Crystal structure of alkyl hydroperoxidase D like protein PA0269 from Pseudomonas aeruginosa: Homology of the AhpD-like structural family

<p>Abstract</p> <p>Background</p> <p>Alkyl hydroperoxidase activity provides an important antioxidant defense for bacterial cells. The catalytic mechanism requires two peroxidases, AhpC and AhpD, where AhpD plays the role of an essential adaptor protein.</p> <p>Results</p> <p>The crystal structure of a putative AhpD from <it>Pseudomonas aeruginosa </it>has been determined at 1.9 Å. The protein has an all-helical fold with a chain topology similar to a known AhpD from <it>Mycobacterium tuberculosis </it>despite a low overall sequence identity of 9%. A conserved two α-helical motif responsible for function is present in both. However, in the <it>P. aeruginosa </it>protein, helices H3, H4 of this motif are located at the N-terminal part of the chain, while in <it>M. tuberculosis </it>AhpD, the corresponding helices H8, H9 are situated at the C-terminus. Residues 24-62 of the putative catalytic region of <it>P. aeruginosa </it>have a higher sequence identity of 33% where the functional activity is supplied by a proton relay system of five residues, Glu36, Cys48, Tyr50, Cys51, and His55, and one structural water molecule. A comparison of five other related hypothetical proteins from various species, assigned to the alkyl hydroperoxidase D-like protein family, shows they contain the same conserved structural motif and catalytic sequence Cys-X-X-Cys. We have shown that AhpD from <it>P. aeruginosa </it>exhibits a weak ability to reduce H₂O₂as tested using a ferrous oxidation-xylenol orange (FOX) assay, and this activity is blocked by thiol alkylating reagents.</p> <p>Conclusion</p> <p>Thus, this hypothetical protein was assigned to the AhpD-like protein family with peroxidase-related activity. The functional relationship of specific oligomeric structures of AhpD-like structural family is discussed.</p

Microfluidics: From Crystallization to Serial Time-Resolved Crystallography

Capturing protein structural dynamics in real-time has tremendous potential in elucidating biological functions and providing information for structure-based drug design. While time-resolved structure determination has long been considered inaccessible for a vast majority of protein targets, serial methods for crystallography have remarkable potential in facilitating such analyses. Here, we review the impact of microfluidic technologies on protein crystal growth and X-ray diffraction analysis. In particular, we focus on applications of microfluidics for use in serial crystallography experiments for the time-resolved determination of protein structural dynamics

Two-Dimensional Infrared Spectroscopy of Antiparallel β-Sheet Secondary Structure

We investigate the sensitivity of femtosecond Fourier transform two-dimensional infrared spectroscopy to protein secondary structure with a study of antiparallel β-sheets. The results show that 2D IR spectroscopy is more sensitive to structural differences between proteins than traditional infrared spectroscopy, providing an observable that allows comparison to quantitative models of protein vibrational spectroscopy. 2D IR correlation spectra of the amide I region of poly-L-lysine, concanavalin A, ribonuclease A, and lysozyme show cross-peaks between the IR-active transitions that are characteristic of amide I couplings for polypeptides in antiparallel hydrogen-bonding registry. For poly-L-lysine, the 2D IR spectrum contains the eight-peak structure expected for two dominant vibrations of an extended, ordered antiparallel β-sheet. In the proteins with antiparallel β-sheets, interference effects between the diagonal and cross-peaks arising from the sheets, combined with diagonally elongated resonances from additional amide transitions, lead to a characteristic “Z”-shaped pattern for the amide I region in the 2D IR spectrum. We discuss in detail how the number of strands in the sheet, the local configurational disorder in the sheet, the delocalization of the vibrational excitation, and the angle between transition dipole moments affect the position, splitting, amplitude, and line shape of the cross-peaks and diagonal peaks.

Spectroscopic evidence for Davydov-like solitons in acetanilide

Detailed measurements of infrared absorption and Raman scattering on crystalline acetanilide [(CH3CONHC6H5)x] at low temperature show a new band close to the conventional amide I band. Equilibrium properties and spectroscopic data rule out explanations based on a conventional assignment, crystal defects, Fermi resonance, and upon frozen kinetics between two different subsystems. Thus we cannot account for this band using the concepts of conventional molecular spectroscopy, but a soliton model, similar to that proposed by Davydov for -helix in protein, is in satisfactory agreement with the experimental data. © 1984 The American Physical Society

Calcium Ions Promote Formation of Amyloid β-Peptide (1–40) Oligomers Causally Implicated in Neuronal Toxicity of Alzheimer's Disease

Amyloid β-peptide (Aβ) is directly linked to Alzheimer's disease (AD). In its monomeric form, Aβ aggregates to produce fibrils and a range of oligomers, the latter being the most neurotoxic. Dysregulation of Ca2+ homeostasis in aging brains and in neurodegenerative disorders plays a crucial role in numerous processes and contributes to cell dysfunction and death. Here we postulated that calcium may enable or accelerate the aggregation of Aβ. We compared the aggregation pattern of Aβ(1–40) and that of Aβ(1–40)E22G, an amyloid peptide carrying the Arctic mutation that causes early onset of the disease. We found that in the presence of Ca2+, Aβ(1–40) preferentially formed oligomers similar to those formed by Aβ(1–40)E22G with or without added Ca2+, whereas in the absence of added Ca2+ the Aβ(1–40) aggregated to form fibrils. Morphological similarities of the oligomers were confirmed by contact mode atomic force microscopy imaging. The distribution of oligomeric and fibrillar species in different samples was detected by gel electrophoresis and Western blot analysis, the results of which were further supported by thioflavin T fluorescence experiments. In the samples without Ca2+, Fourier transform infrared spectroscopy revealed conversion of oligomers from an anti-parallel β-sheet to the parallel β-sheet conformation characteristic of fibrils. Overall, these results led us to conclude that calcium ions stimulate the formation of oligomers of Aβ(1–40), that have been implicated in the pathogenesis of AD

Application of the PM6 semi-empirical method to modeling proteins enhances docking accuracy of AutoDock

<p>Abstract</p> <p>Background</p> <p>Molecular docking methods are commonly used for predicting binding modes and energies of ligands to proteins. For accurate complex geometry and binding energy estimation, an appropriate method for calculating partial charges is essential. AutoDockTools software, the interface for preparing input files for one of the most widely used docking programs AutoDock 4, utilizes the Gasteiger partial charge calculation method for both protein and ligand charge calculation. However, it has already been shown that more accurate partial charge calculation - and as a consequence, more accurate docking- can be achieved by using quantum chemical methods. For docking calculations quantum chemical partial charge calculation as a routine was only used for ligands so far. The newly developed Mozyme function of MOPAC2009 allows fast partial charge calculation of proteins by quantum mechanical semi-empirical methods. Thus, in the current study, the effect of semi-empirical quantum-mechanical partial charge calculation on docking accuracy could be investigated.</p> <p>Results</p> <p>The docking accuracy of AutoDock 4 using the original AutoDock scoring function was investigated on a set of 53 protein ligand complexes using Gasteiger and PM6 partial charge calculation methods. This has enabled us to compare the effect of the partial charge calculation method on docking accuracy utilizing AutoDock 4 software. Our results showed that the docking accuracy in regard to complex geometry (docking result defined as accurate when the RMSD of the first rank docking result complex is within 2 Å of the experimentally determined X-ray structure) significantly increased when partial charges of the ligands and proteins were calculated with the semi-empirical PM6 method.</p> <p>Out of the 53 complexes analyzed in the course of our study, the geometry of 42 complexes were accurately calculated using PM6 partial charges, while the use of Gasteiger charges resulted in only 28 accurate geometries. The binding affinity estimation was not influenced by the partial charge calculation method - for more accurate binding affinity prediction development of a new scoring function for AutoDock is needed.</p> <p>Conclusion</p> <p>Our results demonstrate that the accuracy of determination of complex geometry using AutoDock 4 for docking calculation greatly increases with the use of quantum chemical partial charge calculation on both the ligands and proteins.</p

Protein Engineering vol.9 no.9 pp.745-754, 19% Alternating charge clusters of side chains: new surface structural invariants observed in calf eye lens gamma-crystallins

A detailed stereochemical analysis of the oppositely charged side chains of amino acid residues on the surface of calf eye lens protein gamma-crystallin B has been carried out. The refined structural data of very high quality obtained at 1.47 A resolution have been used. Charge—charge interactions were considered to be valuable for all the charged oxygen and nitrogen atoms situated at distances, d, between 2.4 and 7.0 A. This means we consider short contact ion pairs as those with interchange distances 2.4 &lt; d ^s 4.0 A and distant contact ion pairs as those with distances 4.0 &lt; d = £ 7.0 A. JHydrogen bonding of the charged atomic groups with the structural water molecules also has been considered. We have not looked at the side groups of histidines which are charged only partially at neutral pH. Five clusters of charged side chains which were large enough were observed. The clusters are comprised of four to six residues which compose 543 % of the total charged residues in the protein. The clusters contain from eight to 12 charged atoms and look like the bent chains of oppositely charged atoms. All clusters are of plane geometry and their maximal linear dimensions are from 11 to 18 A. The root mean square deviations of charged atoms from the cluster plane varied from 0.63 to 0.86 A for four clusters and was only 1.85 A for the largest cluster. All clusters include a number of water molecules situated on the cluster boundary and grouped near the cluster plane. It was shown that the amino acid sequence positions of charged residues are conservative for all the proteins of the gamma-crystallin family of vertebrates including fish, frog, mouse, rat, calf and human. The cluster properties were discussed both in their functional aspect for gamma-crystallins and in other aspects common for globular proteins. As a result, the alternating charge clusters should be considered as newly recognized surface structural invariants. The importance of the charged side chain clusters is claimed for the updated concept of the protein surface

Novel approach for structural identification of protein family: glyoxalase I

Glyoxalase is one of two enzymes of the glyoxalase detoxification system against methylglyoxal and other aldehydes, the metabolites derived from glycolysis. The glyoxalase system is found almost in all living organisms: bacteria, protozoa, plants, and animals, including humans, and is related to the class of ‘life essential proteins’. The enzyme belongs to the expanded Glyoxalase/Bleomycin resistance protein/Dioxygenase superfamily. At present the GenBank contains about 700 of amino acid sequences of this enzyme type, and the Protein Data Bank includes dozens of spatial structures. We have offered a novel approach for structural identification of glyoxalase I protein family, which is based on the selecting of basic representative proteins with known structures. On this basis, six new subfamilies of these enzymes have been derived. Most populated subfamilies A1 and A2 were based on representative human Homo sapiens and bacterial Escherichia coli enzymes. We have found that the principle feature, which defines the subfamilies’ structural differences, is conditioned by arrangement of N- and C-domains inside the protein monomer. Finely, we have deduced the structural classification for the glyoxalase I and assigned about 460 protein sequences distributed among six new subfamilies. Structural similarities and specific differences of all the subfamilies have been presented. This approach can be used for structural identification of thousands of the so-called hypothetical proteins with the known PDB structures allowing to identify many of already existing atomic coordinate entrees