| 1. |
- Brodmerkel, Maxim N., et al.
(författare)
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Rehydration Post-orientation : Investigating Field-Induced Structural Changes via Computational Rehydration
- 2023
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Ingår i: The Protein Journal. - : Springer Nature. - 1572-3887 .- 1875-8355. ; 42:3, s. 205-218
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Tidskriftsartikel (refereegranskat)abstract
- Proteins can be oriented in the gas phase using strong electric fields, which brings advantages for structure determination using X-ray free electron lasers. Both the vacuum conditions and the electric-field exposure risk damaging the protein structures. Here, we employ molecular dynamics simulations to rehydrate and relax vacuum and electric-field exposed proteins in aqueous solution, which simulates a refinement of structure models derived from oriented gas-phase proteins. We find that the impact of the strong electric fields on the protein structures is of minor importance after rehydration, compared to that of vacuum exposure and ionization in electrospraying. The structures did not fully relax back to their native structure in solution on the simulated timescales of 200 ns, but they recover several features, including native-like intra-protein contacts, which suggests that the structures remain in a state from which the fully native structure is accessible. Our fndings imply that the electric fields used in native mass spectrometry are well below a destructive level, and suggest that structures inferred from X-ray difraction from gas-phase proteins are relevant for solution and in vivo conditions, at least after in silico rehydration.
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| 2. |
- Greiner, R, et al.
(författare)
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myo-inositol phosphate isomers generated by the action of a phytase from a malaysian waste-water bacterium
- 2007
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Ingår i: Protein Journal. - : Springer Science and Business Media LLC. - 1572-3887 .- 1875-8355. ; 26:8, s. 577-584
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Tidskriftsartikel (refereegranskat)abstract
- Using a combination of High-Performance Ion Chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by a phytase from a Malaysian waste-water bacterium was established. The data demonstrate that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-I(1,2,3,4,5)P-5, D-I(2,3,4,5)P-4, D-I(2,3,4)P-3, D-I(2,3)P-2 to finally I(2)P. It was estimated that more than 90% of phytate hydrolysis occurs via D-I(1,2,3,4,5)P-5. Thus, the phytase from the Malaysian waste-water bacterium has to be considered a 6-phytase (E.C. 3.1.3.26). A second pathway of minor importance could be proposed which is in accordance with the results obtained from analysis of the dephosphorylation products formed by the action of the phytase under investigation on myo-inositol hexakisphosphate. It proceeds via D/L-I(1,2,4,5,6)P-5, D/L-I(1,2,4,5)P-4, D/L-I(1,2,4)P-3, D/L-I(2,4)P-2 to finally I(2)P.
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| 3. |
- Hirschberg, Daniel, et al.
(författare)
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Identification of endothelial proteins by MALDI-MS using a compact disc microfluidic system.
- 2004
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Ingår i: The Protein Journal. - 1572-3887 .- 1875-8355. ; 23:4
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Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial proteins have been analyzed using two-dimensional (2D) gel electrophoresis and subsequent mass spectrometry, with separate methods for the intervening sample preparations. Compact disc (CD) technology was found to be rapid, giving high overall yield both with ordinary Coomassie staining and with Sypro Ruby staining. Combined with automatic in-gel digestion, the CD technology has great capacity for large numbers of protein analysis, although for limited sample numbers, manual methods can give similar sequence coverage. In a test set of 48 samples, 45 proteins were identified using the CD preparation technique, 32 identified with higher sequence coverage using the CD technique, 7 with higher using ZipTips in a robotic workstation, and 5 with higher coverage using dried droplets of unpurified samples. In the process of these methodological comparisons, basic patterns for 116 endothelial proteins were defined, representing 297 separate protein spots on the 2D gels.
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| 4. |
- Hosseini, A. Najla, et al.
(författare)
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Simulations of Amyloid-Forming Peptides in the Crystal State
- 2023
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Ingår i: The Protein Journal. - : Springer. - 1572-3887 .- 1875-8355. ; 42:3, s. 192-204
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Tidskriftsartikel (refereegranskat)abstract
- There still is little treatment available for amyloid diseases, despite their significant impact on individuals and the social and economic implications for society. One reason for this is that the physical nature of amyloid formation is not understood sufficiently well. Therefore, fundamental research at the molecular level remains necessary to support the development of therapeutics. A few structures of short peptides from amyloid-forming proteins have been determined. These can in principle be used as scaffolds for designing aggregation inhibitors. Attempts to this end have often used the tools of computational chemistry, in particular molecular simulation. However, few simulation studies of these peptides in the crystal state have been presented so far. Hence, to validate the capability of common force fields (AMBER19SB, CHARMM36m, and OPLS-AA/M) to yield insight into the dynamics and structural stability of amyloid peptide aggregates, we have performed molecular dynamics simulations of twelve different peptide crystals at two different temperatures. From the simulations, we evaluate the hydrogen bonding patterns, the isotropic B-factors, the change in energy, the Ramachandran plots, and the unit cell parameters and compare the results with the crystal structures. Most crystals are stable in the simulations but for all force fields there is at least one that deviates from the experimental crystal, suggesting more work is needed on these models.
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| 5. |
- Kivi, Rait, et al.
(författare)
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Kinetics of Acrylodan-Labelled cAMP-Dependent Protein Kinase Catalytic Subunit Denaturation
- 2013
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Ingår i: The Protein Journal. - : Springer Science and Business Media LLC. - 1572-3887 .- 1875-8355. ; 32:7, s. 519-525
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescence spectroscopy was used to study denaturation of cAMP-dependent protein kinase catalytic subunit labeled with an acrylodan moiety. The dye was covalently bound to a cystein residue introduced into the enzyme by replacement of arginine in position 326 in the native sequence, located near the enzyme active center. This labeling had no effect on catalytic activity of the enzyme, but provided possibility to monitor changes in protein structure through measuring the fluorescence spectrum of the dye, which is sensitive to changes in its environment. This method was used to monitor denaturation of the protein kinase catalytic subunit and study the kinetics of this process as well as influence of specific ligands on stability of the protein. Stabilization of the enzyme structure was observed in the presence of adenosine triphosphate, peptide substrate RRYSV and inhibitor peptide PKI[5-24].
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| 6. |
- Kivi, Rait, et al.
(författare)
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Thermodynamic Aspects of cAMP Dependent Protein Kinase Catalytic Subunit Allostery
- 2014
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Ingår i: The Protein Journal. - : Springer Science and Business Media LLC. - 1572-3887 .- 1875-8355. ; 33:4, s. 386-393
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Tidskriftsartikel (refereegranskat)abstract
- Kinetics of thermal inactivation of acrylodan-labeled cAMP dependent protein kinase catalytic subunit, its binary complexes with ATP and peptide inhibitor PKI[5-24], respectively, and the ternary complex involving both of these ligands were studied at different temperatures (5-50 A degrees C). The thermodynamic parameters Delta H and Delta S for ligand binding equilibria as well as for the allosteric interaction between the binding sites of these ligands were obtained by using the Van't Hoff analysis. The results indicated that more inter- and intra-molecular non-covalent bonds were involved in ATP binding with the protein when compared to the peptide binding. Similarly, nucleotide and peptide binding steps were accompanied with different entropy effects, while almost no entropy change accompanied PKI[5-24] binding, suggesting that the protein flexibility was not affected in this case. Differently from the binary complex formation the ternary complex formation was accompanied by a significant entropy change and with intensive formation of new non-covalent interactions (Delta H). At the same time both ligand binding steps as well as the allosteric interaction between ligand binding sites could be described by a common entropy-enthalpy compensation plot, pointing to a similar mechanism of these phenomena. It was concluded that numerous weak interactions govern the allostery of cAMP dependent protein kinase catalytic subunit.
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| 7. |
- Lundborg, Magnus, et al.
(författare)
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On the Path to Optimal Alchemistry
- 2023
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Ingår i: The Protein Journal. - : Springer Nature. - 1572-3887 .- 1875-8355. ; 42:5, s. 477-489
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Tidskriftsartikel (refereegranskat)abstract
- Alchemical free energy calculations have become a standard and widely used tool, in particular for calculating and comparing binding affinities of drugs. Although methods to compute such free energies have improved significantly over the last decades, the choice of path between the end states of interest is usually still the same as two decades ago. We will show that there is a fundamentally arbitrary, implicit choice of parametrization of this path. To address this, the notion of the length of a path or a metric is required. A metric recently introduced in the context of the accelerated weight histogram method also proves to be very useful here. We demonstrate that this metric can not only improve the efficiency of sampling along a given path, but that it can also be used to improve the actual choice of path. For a set of relevant use cases, the combination of these improvements can increase the efficiency of alchemical free energy calculations by up to a factor 16.
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| 8. |
- Mahdi, Rabab, et al.
(författare)
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Heterologous Expression of the Barley (Hordeum vulgare L.) Xantha-f, -g and -h Genes that Encode Magnesium Chelatase Subunits
- 2020
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Ingår i: Protein Journal. - : Springer Science and Business Media LLC. - 1572-3887 .- 1875-8355. ; 39:5, s. 554-562
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Tidskriftsartikel (refereegranskat)abstract
- Biosynthesis of chlorophyll involves several enzymatic reactions of which many are shared with the heme biosynthesis pathway. Magnesium chelatase is the first specific enzyme in the chlorophyll pathway. It catalyzes the formation of Mg-protoporphyrin IX from the insertion of Mg2+ into protoporphyrin IX. The enzyme consists of three subunits encoded by three genes. The three genes are named Xantha-h, Xantha-g and Xantha-f in barley (Hordeum vulgare L.). The products of the genes have a molecular weight of 38, 78 and 148 kDa, respectively, as mature proteins in the chloroplast. Most studies on magnesium chelatase enzymes have been performed using recombinant proteins of Rhodobacter capsulatus, Synechocystis sp. PCC6803 and Thermosynechococcus elongatus, which are photosynthetic bacteria. In the present study we established a recombinant expression system for barley magnesium chelatase with the long-term goal to obtain structural information of this enigmatic enzyme complex from a higher plant. The genes Xantha-h, -g and -f were cloned in plasmid pET15b, which allowed the production of the three subunits as His-tagged proteins in Escherichia coli BL21(DE3)pLysS. The purified subunits stimulated magnesium chelatase activity of barley plastid extracts and produced activity in assays with only recombinant proteins. In preparation for future structural analyses of the barley magnesium chelatase, stability tests were performed on the subunits and activity assays were screened to find an optimal buffer system and pH.
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| 9. |
- Nielsen, Henrik, et al.
(författare)
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A Brief History of Protein Sorting Prediction
- 2019
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Ingår i: The Protein Journal. - : Springer Science and Business Media LLC. - 1572-3887 .- 1875-8355. ; 38:3, s. 200-216
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Forskningsöversikt (refereegranskat)abstract
- Ever since the signal hypothesis was proposed in 1971, the exact nature of signal peptides has been a focus point of research. The prediction of signal peptides and protein subcellular location from amino acid sequences has been an important problem in bioinformatics since the dawn of this research field, involving many statistical and machine learning technologies. In this review, we provide a historical account of how position-weight matrices, artificial neural networks, hidden Markov models, support vector machines and, lately, deep learning techniques have been used in the attempts to predict where proteins go. Because the secretory pathway was the first one to be studied both experimentally and through bioinformatics, our main focus is on the historical development of prediction methods for signal peptides that target proteins for secretion; prediction methods to identify targeting signals for other cellular compartments are treated in less detail.
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| 10. |
- Singh, Amit K, et al.
(författare)
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Bovine Carbonyl Lactoperoxidase Structure at 2.0Å Resolution and Infrared Spectra as a Function of pH.
- 2012
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Ingår i: The protein journal. - : Springer Science and Business Media LLC. - 1875-8355 .- 1572-3887. ; 31:7, s. 598-608
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Tidskriftsartikel (refereegranskat)abstract
- Lactoperoxidase (LPO) is a hemeprotein catalyzing the oxidation of thiocyanate and I(-) into antimicrobials and small aromatic organics after being itself oxidized by H(2)O(2). LPO is excreted by the lungs, mammary glands, found in saliva and tears and protects mammals against bacterial, fungal and viral invasion. The Fe(II) form binds CO which inactivates LPO like many other hemeproteins. We present the 3-dimensional structure of CO-LPO at 2.0Å resolution and infrared (IR) spectra of the iron-bound CO stretch from pH 3 to 8.8 at 1 cm(-1) resolution. The observed Fe-C-O bond angle of 132° is more acute than the electronically related Fe(III), CN-LPO with a Fe-C-N angle of 161°. The orientations of the two ligands are different with the oxygen of CO pointing towards the imidazole of distal His109 while the nitrogen of CN points away, the Fe(II) moves towards His109 while the Fe(III) moves away; both movements are consistent with a hydrogen bond between the distal His109 and CO, but not to the nitrogen of CN-LPO. The IR spectra of CO-LPO exhibit two major CO absorbances with pH dependent relative intensities. Both crystallographic and IR data suggest proton donation to the CO oxygen by His109 with a pK ≈ 4; close to the pH of greatest enzyme turnover. The IR absorbance maxima are consistent with a first order correlation between frequency and Fe(III)/Fe(II) reduction potential at pH 7; both band widths at half-height correlate with electron density donation from Fe(II) to CO as gauged by the reduction potential.
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