Electron Density Sharpening as a General Technique in Crystallographic Studies

Sharpening is a powerful method to restore the details from blurred electron density in crystals with high overall temperature factors (B-factors). This valuable technique is currently not optimally used because of the uncertainty in the scope of its application and ambiguities in practice. We performed an analysis of ~ 2000 crystal data sets deposited in the Protein Data Bank and show that sharpening improves the electron density map in many cases across all resolution ranges, often with dramatic enhancement for mid- and low-resolution structures. It is effective when used with either experimental or model phases without introducing additional bias. Our tests also provide a practical guide for optimal sharpening. We further show that anisotropic diffraction correction improves electron density in many cases but should be used with caution. Our study demonstrates that a routine practice of electron density sharpening may have a broad impact on the outcomes of structural biology studies.     

Structural and Functional Studies on the Overproduced L11 Protein from Thermus thermophilus

The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol. The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli. Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus. The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms. Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion. The similar structural and biochemical properties as well as the significant homology between L11 from E. coli and B. stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function.     

A comparative study of molecular and morphological methods of describing relationships between perennial ryegrass (Lolium perenne L.) varieties

A sample set of registered perennial ryegrass varieties was used to compare how morphological characterisation and AFLP^® (AFLP^® is a registered trademark of Keygene N.V.) and STS molecular markers described variety relationships. All the varieties were confirmed as morphologically distinct, and both the STS and AFLP markers exposed sufficient genetic diversity to differentiate these registered ryegrass varieties. Distances obtained by each of the approaches were compared, with special attention given to the coincidences and divergences between the methods. When correlations between morphological, AFLP and STS distances were calculated and the corresponding scatter-plots constructed, the variety relationships appeared to be rather inconsistent across the methods, especially between morphology and the molecular markers. However, some consistencies were found for closely related material. An implication could be that these molecular-marker techniques, while not yet suited to certain operations in the traditional registration of new varieties, could be suitable methods for investigating disputable distinctness situations or possible EDV (EDV= essentially derived variety. An EDV is a variety being clearly distinct from, but conforming in the expression of the essential characteristics of, an ’initial variety’ (IV) from which it is found to have been predominantly derived) relationships, subject to establishing standardised protocols and statistical techniques. Some suggestions for such a protocol, including a statistical test for distinctness, are given.     

Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Contributions of water transfer energy to protein‐ligand association and dissociation barriers: Watermap analysis of a series of p38α MAP kinase inhibitors

In our previous work, we proposed that desolvation and resolvation of the binding sites of proteins can serve as the slowest steps during ligand association and dissociation, respectively, and tested this hypothesis on two protein‐ligand systems with known binding kinetics behavior. In the present work, we test this hypothesis on another kinetically‐determined protein‐ligand system—that of p38α and eight Type II BIRB 796 inhibitor analogs. The k_on values among the inhibitor analogs are narrowly distributed (10⁴ ≤ k_on ≤ 10⁵ M^?1 s^?1), suggesting a common rate‐determining step, whereas the k_off values are widely distributed (10^?1 ≤ k_off ≤ 10^?6 s^?1), suggesting a spectrum of rate‐determining steps. We calculated the solvation properties of the DFG‐out protein conformation using an explicit solvent molecular dynamics simulation and thermodynamic analysis method implemented in WaterMap to predict the enthalpic and entropic costs of water transfer to and from bulk solvent incurred upon association and dissociation of each inhibitor. The results suggest that the rate‐determining step for association consists of the transfer of a common set of enthalpically favorable solvating water molecules from the binding site to bulk solvent. The rate‐determining step for inhibitor dissociation consists of the transfer of water from bulk solvent to specific binding site positions that are unfavorably solvated in the apo protein, and evacuated during ligand association. Different sets of unfavorable solvation are evacuated by each ligand, and the observed dissociation barriers are qualitatively consistent with the calculated solvation free energies of those sets.     

Redesign of LAOBP to bind novel l‐amino acid ligands

Computational protein design is still a challenge for advancing structure‐function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported.     

Anaerobic/Aerobic Composting of Soil Contaminated with 2,4,6-Trinitrotoluene

A loam soil from Pennsylvania without a history of exposure to explosives was incubated with 5 g kg^-1 of ¹⁵N-labeled 2,4,6-trinitrotoluene (TNT) and 200 μCi kg^-1 of ¹⁴C-TNT for 3 days and then amended with compost at a 1:2 soil to compost ratio. The compost was prepared by mixing 40% alfalfa hay, 40% grass hay, 10% spent mushroom compost, and 10% municipal biosolids. The mixture of soil and compost was inoculated with methanogens from cattle manure, amended with glucose and starch, and incubated for 37 days under anaerobic conditions. The anaerobic incubation was followed by 26 days of forced aerobic incubation. At the end of the aerobic phase, most of the radioactivity was associated with organic matter; only 8.7% could be extracted with water and methanol, but no TNT was present in the extracts as determined by high-performance liquid chromatography. The unextractable radioactivity was associated with humic acid (40.0±1.0%), fulvic acid (14.3±1.4%), and humin (28.2±0.5%). Radioactive materials associated with humic acid and humin were analyzed by solid-state ¹⁵N-nuclear magnetic resonance (NMR) spectrometry. The NMR spectra indicated that nitro groups of TNT had been reduced to amino groups thatwere subsequently involved in the formation of covalent bonds with soil organic matter.