| 1. |
- Lehtio, J., et al.
(författare)
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Alpha-amylase inhibitors selected from a combinatorial library of a cellulose binding domain scaffold
- 2000
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Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; 41:3, s. 316-322
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Tidskriftsartikel (refereegranskat)abstract
- A disulfide bridge-constrained cellulose binding domain (CBD,) derived from the cellobiohydrolase Ce17A from Trichoderma reesei has been investigated for use in scaffold engineering to obtain novel binding proteins. The gene encoding the wild-type 36 aa CBDWT domain was first inserted into a phagemid vector and shown to be functionally displayed on M13 filamentous phage as a protein III fusion protein with retained cellulose binding activity. A combinatorial library comprising 46 million variants of the CBD domain was constructed through randomization of 11 positions located at the domain surface and distributed over three separate beta -sheets of the domain. Using the enzyme porcine alpha-amylase (PPA) as target in biopannings, two CBD variants showing selective binding to the enzyme were characterized. Reduction and iodoacetamide blocking of cysteine residues in selected CBD variants resulted in a loss of binding activity, indicating a conformation dependent binding. Interestingly, further studies showed that the selected CBD variants were capable of competing with the binding of the amylase inhibitor acarbose to the enzyme. In addition, the enzyme activity could be partially inhibited by addition of soluble protein, suggesting that the selected CBD variants bind to the active site of the enzyme.
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| 2. |
- Andersson, K, et al.
(författare)
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Kinetic characterization of the interaction of the Z-fragment of protein A with mouse-IgG3 in a volume in chemical space.
- 1999
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Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; 37:3
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Tidskriftsartikel (refereegranskat)abstract
- The kinetic rate parameters for the interaction between a single domain analogue of staphylococcal protein A (Z) and a mouse-IgG3 monoclonal antibody (MAb) were measured in Hepes buffer with different chemical additives. Five buffer ingredients (pH, NaCl, DMSO, EDTA, and KSCN) were varied simultaneously in 16 experiments following a statistical experimental plan. The 16 buffers thus spanned a volume in chemical space. A mathematical model, using data from the buffer composition, was developed and used to predict apparent kinetic parameters in five new buffers within the spanned volume. Association and dissociation parameters were measured in the new buffers, and these agreed with the predicted values, indicating that the model was valid within the spanned volume. The pattern of variation of the kinetic parameters in relation to buffer composition was different for association and dissociation, such that pH influenced both association and dissociation and NaCl influenced only dissociation. This indicated that the recognition mechanism (association) and the stability of the formed complex (dissociation) involve different binding forces, which can be further investigated by kinetic studies in systematically varied buffers.
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| 3. |
- Berndt, Kurt D, et al.
(författare)
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Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long-time molecular dynamics calculations in explicit water
- 1996
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Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; 24, s. 304-313
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Tidskriftsartikel (refereegranskat)abstract
- The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using "realistic" potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.
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| 4. |
- Hargbo, Jeanette, et al.
(författare)
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Hidden Markov Models That Use Predicted Secondary Structures For Fold Recognition
- 1999
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Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; 36:1, s. 68-76
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Tidskriftsartikel (refereegranskat)abstract
- There are many proteins that share the same fold but have no clear sequence similarity. To predict the structure of these proteins, so called protein fold recognition methods have been developed. During the last few years, improvements of protein fold recognition methods have been achieved through the use of predicted secondary structures (Rice and Eisenberg, J Mol Biol 1997;267:1026-1038), as well as by using multiple sequence alignments in the form of hidden Markov models (HMM) (Karplus et al., Proteins Suppl 1997;1:134-139). To test the performance of different fold recognition methods, we have developed a rigorous benchmark where representatives for all proteins of known structure are matched against each other. Using this benchmark, we have compared the performance of automatically-created hidden Markov models with standard-sequence-search methods. Further, we combine the use of predicted secondary structures and multiple sequence alignments into a combined method that performs better than methods that do not use this combination of information. Using only single sequences, the correct fold of a protein was detected for 10% of the test cases in our benchmark. Including multiple sequence information increased this number to 16%, and when predicted secondary structure information was included as well, the fold was correctly identified in 20% of the cases. Moreover, if the correct secondary structure was used, 27% of the proteins could be correctly matched to a fold. For comparison, blast2, fasta, and ssearch identifies the fold correctly in 13-17% of the cases. Thus, standard pairwise sequence search methods perform almost as well as hidden Markov models in our benchmark. This is probably because the automatically-created multiple sequence alignments used in this study do not contain enough diversity and because the current generation of hidden Markov models do not perform very well when built from a few sequences. Proteins 1999;36:68-76
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| 5. |
- Knapp, S, et al.
(författare)
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Thermal unfolding of small proteins with SH3 domain folding pattern
- 1998
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Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; 31:3, s. 309-319
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Tidskriftsartikel (refereegranskat)abstract
- The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy, The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec), At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities. (C) 1998 Wiley-Liss, Inc.
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| 6. |
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| 7. |
- Schneider, Gisbert, et al.
(författare)
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Feature-extraction from endopeptidase cleavage sites in mitochondrial targeting peptides
- 1998
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Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; 30:1, s. 49-60
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Tidskriftsartikel (refereegranskat)abstract
- Cleavage sites in nuclear-encoded mitochondrial protein targeting peptides (mTPs) from mammals, yeast, and plants have been analysed for characteristic physicochemical features using statistical methods, perceptrons, multilayer neural networks, and self-organizing feature maps, Three different sequence motifs were found, revealing loosely defined arginine motifs with Arg in positions -10, -3, and -2. A self-organizing feature map was able to cluster these three types of endopeptidase target sites but did not identify any species-specific characteristics in mTPs, Neural networks were used to define local sequence features around precursor cleavage sites.
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| 8. |
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| 9. |
- Agullo, Luis, et al.
(författare)
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Computational exploration of the binding mode of heme-dependent stimulators into the active catalytic domain of soluble guanylate cyclase
- 2016
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 84:10, s. 1534-1548
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Tidskriftsartikel (refereegranskat)abstract
- Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction, and myocardial infarction. One of its agonists, BAY 41-2272 (Riociguat), has been recently approved for treatment of pulmonary arterial hypertension (PHA), while some others are in clinical phases of development. However, the location of the binding sites for the two known types of agonists, heme-dependent stimulators and heme-independent activators, is a matter of debate, particularly for the first group where both a location on the regulatory (H-NOX) and on the catalytic domain have been suggested by different authors. Here, we address its potential location on the catalytic domain, the unique well characterized at the structural level, by an in silico approach. Homology models of the catalytic domain of sGC in inactive or active conformations were constructed using the structure of previously described crystals of the catalytic domains of inactive sGCs (2WZ1, 3ET6) and of active adenylate cyclase (1CJU). Each model was submitted to six independent molecular dynamics simulations of about 1 s. Docking of YC-1, a classic heme-dependent stimulator, to all frames of representative trajectories of inactive and active conformations, followed by calculation of absolute binding free energies with the linear interaction energy (LIE) method, revealed a potential high-affinity binding site on the active structure. The site, located between the pseudo-symmetric and the catalytic site just over the loop (2)-(3), does not overlap with the forskolin binding site on adenylate cyclases.
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| 10. |
- Aifa, Sami, 1967-, et al.
(författare)
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Electrostatic interactions of peptides flanking the tyrosine kinase domain in the epidermal growth factor receptor provides a model for intracellular dimerization and autophosphorylation
- 2006
-
Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 62:4, s. 1036-1043
-
Tidskriftsartikel (refereegranskat)abstract
- The mechanism by which ligand-activated EGFR induces autophosphorylation via dimerization is not fully understood. Structural studies have revealed an extracellular loop mediated receptor dimerization. We have previously presented experimental data showing the involvement of a positive 13 amino acid peptide (R645-R657, P13+) from the intracellular juxtamembrane domain (JM) of EGFR important for intracellular dimerization and autophosphorylation. A model was presented that suggest that P13+ interacts with a negative peptide (D979-E991, P13-) positioned distal to the tyrosine kinase domain in the opposite EGFR monomer. The present work shows additional data strengthening this model. In fact, by analyzing protein sequences of 21 annotated ErbB proteins from 9 vertebrate genomes, we reveal the high conservation of peptides P13+ and P13- with regard to their sequence as well as their position relative to the tyrosine kinase (TK) domain. Moreover in silico structure modeling of these ErbB intracellular domains supports a general electrostatic P13+/P13- interaction, implying that the C-terminal of one receptor monomer is facing the TK domain of the other monomer in the receptor dimer and vice versa. This model provides new insights into the molecular mechanism of ErbB receptor activation and suggests a new strategy to pharmacologically interfering with ErbB receptor activity. © 2005 Wiley-Liss, Inc.
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| 11. |
- Andersson, David C., 1978-, et al.
(författare)
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Mapping of ligand-binding cavities in proteins
- 2010
-
Ingår i: Proteins. - : John Wiley & Sons, Inc. - 0887-3585 .- 1097-0134. ; 78:6, s. 1408-1422
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Tidskriftsartikel (refereegranskat)abstract
- The complex interactions between proteins and small organic molecules (ligands) are intensively studied because they play key roles in biological processes and drug activities. Here, we present a novel approach to characterize and map the ligand-binding cavities of proteins without direct geometric comparison of structures, based on Principal Component Analysis of cavity properties (related mainly to size, polarity, and charge). This approach can provide valuable information on the similarities and dissimilarities, of binding cavities due to mutations, between-species differences and flexibility upon ligand-binding. The presented results show that information on ligand-binding cavity variations can complement information on protein similarity obtained from sequence comparisons. The predictive aspect of the method is exemplified by successful predictions of serine proteases that were not included in the model construction. The presented strategy to compare ligand-binding cavities of related and unrelated proteins has many potential applications within protein and medicinal chemistry, for example in the characterization and mapping of "orphan structures", selection of protein structures for docking studies in structure-based design, and identification of proteins for selectivity screens in drug design programs.
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| 12. |
- Andre, Ingemar, et al.
(författare)
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Computational assessment of folding energy landscapes in heterodimeric coiled coils
- 2018
-
Ingår i: Proteins. - : Wiley-Blackwell. - 0887-3585 .- 1097-0134. ; 86:7, s. 790-801
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Tidskriftsartikel (refereegranskat)abstract
- The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs-into-holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes.
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| 13. |
- Berglund, Anders, et al.
(författare)
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ProVal : A protein-scoring function for the selection of native and near-native folds
- 2004
-
Ingår i: Proteins. - : Wiley-Liss, Inc. - 0887-3585 .- 1097-0134. ; 54:2, s. 289-302
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Tidskriftsartikel (refereegranskat)abstract
- A low-resolution scoring function for the selection of native and near-native structures from a set of predicted structures for a given protein sequence has been developed. The scoring function, ProVal (Protein Validate), used several variables that describe an aspect of protein structure for which the proximity to the native structure can be assessed quantitatively. Among the parameters included are a packing estimate, surface areas, and the contact order. A partial least squares for latent variables (PLS) model was built for each candidate set of the 28 decoy sets of structures generated for 22 different proteins using the described parameters as independent variables. The C(alpha) RMS of the candidate structures versus the experimental structure was used as the dependent variable. The final generalized scoring function was an average of all models derived, ensuring that the function was not optimized for specific fold classes or method of structure generation of the candidate folds. The results show that the crystal structure was scored best in 64% of the 28 test sets and was clearly separated from the decoys in many examples. In all the other cases in which the crystal structure did not rank first, it ranked within the top 10%. Thus, although ProVal could not distinguish between predicted structures that were similar overall in fold quality due to its inherently low resolution, it can clearly be used as a primary filter to eliminate approximately 90% of fold candidates generated by current prediction methods from all-atom modeling and further evaluation. The correlation between the predicted and actual C(alpha) RMS values varies considerably between the candidate fold sets.
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| 14. |
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| 15. |
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| 16. |
- Bredenberg, Johan, et al.
(författare)
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Conformational states of the glucocorticoid receptor DNA-binding domain from molecular dynamics simulations
- 2002
-
Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 49:1, s. 24-36
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Tidskriftsartikel (refereegranskat)abstract
- Molecular dynamics simulations (MD) have been performed on variant crystal and NMR-derived structures of the glucocorticoid receptor DNA-binding domain (GR DBD). A loop region five residues long, the so-called D-box, exhibits significant flexibility, and transient perturbations of the tetrahedral geometry of two structurally important Cys4 zinc finger are seen, coupled to conformational changes in the D-box. In some cases, one of the Cys ligands to zinc exchanges with water, although no global distortion of the protein structure is observed. Thus, from MD simulation, dynamics of the D-box could partly be explained by solvent effects in conjunction with structural reformation of the zinc finger.
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| 17. |
- Brunne, R M, et al.
(författare)
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Structure and internal dynamics of the bovine pancreatic trypsin inhibitor in aqueous solution from long-time molecular dynamics simulations
- 1995
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 23:1, s. 49-62
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Tidskriftsartikel (refereegranskat)abstract
- Structural and dynamic properties of bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution are investigated using two molecular dynamics (MD) simulations: one of 1.4 ns length and one of 0.8 ns length in which atom-atom distance bounds derived from NMR spectroscopy are included in the potential energy function to make the trajectory satisfy these experimental data more closely. The simulated properties of BPTI are compared with crystal and solution structures of BPTI, and found to be in agreement with the available experimental data. The best agreement with experiment was obtained when atom-atom distance restraints were applied in a time-averaged manner in the simulation. The polypeptide segments found to be most flexible in the MD simulations coincide closely with those showing differences between the crystal and solution structures of BPTI.
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| 18. |
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| 19. |
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| 20. |
- Cheng, Jianlin, et al.
(författare)
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Estimation of model accuracy in CASP13
- 2019
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 87:12, s. 1361-1377
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Tidskriftsartikel (refereegranskat)abstract
- Methods to reliably estimate the accuracy of 3D models of proteins are both a fundamental part of most protein folding pipelines and important for reliable identification of the best models when multiple pipelines are used. Here, we describe the progress made from CASP12 to CASP13 in the field of estimation of model accuracy (EMA) as seen from the progress of the most successful methods in CASP13. We show small but clear progress, that is, several methods perform better than the best methods from CASP12 when tested on CASP13 EMA targets. Some progress is driven by applying deep learning and residue‐residue contacts to model accuracy prediction. We show that the best EMA methods select better models than the best servers in CASP13, but that there exists a great potential to improve this further. Also, according to the evaluation criteria based on local similarities, such as lDDT and CAD, it is now clear that single model accuracy methods perform relatively better than consensus‐based methods.
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| 21. |
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| 22. |
- Di Matteo, Adele, et al.
(författare)
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Structural and functional characterization of CcmG from Pseudomonas aeruginosa, a key component of the bacterial cytochrome c maturation apparatus
- 2010
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 78:10, s. 2213-2221
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Tidskriftsartikel (refereegranskat)abstract
- The cytochrome c maturation process is carried out in the bacterial periplasm, where some specialized thiol-disulfide oxidoreductases work in close synergy for the correct reduction of oxidized apocytochrome before covalent heme attachment. We present a structural and functional characterization of the soluble periplasmic domain of CcmG from the opportunistic pathogen P. aeruginosa (Pa-CcmG), a component of the protein machinery involved in cyt c maturation in gram-negative bacteria. X-ray crystallography reveals that Pa-CcmG is a TRX-like protein; high-resolution crystal structures show that the oxidized and the reduced forms of the enzyme are identical except for the active-site disulfide. The standard redox potential was calculated to be E-0' = -0.213 V at pH 7.0; the pK(a) of the active site thiols were pK(a) = 6.13 +/- 0.05 for the N-terminal Cys74 and pK(a) = 10.5 +/- 0.17 for the C-terminal Cys77. Experiments were carried out to characterize and isolate the mixed disulfide complex between Pa-CcmG and Pa-CcmH (the other redox active component of System I in P. aeruginosa). Our data indicate that the target disulfide of this TRX-like protein is not the intramolecular disulfide of oxidized Pa-CcmH, but the intermolecular disulfide formed between Cys28 of Pa-CcmH and DTNB used for the in vitro experiments. This observation suggests that, in vivo, the physiological substrate of Pa-CcmG may be the mixed-disulfide complex between Pa-CcmH and apo-cyt. Proteins 2010; 78:2213-2221. (C) 2010 Wiley-Liss, Inc.
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| 23. |
- Dourado, Daniel F. A. R., et al.
(författare)
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A multiscale approach to predicting affinity changes in protein-protein interfaces
- 2014
-
Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 82:10, s. 2681-2690
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Tidskriftsartikel (refereegranskat)abstract
- Substitution mutations in protein-protein interfaces can have a substantial effect on binding, which has consequences in basic and applied biomedical research. Experimental expression, purification, and affinity determination of protein complexes is an expensive and time-consuming means of evaluating the effect of mutations, making a fast and accurate in silico method highly desirable. When the structure of the wild-type complex is known, it is possible to economically evaluate the effect of point mutations with knowledge based potentials, which do not model backbone flexibility, but these have been validated only for single mutants. Substitution mutations tend to induce local conformational rearrangements only. Accordingly, ZEMu (Zone Equilibration of Mutants) flexibilizes only a small region around the site of mutation, then computes its dynamics under a physics-based force field. We validate with 1254 experimental mutants (with 1-15 simultaneous substitutions) in a wide variety of different protein environments (65 protein complexes), and obtain a significant improvement in the accuracy of predicted Delta Delta G.
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| 24. |
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| 25. |
- Eklund, M., et al.
(författare)
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Anti-idiotypic protein domains selected from protein A-based affibody libraries
- 2002
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 48:3, s. 454-462
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Tidskriftsartikel (refereegranskat)abstract
- Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fe binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K.) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.
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| 26. |
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| 27. |
- Elofsson, Arne, et al.
(författare)
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Methods for estimation of model accuracy in CASP12
- 2018
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 86:S1, s. 361-373
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Tidskriftsartikel (refereegranskat)abstract
- Methods to reliably estimate the quality of 3D models of proteins are essential drivers for the wide adoption and serious acceptance of protein structure predictions by life scientists. In this article, the most successful groups in CASP12 describe their latest methods for estimates of model accuracy (EMA). We show that pure single model accuracy estimation methods have shown clear progress since CASP11; the 3 top methods (MESHI, ProQ3, SVMQA) all perform better than the top method of CASP11 (ProQ2). Although the pure single model accuracy estimation methods outperform quasi-single (ModFOLD6 variations) and consensus methods (Pcons, ModFOLDclust2, Pcomb-domain, and Wallner) in model selection, they are still not as good as those methods in absolute model quality estimation and predictions of local quality. Finally, we show that when using contact-based model quality measures (CAD, lDDT) the single model quality methods perform relatively better.
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| 28. |
- Friedman, Ran, et al.
(författare)
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On the orientation of the catalytic dyad in aspartic proteases
- 2010
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 78:6, s. 1575-1582
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Tidskriftsartikel (refereegranskat)abstract
- The recent re-refinement of the X-ray structure of apo plasmepsin II from Plasmodium falciparum suggests that the two carboxylate groups in the catalytic dyad are noncoplanar, (Robbins et al., Acta Crystallogr D Biol Crystallogr 2009;65: 294–296) in remarkable contrast with the vast majority of structures of aspartic proteases. Here, evidence for the noncoplanarity of the catalytic aspartates is provided by analysis of multiple explicit water molecular dynamics (MD) simulations of plasmepsin II, human β-secretase, and HIV-protease. In the MD runs of plasmepsin II, the angle between the planes of the two carboxylates of the catalytic dyad is almost always in the range 60°–120°, in agreement with the perpendicular orientation in the re-refined X-ray structure. The noncoplanar arrangement is prevalent also in the β-secretase simulations, as well as in the runs with the inhibitor-bound proteases. Quantum-mechanics calculations provide further evidence that before catalysis the noncoplanar arrangement is favored energetically in eukaryotic aspartic proteases. Remarkably, the coplanar orientation of the catalytic dyad is observed in MD simulations of HIV-protease at 100 K but not at 300 K, which indicates that the noncoplanar arrangement is favored by conformational entropy. This finding suggests that the coplanar orientation in the crystal structures of apo aspartic proteases is promoted by the very low temperature used for data collection (usually around 100 K).
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| 29. |
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| 30. |
- Friedman, Ran
(författare)
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The molecular mechanism behind resistance of the kinase FLT3 to the inhibitor quizartinib
- 2017
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Ingår i: Proteins. - : John Wiley & Sons. - 0887-3585 .- 1097-0134. ; 85:11, s. 2143-2152
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Tidskriftsartikel (refereegranskat)abstract
- Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is a drug target for leukemias. Several potent inhibitors of FLT3 exist, and bind to the inactive form of the enzyme. Unfortunately, resistance due to mutations in the kinase domain of FLT3 limits the therapeutic effects of these inhibitors. As in many other cases, it is not straightforward to explain why certain mutations lead to drug resistance. Extensive fully atomistic molecular dynamics (MD) simulations of FLT3 were carried out with an inhibited form (FLT-quizartinib complex), a free (apo) form, and an active conformation. In all cases, both the wild type (wt) proteins and two resistant mutants (D835F and Y842H) were studied. Analysis of the simulations revealed that impairment of protein-drug interactions cannot explain the resistance mutations in question. Rather, it appears that the active state of the mutant forms is perturbed by the mutations. It is therefore likely that perturbation of deactivation of the protein (which is necessary for drug binding) is responsible for the reduced affinity of the drug to the mutants. Importantly, this study suggests that it is possible to explain the source of resistance by mutations in FLT3 by an analysis of unbiased MD simulations.
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| 31. |
- Friedman, Ran, et al.
(författare)
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Wild type and mutants of the HET-s(218-289) prion show different flexibility at fibrillar ends : A simulation study
- 2014
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 82:3, s. 399-404
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Tidskriftsartikel (refereegranskat)abstract
- The C-terminal segment (residues 218–289) of the HET-s protein of the filamentous fungus Podosporina anserina is a prion-forming domain. The structural model of the HET-s(218–289) amyloid fibril based on solid-state nuclear magnetic resonance (NMR) restraints shows a β solenoid topology which is comprised of a β-sheet core and interconnecting loops. For the single-point mutants Phe286Ala and Trp287Ala, slower aggregation rates in vitro and loss of prionic infectivity have been reported recently. Here we have used molecular dynamics to compare the flexibility of the mutants and wild type. The simulations, initiated from a trimeric aggregate extracted from the NMR structural model, show structural stability on a 100-ns time scale for wild type and mutants. Analysis of the fluctuations along the simulations reveals that the mutants are less flexible than the wild type in the C-terminal segment at only one of the two external monomers. Analysis of interaction energy and buried accessible surface indicates that residue Phe286 in particular is stabilized in the Trp287Ala mutant. The simulation results provide an atomistic explanation of the suggestion (based on indirect experimental evidence) that flexibility at the protofibril end(s) is required for fibril elongation. Moreover, they provide further evidence that the growth of the HET-s amyloid fibril is directional.
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| 32. |
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| 33. |
- Griese, Julia J., et al.
(författare)
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Structure and DNA-binding activity of the Pyrococcus furiosus SMC protein hinge domain
- 2011
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 79:2, s. 558-568
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Tidskriftsartikel (refereegranskat)abstract
- Structural Maintenance of Chromosomes (SMC) proteins are essential for a wide range of processes including chromosome structure and dynamics, gene regulation, and DNA repair. While bacteria and archaea have one SMC protein that forms a homodimer, eukaryotes possess three distinct SMC complexes, consisting of heterodimeric pairs of six different SMC proteins. SMC holocomplexes additionally contain several specific regulatory subunits. The bacterial SMC complex is required for chromosome condensation and segregation. In eukaryotes, this function is carried out by the condensin (SMC2-SMC4) complex. SMC proteins consist of N-terminal and C-terminal domains that fold back onto each other to create an ATPase "head" domain, connected to a central "hinge" domain via a long coiled-coil region. The hinge domain mediates dimerization of SMC proteins and binds DNA. This activity implicates a direct involvement of the hinge domain in the action of SMC proteins on DNA. We studied the SMC hinge domain from the thermophilic archaeon Pyrococcus furiosus. Its crystal structure shows that the SMC hinge domain fold is largely conserved between archaea and bacteria as well as eukarya. Like the eukaryotic condensin hinge domain, the P. furiosus SMC hinge domain preferentially binds single-stranded DNA (ssDNA), but its affinity for DNA is weaker than that of its eukaryotic counterpart, and point mutations reveal that its DNA-binding surface is more confined. The ssDNA-binding activity of its hinge domain might play a role in the DNA-loading process of the prokaryotic SMC complex during replication.
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| 34. |
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| 35. |
- Gurmu, Daniel, et al.
(författare)
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The crystal structure of the protein YhaK from Escherichia coli reveals a new subclass of redox sensitive enterobacterial bicupins
- 2009
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 74:1, s. 18-31
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Tidskriftsartikel (refereegranskat)abstract
- YhaK is a protein of unknown function found in low abundance in the cytosol of Escherichia coli. DNA array studies have revealed that YhaK is strongly up-regulated by nitroso-glutathione (GSNO) and also displays a 12-fold increase in expression during biofilm growth of E. coli 83972 and VR50 in human urine. We have determined the YhaK crystal structure and demonstrated that in vitro YhaK is a good marker for monitoring oxidative stresses in E. coli. The YhaK protein structure shows a bicupin fold where the two cupin domains are crosslinked with one intramolecular disulfide bond (Cys10 to Cys204). We found that the third cysteine in YhaK, Cys122, is oxidized to a sulfenic acid. Two chloride ions are found in the structure, one close to the reactive Cys122, and the other on a hydrophobic surface close to a symmetry-related molecule. There are major structural differences at the N-terminus of YhaK compared with similar structures that also display the bicupin fold (YhhW and hPirin). YhaK showed no quercetinase and peroxidase activity. However, reduced YhaK was very sensitive to reactive oxygen species (ROS). The complete, functional E. coli glutaredoxin or thioredoxin systems protected YhaK from oxidation. E. coli thioredoxin reductase and NADPH produced ROS and caused oxidation and oligomerization of reduced YhaK. Taken together, we propose that YhaK is the first of a new sub-class of bicupins that lack the canonical cupin metal-binding residues of pirins and may be involved in chloride binding and/or sensing of oxidative stress in enterobacteria.
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| 36. |
- Gutmanas, Aleksandras, et al.
(författare)
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Specific DNA recognition by the Antp homeodomain: MD simulations of specific and nonspecific complexes.
- 2004
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Ingår i: Proteins. - : Wiley. - 1097-0134. ; 57:4, s. 772-82
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Tidskriftsartikel (refereegranskat)abstract
- Four molecular dynamics simulation trajectories of complexes between the wild-type or a mutant Antennapedia homeodomain and 2 DNA sequences were generated in order to probe the mechanisms governing the specificity of DNA recognition. The starting point was published affinity measurements showing that a single protein mutation combined with a replacement of 2 base pairs yields a new high-affinity complex, whereas the other combinations, with changes on only 1 macromolecule, exhibited lower affinity. The simulations of the 4 complexes yielded fluctuating networks of interaction. On average, these networks differ significantly, explaining the switch of affinity caused by the alterations in the macromolecules. The network of mostly hydrogen-bonding interactions involving several water molecules, which was suggested both by X-ray and NMR structures of the wild-type homeodomain and its DNA operator sequence, could be reproduced in the trajectory. More interestingly, the high-affinity complex with alterations in both the protein and the DNA yielded again a dynamic but very tight network of intermolecular interactions, however, attributing a significantly stronger role to direct hydrophobic interactions at the expense of water bridges. The other 2 homeodomain-DNA complexes, with only 1 molecule altered, show on average over the trajectories a clearly reduced number of protein-DNA interactions. The observations from these simulations suggest specific experiments and thus close the circle formed by biochemical, structural, and computational studies. The shift from a water-dominated to a more "dry" interface may prove important in the design of proteins binding DNA in a specific manner.
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| 37. |
- Hansson, Mats, et al.
(författare)
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Purification, crystallization, and preliminary X-ray analysis of Bacillus subtilis ferrochelatase
- 1995
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Ingår i: Proteins. - : Wiley. - 0887-3585. ; 23:4, s. 607-609
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Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis ferrochelatase (EC 4.99.1.1), the final enzyme in protoheme IX biosynthesis, was produced with an inducible T7 RNA polymerase expression system in Escherichia coli and purified from the soluble cell fraction. It was crystallized from polyethylene glycol solution using the microseeding technique. The crystals diffract to a minimum Bragg spacing of 2.1 A. The space group is P4(2) with unit cell dimensions a = b = 50.2 A, c = 120.1 A.
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| 38. |
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| 39. |
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| 40. |
- Hassan, Sameer, et al.
(författare)
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Elucidation of ligand binding and dimerization of NADPH:protochlorophyllide (Pchlide) oxidoreductase from pea (Pisum sativum L.) by structural analysis and simulations
- 2021
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Ingår i: Proteins-Structure Function and Bioinformatics. - : Wiley. - 0887-3585 .- 1097-0134. ; 89:10, s. 1300-1314
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Tidskriftsartikel (refereegranskat)abstract
- NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. It is one of few known photoenzymes, which catalyzes the light-activated trans-reduction of the C17-C18 double bond of Pchlide's porphyrin ring. Due to the light requirement, dark-grown angiosperms cannot synthesize chlorophyll. No crystal structure of POR is available, so to improve understanding of the protein's three-dimensional structure, its dimerization, and binding of ligands (both the cofactor NADPH and substrate Pchlide), we computationally investigated the sequence and structural relationships among homologous proteins identified through database searches. The results indicate that alpha 4 and alpha 7 helices of monomers form the interface of POR dimers. On the basis of conserved residues, we predicted 11 functionally important amino acids that play important roles in POR binding to NADPH. Structural comparison of available crystal structures revealed that they participate in formation of binding pockets that accommodate the Pchlide ligand, and that five atoms of the closed tetrapyrrole are involved in non-bonding interactions. However, we detected no clear pattern in the physico-chemical characteristics of the amino acids they interact with. Thus, we hypothesize that interactions of these atoms in the Pchlide porphyrin ring are important to hold the ligand within the POR binding site. Analysis of Pchlide binding in POR by molecular docking and PELE simulations revealed that the orientation of the nicotinamide group is important for Pchlide binding. These findings highlight the complexity of interactions of porphyrin-containing ligands with proteins, and we suggest that fit-inducing processes play important roles in POR-Pchlide interactions.
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| 41. |
- Hassan, Sameer, et al.
(författare)
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Ligand Binding Site Comparison - LiBiSCo - a web-based tool for analyzing interactions between proteins and ligands to explore amino acid specificity within active sites
- 2021
-
Ingår i: Proteins-Structure Function and Bioinformatics. - : Wiley. - 0887-3585 .- 1097-0134. ; 89:11, s. 1530-1540
-
Tidskriftsartikel (refereegranskat)abstract
- Interaction between protein and ligands are ubiquitous in a biological cell, and understanding these interactions at the atom level in protein-ligand complexes is crucial for structural bioinformatics and drug discovery. Here, we present a web-based protein-ligand interaction application named Ligand Binding Site Comparison (LiBiSCo) for comparing the amino acid residues interacting with atoms of a ligand molecule between different protein-ligand complexes available in the Protein Data Bank (PDB) database. The comparison is performed at the ligand atom level irrespectively of having binding site similarity or not between the protein structures of interest. The input used in LiBiSCo is one or several PDB IDs of protein-ligand complex(es) and the tool returns a list of identified interactions at ligand atom level including both bonded and non-bonded interactions. A sequence profile for the interaction for each ligand atoms is provided as a WebLogo. The LiBiSco is useful in understanding ligand binding specificity and structural promiscuity among families that are structurally unrelated. The LiBiSCo tool can be accessed through .
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| 42. |
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| 43. |
- Hvidsten, Torgeir R., et al.
(författare)
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Local descriptors of protein structure : A systematic analysis of the sequence-structure relationship in proteins using short- and long-range interactions
- 2009
-
Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 75:4, s. 870-884
-
Tidskriftsartikel (refereegranskat)abstract
- Local protein structure representations that incorporate long-range contacts between residues are often considered in protein structure comparison but have found relatively little use in structure prediction where assembly from single backbone fragments dominates. Here, we introduce the concept of local descriptors of protein structure to characterize local neighborhoods of amino acids including short- and long-range interactions. We build a library of recurring local descriptors and show that this library is general enough to allow assembly of unseen protein structures. The library could on average re-assemble 83% of 119 unseen structures, and showed little or no performance decrease between homologous targets and targets with folds not represented among domains used to build it. We then systematically evaluate the descriptor library to establish the level of the sequence signal in sets of protein fragments of similar geometrical conformation. In particular, we test whether that signal is strong enough to facilitate correct assignment and alignment of these local geometries to new sequences. We use the signal to assign descriptors to a test set of 479 sequences with less than 40% sequence identity to any domain used to build the library, and show that on average more than 50% of the backbone fragments constituting descriptors can be correctly aligned. We also use the assigned descriptors to infer SCOP folds, and show that correct predictions can be made in many of the 151 cases where PSI-BLAST was unable to detect significant sequence similarity to proteins in the library. Although the combinatorial problem of simultaneously aligning several fragments to sequence is a major bottleneck compared with single is that correct alignments imply correct long range distance constraints. The lack of these constraints is most likely the major reason why structure prediction methods fail to consistently produce adequate models when good templates are unavailable or undetectable. Thus, we believe that the current study offers new and valuable insight into the prediction of sequence-structure relationships in proteins.
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| 44. |
- Illergård, Kristoffer, et al.
(författare)
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Structure is three to ten times more conserved than sequence-A study of structural response in protein cores
- 2009
-
Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 77:3, s. 499-508
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Tidskriftsartikel (refereegranskat)abstract
- Protein structures change during evolution in response to mutations. Here, we analyze the mapping between sequence and structure in a set of structurally aligned protein domains. To avoid artifacts, we restricted our attention only to the core components of these structures. We found that on average, using different measures of structural change, protein cores evolve linearly with evolutionary distance (amino acid substitutions per site). This is true irrespective of which measure of structural change we used, whether RMSD or discrete structural descriptors for secondary structure, accessibility, or contacts. This linear response allows us to quantify the claim that structure is more conserved than sequence. Using structural alphabets of similar cardinality to the sequence alphabet, structural cores evolve three to ten times slower than sequences. Although we observed an average linear response, we found a wide variance. Different domain families varied fivefold in structural response to evolution. An attempt to categorically analyze this variance among subgroups by structural and functional category revealed only one statistically significant trend. This trend can be explained by the fact that beta-sheets change faster than alpha-helices, most likely due to that they are shorter and that change occurs at the ends of the secondary structure elements. Proteins 2009; 77:499-508. (C) 2009 Wiley-Liss, Inc.
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| 45. |
- Illergård, Kristoffer, et al.
(författare)
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Why are polar residues within the membrane core evolutionary conserved?
- 2011
-
Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 79:1, s. 79-91
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Tidskriftsartikel (refereegranskat)abstract
- Here, we present a study of polar residues within the membrane core of alpha-helical membrane proteins. As expected, polar residues are less frequent in the membrane than expected. Further, most of these residues are buried within the interior of the protein and are only rarely exposed to lipids. However, the polar groups often border internal water filled cavities, even if the rest of the sidechain is buried. A survey of their functional roles in known structures showed that the polar residues are often directly involved in binding of small compounds, especially in channels and transporters, but other functions including proton transfer, catalysis, and selectivity have also been attributed to these proteins. Among the polar residues histidines often interact with prosthetic groups in photosynthetic-and oxidoreductase-related proteins, whereas pro-lines often are required for conformational changes of the proteins. Indeed, the polar residues in the membrane core are more conserved than other residues in the core, as well as more conserved than polar residues outside the membrane. The reason is twofold; they are often (i) buried in the interior of the protein and (ii) directly involved in the function of the proteins. Finally, a method to identify which polar residues are present within the membrane core directly from protein sequences was developed. Applying the method to the set of all human membrane proteins the prediction indicates that polar residues were most frequent among active transporter proteins and GPCRs, whereas infrequent in families with few transmembrane regions, such as non-GPCR receptors. Proteins 2011; 79: 79-91.
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| 46. |
- Irbäck, Anders, et al.
(författare)
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Spontaneous beta-barrel formation: an all-atom study of Abeta(16-22) oligomerization
- 2008
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Ingår i: Proteins. - : Wiley. - 0887-3585. ; 71:1, s. 207-214
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Tidskriftsartikel (refereegranskat)abstract
- Using all-atom Monte Carlo simulations with implicit water, combined with a cluster size analysis, we study the aggregation of A16-22, a peptide capable of forming amyloid fibrils. We consider a system of six initially randomly oriented A16-22 peptides, and investigate the thermodynamics and structural properties of aggregates formed by this system. The system is unaggregated without ordered secondary structure at high temperature, and forms -sheet rich aggregates at low temperature. At the crossover between these two regimes, we find that clusters of all sizes occur, whereas the -strand content is low. In one of several runs, we observe the spontaneous formation of a -barrel with six antiparallel strands. The -barrel stands out as the by far most long-lived aggregate seen in our simulations.
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| 47. |
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| 48. |
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| 49. |
- Jones, TA, et al.
(författare)
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CASP3 comparative modeling evaluation
- 1999
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Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; , s. 30-46
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Tidskriftsartikel (refereegranskat)abstract
- We report our evaluation of the CASP3 comparative modelling competition. Our analysis covers the accuracy of the over-all fold, the bridging of insertions and deletions, and the adding of side-chains. We describe our attempts at automating aspects of the evaluation.
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| 50. |
- Julenius, Karin, et al.
(författare)
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Coupling of ligand binding and dimerization of helix-loop-helix peptides: Spectroscopic and sedimentation analyses of calbindin D-9k EF-hands
- 2002
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Ingår i: Proteins. - : Wiley. - 0887-3585. ; 47:3, s. 323-333
-
Tidskriftsartikel (refereegranskat)abstract
- Isolated Ca2+-binding EF-hand peptides have a tendency to dimerize. This study is an attempt to account for the coupled equilibria of Ca2+-binding and peptide association for two EF-hands with strikingly different loop sequence and net charge. We have studied each of the two separate EF-hand fragments from calbindin D-9k. A series of Ca2+-titrations at different peptide concentrations were monitored by CD and fluorescence spectroscopy. All data were fitted simultaneously to both a complete model of all possible equilibrium intermediates and a reduced model not including dimerization in the absence of Ca2+. Analytical ultracentrifugation shows that the peptides may occur as monomers or dimers depending on the solution conditions. Our results show strikingly different behavior for the two EF-hands. The fragment containing the N-terminal EF-hand shows a strong tendency to dimerize in the Ca2+-bound state. The average Ca2+-affinity is 3.5 orders of magnitude lower than for the intact protein. We observe a large apparent cooperativity of Ca2+ binding for the overall process from Ca2+-free monomer to fully loaded dimer, showing that a Ca2+-free EF-hand folds upon dimerization to a Ca2+-bound EF-hand, thereby presenting a preformed binding site to the second Ca2+-ion. The C-terminal EF-hand shows a much smaller tendency to dimerize, which may be related to its larger net negative charge. In spite of the differences in dimerization behavior, the Ca2+ affinities of both EF-hand fragments are similar and in the range IgK = 4.6-5.3.
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