis, the single-wavelength and multi-wavelength anomalous diffraction (SAD and MAD) methods have been widely used in order to solve the phase problem. Selenium-labelled methionine has been shown to be very effective for anomalous dispersion phasing, and at least one selenomethionine is required for every 100 amino acids. Some proteins, such as the Arabidopsis thaliana thylakoid lumen protein AtTLP18.3, can be overexpressed in an Escherichia coli system and high-qua ...[more]

PMID: 24598932

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Project description:BACKGROUND: It is well known that most of the binding free energy of protein interaction is contributed by a few key hot spot residues. These residues are crucial for understanding the function of proteins and studying their interactions. Experimental hot spots detection methods such as alanine scanning mutagenesis are not applicable on a large scale since they are time consuming and expensive. Therefore, reliable and efficient computational methods for identifying hot spots are greatly desired and urgently required. RESULTS: In this work, we introduce an efficient approach that uses support vector machine (SVM) to predict hot spot residues in protein interfaces. We systematically investigate a wide variety of 62 features from a combination of protein sequence and structure information. Then, to remove redundant and irrelevant features and improve the prediction performance, feature selection is employed using the F-score method. Based on the selected features, nine individual-feature based predictors are developed to identify hot spots using SVMs. Furthermore, a new ensemble classifier, namely APIS (A combined model based on Protrusion Index and Solvent accessibility), is developed to further improve the prediction accuracy. The results on two benchmark datasets, ASEdb and BID, show that this proposed method yields significantly better prediction accuracy than those previously published in the literature. In addition, we also demonstrate the predictive power of our proposed method by modelling two protein complexes: the calmodulin/myosin light chain kinase complex and the heat shock locus gene products U and V complex, which indicate that our method can identify more hot spots in these two complexes compared with other state-of-the-art methods. CONCLUSION: We have developed an accurate prediction model for hot spot residues, given the structure of a protein complex. A major contribution of this study is to propose several new features based on the protrusion index of amino acid residues, which has been shown to significantly improve the prediction performance of hot spots. Moreover, we identify a compact and useful feature subset that has an important implication for identifying hot spot residues. Our results indicate that these features are more effective than the conventional evolutionary conservation, pairwise residue potentials and other traditional features considered previously, and that the combination of our and traditional features may support the creation of a discriminative feature set for efficient prediction of hot spot residues. The data and source code are available on web site http://home.ustc.edu.cn/~jfxia/hotspot.html.

Project description:Although most proteins conform to the classical one-structure/one-function paradigm, an increasing number of proteins with dual structures and functions have been discovered. In response to cellular stimuli, such proteins undergo structural changes sufficiently dramatic to remodel even their secondary structures and domain organization. This "fold-switching" capability fosters protein multi-functionality, enabling cells to establish tight control over various biochemical processes. Accurate predictions of fold-switching proteins could both suggest underlying mechanisms for uncharacterized biological processes and reveal potential drug targets. Recently, we developed a prediction method for fold-switching proteins using structure-based thermodynamic calculations and discrepancies between predicted and experimentally determined protein secondary structure (Porter and Looger, Proc Natl Acad Sci U S A 2018; 115:5968-5973). Here we seek to leverage the negative information found in these secondary structure prediction discrepancies. To do this, we quantified secondary structure prediction accuracies of 192 known fold-switching regions (FSRs) within solved protein structures found in the Protein Data Bank (PDB). We find that the secondary structure prediction accuracies for these FSRs vary widely. Inaccurate secondary structure predictions are strongly associated with fold-switching proteins compared to equally long segments of non-fold-switching proteins selected at random. These inaccurate predictions are enriched in helix-to-strand and strand-to-coil discrepancies. Finally, we find that most proteins with inaccurate secondary structure predictions are underrepresented in the PDB compared with their alternatively folded cognates, suggesting that unequal representation of fold-switching conformers within the PDB could be an important cause of inaccurate secondary structure predictions. These results demonstrate that inconsistent secondary structure predictions can serve as a useful preliminary marker of fold switching.

Dataset Information

Combining secondary-structure and protein solvent-accessibility predictions in methionine substitution for anomalous dispersion.

Publications

Combining secondary-structure and protein solvent-accessibility predictions in methionine substitution for anomalous dispersion.

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