1,436 results on '"protein structures"'
Search Results
102. Cryo‐EM Resolves Molecular Recognition Of An Optojasp Photoswitch Bound To Actin Filaments In Both Switch States.
- Author
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Pospich, Sabrina, Küllmer, Florian, Nasufović, Veselin, Funk, Johanna, Belyy, Alexander, Bieling, Peter, Arndt, Hans‐Dieter, and Raunser, Stefan
- Subjects
- *
ACTIN , *F-actin , *CYTOSKELETON , *EUKARYOTIC cells , *BINDING sites , *MOLECULAR recognition - Abstract
Actin is essential for key processes in all eukaryotic cells. Cellpermeable optojasps provide spatiotemporal control of the actin cytoskeleton, confining toxicity and potentially rendering F‐actin druggable by photopharmacology. Here, we report cryo electron microscopy (cryo‐EM) structures of both isomeric states of one optojasp bound to actin filaments. The high‐resolution structures reveal for the first time the pronounced effects of photoswitching a functionalized azobenzene. By characterizing the optojasp binding site and identifying conformational changes within F‐actin that depend on the optojasp isomeric state, we refine determinants for the design of functional F‐actin photoswitches. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
103. A missense mutation sheds light on a novel structure–function relationship of RANKL.
- Author
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Qiu, Heng, Qin, An, Cheng, Taksum, Chim, Shek M., Smithers, Luke, Chen, Kai, Song, Dezhi, Liu, Qian, Zhao, Jinmin, Wang, Chao, Teguh, Dian, Zhang, Ge, Tickner, Jennifer, Vrielink, Alice, Pavlos, Nathan J., and Xu, Jiake
- Subjects
- *
TRANCE protein , *MISSENSE mutation , *OSTEOCLASTS , *TUMOR necrosis factors , *RECESSIVE genes , *GLUTAMIC acid , *WESTERN immunoblotting , *LYSINE - Abstract
The tumor necrosis factor (TNF)‐like core domain of receptor activator of nuclear factor‐κB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast‐poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF‐like core domain. However, the structure–function relationship of this mutation is not clear. Sequence‐based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site‐directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcP staining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast‐related signal transduction. However, no antagonistic effect was found in M200s against wild‐type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor–ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199‐induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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104. Conformation Transition Kinetics of Silk Fibroin in Aqueous Solution Explored Using Circular Dichroism Spectroscopy.
- Author
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Lee, Sora, Kim, Soo Hyun, Jo, You‐Young, Ju, Wan‐Taek, Kim, Hyun‐Bok, and Kweon, HaeYong
- Subjects
- *
SILK fibroin , *AQUEOUS solutions , *CHEMICAL stability , *EXPONENTIAL functions , *SPECTROMETRY , *CIRCULAR dichroism , *MODULATIONAL instability - Abstract
Silk fibroin (SF) solution is characterized by its structural instability. Despite many efforts to uncover the structural features of SF, the time‐dependent conformation transition behaviors of SF solutions prepared under different conditions have not yet been investigated in detail. Therefore, in this study, the conformation transition changes of SF were dynamically monitored in an aqueous state using circular dichroism (CD) spectroscopic analysis. CD spectra of SF continuously change during ethanol treatment and storage, showing growth with a positive peak at 197 nm and a negative peak at 218 nm, consistent with β‐sheet development. Additionally, the time points for SF to begin and complete changing structures, including its changing rates, were confirmed. In particular, the transition kinetics follow the one‐phase exponential decay function, quantitatively showing a longer dissolution time and lower storage temperature delay for the development of β‐sheets with lower rate constants. In conclusion, our investigation provides useful information on the intrinsic properties of SF and environmental factors that can affect the structural stability of SF. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
105. 3D proteomics : analysis of proteins and protein complexes by chemical cross-linking and mass spectrometry
- Author
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Chen, Zhuo A., Barlow, Paul, and Rappsilber, Juri
- Subjects
572 ,3D proteomics ,Chemical cross-linking ,Mass spectrometry ,Protein structures ,Protein-protein interactions - Abstract
The concept of 3D proteomics is a technique that couples chemical cross-linking with mass spectrometry and has emerged as a tool to study protein conformations and protein-protein interactions. In this thesis I present my work on improving the analytical workflow and developing applications for 3D proteomics in the structural analysis of proteins and protein complexes through four major tasks. I. As part of the technical development of an analytical workflow for 3D proteomics, a cross-linked peptide library was created by cross-linking a mixture of synthetic peptides. Analysis of this library generated a large dataset of cross-linked peptides. Characterizing the general features of cross-linked peptides using this dataset allowed me to optimize the settings for mass spectrometric analysis and to establish a charge based enrichment strategy for cross-linked peptides. In addition to this, 1185 manually validated high resolution fragmentation spectra gave an insight into general fragmentation behaviours of cross-linked peptides and facilitated the development of a cross-linked peptide search algorithm. II. The advanced 3D proteomics workflow was applied to study the architecture of the 670 kDa 15-subunit Pol II-TFIIF complex. This work established 3D proteomics as a structure analysis tool for large multi-protein complexes. The methodology was validated by comparing 3D proteomics analysis results and the X-ray crystallographic data on the 12-subunit Pol II core complex. Cross-links observed from the Pol II–TFIIF complex revealed interactions between the Pol II and TFIIF at the peptide level, which also reflected the dynamic nature of Pol II-TFIIF structure and implied possible Pol II conformational changes induced by TFIIF binding. III. Conformational changes of flexible protein molecules are often associated with specific functions of proteins or protein complexes. To quantitatively measure the differences between protein conformations, I developed a quantitative 3D proteomics strategy which combines isotope labelling and cross-linking with mass spectrometry and database searching. I applied this approach to detect in solution the conformational differences between complement component C3 and its active form C3b in solution. The quantitative cross-link data confirmed the previous observation made by X-ray crystallography. Moreover, this analysis detected the spontaneous hydrolysis of C3 in both C3 and C3b samples. The architecture of hydrolyzed C3-C3(H2O) was proposed based on the quantified cross-links and crystal structure of C3 and C3b, which revealed that C3(H2O) adopted the functional domain arrangement of C3b. This work demonstrated that quantitative 3D proteomics is a valuable tool for conformational analysis of proteins and protein complexes. IV. Encouraged by the achievements in the above applications with relatively large amounts of highly purified material, I explored the application of 3D proteomics on affinity purified tagged endogenous protein complexes. Using an on-beads process which connected cross-linking and an affinity purification step directly, provided increased sensitivity through minimized sample handling. A charge-based enrichment step was carried out to improve the detection of cross-linked peptides. The occurrence of cross-links between complexes was monitored by a SILAC based control. Cross-links observed from low micro-gram amounts of single-step purified endogenous protein complexes provided insights into the structural organization of the S. cerevisiae Mad1-Mad2 complex and revealed a conserved coiled-coil interruption in the S. cerevisiae Ndc80 complex. With this endeavour I have demonstrated that 3D proteomics has become a valuable tool for studying structure of proteins and protein complexes.
- Published
- 2011
106. Protein-Based Hydrogels: Promising Materials for Tissue Engineering
- Author
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Niyousha Davari, Negar Bakhtiary, Mehran Khajehmohammadi, Soulmaz Sarkari, Hamidreza Tolabi, Farnaz Ghorbani, and Behafarid Ghalandari
- Subjects
tissue engineering ,protein-based hydrogel ,protein structures ,unfolding ,interaction ,Organic chemistry ,QD241-441 - Abstract
The successful design of a hydrogel for tissue engineering requires a profound understanding of its constituents’ structural and molecular properties, as well as the proper selection of components. If the engineered processes are in line with the procedures that natural materials undergo to achieve the best network structure necessary for the formation of the hydrogel with desired properties, the failure rate of tissue engineering projects will be significantly reduced. In this review, we examine the behavior of proteins as an essential and effective component of hydrogels, and describe the factors that can enhance the protein-based hydrogels’ structure. Furthermore, we outline the fabrication route of protein-based hydrogels from protein microstructure and the selection of appropriate materials according to recent research to growth factors, crucial members of the protein family, and their delivery approaches. Finally, the unmet needs and current challenges in developing the ideal biomaterials for protein-based hydrogels are discussed, and emerging strategies in this area are highlighted.
- Published
- 2022
- Full Text
- View/download PDF
107. Temperature as an Extra Dimension in Multidimensional Protein NMR Spectroscopy.
- Author
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Shchukina, Alexandra, Małecki, Paweł, Mateos, Borja, Nowakowski, Michał, Urbańczyk, Mateusz, Kontaxis, Georg, Kasprzak, Paweł, Conrad‐Billroth, Clara, Konrat, Robert, and Kazimierczuk, Krzysztof
- Subjects
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NUCLEAR magnetic resonance spectroscopy , *RADON transforms , *PROTEIN structure , *COMPRESSED sensing , *PROTEINS - Abstract
NMR spectroscopy is a particularly informative method for studying protein structures and dynamics in solution; however, it is also one of the most time‐consuming. Modern approaches to biomolecular NMR spectroscopy are based on lengthy multidimensional experiments, the duration of which grows exponentially with the number of dimensions. The experimental time may even be several days in the case of 3D and 4D spectra. Moreover, the experiment often has to be repeated under several different conditions, for example, to measure the temperature‐dependent effects in a spectrum (temperature coefficients (TCs)). Herein, a new approach that involves joint sampling of indirect evolution times and temperature is proposed. This allows TCs to be measured through 3D spectra in even less time than that needed to acquire a single spectrum by using the conventional approach. Two signal processing methods that are complementary, in terms of sensitivity and resolution, 1) dividing data into overlapping subsets followed by compressed sensing reconstruction, and 2) treating the complete data set with a variant of the Radon transform, are proposed. The temperature‐swept 3D HNCO spectra of two intrinsically disordered proteins, osteopontin and CD44 cytoplasmic tail, show that this new approach makes it possible to determine TCs and their non‐linearities effectively. Non‐linearities, which indicate the presence of a compact state, are particularly interesting. The complete package of data acquisition and processing software for this new approach are provided. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
108. Structural dissection of two redox proteins from the shipworm symbiont Teredinibacter turnerae.
- Author
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Rajagopal BS, Yates N, Smith J, Paradisi A, Tétard-Jones C, Willats WGT, Marcus S, Knox JP, Firdaus-Raih M, Henrissat B, Davies GJ, Walton PH, Parkin A, and Hemsworth GR
- Subjects
- Oxidation-Reduction, Mixed Function Oxygenases, Polysaccharides, Gammaproteobacteria
- Abstract
The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria., (open access.)
- Published
- 2024
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109. Solving protein structures by combining structure prediction, molecular replacement and direct-methods-aided model completion.
- Author
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Li Z, Fan H, and Ding W
- Subjects
- Crystallography, X-Ray, Proteins chemistry, Hydrolases
- Abstract
Highly accurate protein structure prediction can generate accurate models of protein and protein-protein complexes in X-ray crystallography. However, the question of how to make more effective use of predicted models for completing structure analysis, and which strategies should be employed for the more challenging cases such as multi-helical structures, multimeric structures and extremely large structures, both in the model preparation and in the completion steps, remains open for discussion. In this paper, a new strategy is proposed based on the framework of direct methods and dual-space iteration, which can greatly simplify the pre-processing steps of predicted models both in normal and in challenging cases. Following this strategy, full-length models or the conservative structural domains could be used directly as the starting model, and the phase error and the model bias between the starting model and the real structure would be modified in the direct-methods-based dual-space iteration. Many challenging cases (from CASP14) have been tested for the general applicability of this constructive strategy, and almost complete models have been generated with reasonable statistics. The hybrid strategy therefore provides a meaningful scheme for X-ray structure determination using a predicted model as the starting point., (open access.)
- Published
- 2024
- Full Text
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110. A Two‐Armed Probe for In‐Cell DEER Measurements on Proteins*.
- Author
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Miao, Qing, Zurlo, Enrico, Bruin, Donny, Wondergem, Joeri A. J., Timmer, Monika, Blok, Anneloes, Heinrich, Doris, Overhand, Mark, Huber, Martina, and Ubbink, Marcellus
- Subjects
- *
DEER , *DICTYOSTELIUM discoideum , *SPIN labels , *ETHERS , *ELECTRON paramagnetic resonance spectroscopy , *PROTEIN structure - Abstract
The application of double electron‐electron resonance (DEER) with site‐directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin‐label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two‐armed GdIII complexes, GdIII‐CLaNP13a/b/c were synthesized. Rather than the disulfide linkage of most other CLaNP molecules, a thioether linkage was used to avoid reductive dissociation of the linker. The doubly GdIII labeled N55C/V57C/K147C/T151C variants of T4Lysozyme were measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent with results from paramagnetic NMR. A narrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
111. Probing Cooperativity of N‐Terminal Domain Orientations in the p97 Molecular Machine: Synergy Between NMR Spectroscopy and Cryo‐EM.
- Author
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Huang, Rui, Ripstein, Zev A., Rubinstein, John L., and Kay, Lewis E.
- Subjects
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NUCLEAR magnetic resonance spectroscopy , *MOLECULAR orientation , *PROTEIN structure - Abstract
The hexameric p97 enzyme plays an integral role in cellular homeostasis. Large changes to the orientation of its N‐terminal domains (NTDs), corresponding to NTD‐down (p97‐ADP) or NTD‐up (p97‐ATP), accompany ATP hydrolysis. The NTDs in a series of p97 disease mutants interconvert rapidly between up and down conformations when p97 is in the ADP‐bound state. While the populations of up and down NTDs can be determined from bulk measurements, information about the cooperativity of the transition between conformations is lacking. Here we use cryo‐EM to determine populations of the 14 unique up/down NTD states of the homo‐hexameric R95G disease‐causing p97 ring, showing that NTD orientations do not depend on those of neighboring subunits. In contrast, NMR studies establish that inter‐protomer cooperativity is important for regulating the orientation of NTDs in p97 particles comprising mixtures of different subunits, such as wild‐type and R95G, emphasizing the synergy between cryo‐EM and NMR in establishing how the components of p97 function. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
112. Mechanistic Insights into the Interactions of Ras Subfamily GTPases with the SPN Domain of Autism‐associated SHANK3†.
- Author
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Xu, Xiaolong, Liu, Jianping, Wang, Yingli, Wang, Yaru, Gong, Xinyu, and Pan, Lifeng
- Subjects
- *
RAS proteins , *ETIOLOGY of diseases , *AUTISM spectrum disorders , *CRYSTAL structure , *INSIGHT - Abstract
Summary of main observation and conclusion: The active Ras subfamily GTPases, Rap1 and Ras, can be specifically recognized by the SPN domain of SHANK3, mutations of which are associated with many neuropsychiatric diseases such as autism spectrum disorder (ASD). However, the mechanistic bases underlying the interactions of SHANK3 SPN and those Ras subfamily proteins are still elusive. Here, we reported the crystal structures of SHANK3 SPN in complex with the GTP‐bound Rap1b and the Ras‐mimetic Rap1b E30D/K31E double mutant. In addition to uncovering the detailed molecular mechanism governing the specific interactions of SHANK3 SPN with those Ras subfamily proteins, the determined structures also reveal a general binding mode between SHANK3 SPN and its associated Ras subfamily proteins. Finally, our study also provides mechanistic insights into two ASD‐causing R12C and L68P mutations found in the SPN domain of SHANK3, and expands our understanding of the etiology of neuropsychiatric diseases caused by defective SHANK3. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
113. Impact of protein dynamics on secondary structure prediction.
- Author
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de Brevern, Alexandre G.
- Subjects
- *
FORECASTING , *PROTEIN structure , *AMINO acid sequence , *PROTEIN stability , *MOLECULAR dynamics , *PROTEIN conformation - Abstract
Protein 3D structures support their biological functions. As the number of protein structures is negligible in regards to the number of available protein sequences, prediction methodologies relying only on protein sequences are essential tools. In this field, protein secondary structure prediction (PSSPs) is a mature area, and is considered to have reached a plateau. Nonetheless, proteins are highly dynamical macromolecules, a property that could impact the PSSP methods. Indeed, in a previous study, the stability of local protein conformations was evaluated demonstrating that some regions easily changed to another type of secondary structure. The protein sequences of this dataset were used by PSSPs and their results compared to molecular dynamics to investigate their potential impact on the quality of the secondary structure prediction. Interestingly, a direct link is observed between the quality of the prediction and the stability of the assignment to the secondary structure state. The more stable a local protein conformation is, the better the prediction will be. The secondary structure assignment not taken from the crystallized structures but from the conformations observed during the dynamics slightly increase the quality of the secondary structure prediction. These results show that evaluation of PSSPs can be done differently, but also that the notion of dynamics can be included in development of PSSPs and other approaches such as de novo approaches. Image 1 • Assessment of protein secondary structure prediction was done in the light of protein dynamics. • A direct link was observed between the quality of the prediction and the stability of the assignment to the secondary structure state. • More a local protein conformation was stable, better was the prediction. • The notion of dynamics can be included in development of protein secondary structure prediction and other approaches. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
114. Mechanistic Insights into the Interactions of Ras Subfamily GTPases with the SPN Domain of Autism‐associated SHANK3†.
- Author
-
Xu, Xiaolong, Liu, Jianping, Wang, Yingli, Wang, Yaru, Gong, Xinyu, and Pan, Lifeng
- Subjects
RAS proteins ,ETIOLOGY of diseases ,AUTISM spectrum disorders ,CRYSTAL structure ,INSIGHT - Abstract
Summary of main observation and conclusion: The active Ras subfamily GTPases, Rap1 and Ras, can be specifically recognized by the SPN domain of SHANK3, mutations of which are associated with many neuropsychiatric diseases such as autism spectrum disorder (ASD). However, the mechanistic bases underlying the interactions of SHANK3 SPN and those Ras subfamily proteins are still elusive. Here, we reported the crystal structures of SHANK3 SPN in complex with the GTP‐bound Rap1b and the Ras‐mimetic Rap1b E30D/K31E double mutant. In addition to uncovering the detailed molecular mechanism governing the specific interactions of SHANK3 SPN with those Ras subfamily proteins, the determined structures also reveal a general binding mode between SHANK3 SPN and its associated Ras subfamily proteins. Finally, our study also provides mechanistic insights into two ASD‐causing R12C and L68P mutations found in the SPN domain of SHANK3, and expands our understanding of the etiology of neuropsychiatric diseases caused by defective SHANK3. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
115. Interaction between Soluble and Membrane-Embedded Potassium Channel Peptides Monitored by Fourier Transform Infrared Spectroscopy
- Author
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Abbott, Geoffrey W, Ramesh, Bala, Srai, Surjit K, and Hinderberger, Dariush
- Subjects
dependent k+ channel ,inactivation gate ,voltage-sensor ,secondary structure ,protein structures ,putative receptor ,sodium-channel ,domain ,conformation ,information - Published
- 2012
116. Biochemical, Structural and Molecular Dynamics Analyses of the Potential Virulence Factor RipA from Yersinia pestis
- Author
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Torres, Rodrigo, Swift, Robert V., Chim, Nicholas, Wheatley, Nicole, Lan, Benson, Atwood, Brian R., Pujol, Celine, Sankaran, Banu, Bliska, James B., Amaro, Rommie E., and Goulding, Celia W.
- Subjects
coenzyme-a-transferase ,glutaconate coa-transferase ,pig-heart ,acidaminococcus-fermentans ,protein structures ,identification ,simulation ,enzyme ,intermediate ,purification - Abstract
Human diseases are attributed in part to the ability of pathogens to evade the eukaryotic immune systems. A subset of these pathogens has developed mechanisms to survive in human macrophages. Yersinia pestis, the causative agent of the bubonic plague, is a predominately extracellular pathogen with the ability to survive and replicate intracellularly. A previous study has shown that a novel rip (required for intracellular proliferation) operon (ripA, ripB and ripC) is essential for replication and survival of Y. pestis in postactivated macrophages, by playing a role in lowering macrophage-produced nitric oxide (NO) levels. A bioinformatics analysis indicates that the rip operon is conserved among a distally related subset of macrophage-residing pathogens, including Burkholderia and Salmonella species, and suggests that this previously uncharacterized pathway is also required for intracellular survival of these pathogens. The focus of this study is ripA, which encodes for a protein highly homologous to 4-hydroxybutyrate-CoA transferase; however, biochemical analysis suggests that RipA functions as a butyryl-CoA transferase. The 1.9 Å X-ray crystal structure reveals that RipA belongs to the class of Family I CoA transferases and exhibits a unique tetrameric state. Molecular dynamics simulations are consistent with RipA tetramer formation and suggest a possible gating mechanism for CoA binding mediated by Val227. Together, our structural characterization and molecular dynamic simulations offer insights into acyl-CoA specificity within the active site binding pocket, and support biochemical results that RipA is a butyryl-CoA transferase. We hypothesize that the end product of the rip operon is butyrate, a known anti-inflammatory, which has been shown to lower NO levels in macrophages. Thus, the results of this molecular study of Y. pestis RipA provide a structural platform for rational inhibitor design, which may lead to a greater understanding of the role of RipA in this unique virulence pathway.
- Published
- 2011
117. Development of genetic algorithm for optimisation of predicted membrane protein structures
- Author
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Minaji-Moghaddam, Noushin
- Subjects
572 ,C710 Applied Molecular Biology ,Biophysics and Biochemistry ,protein structures ,membrane protein structure ,drug discovery ,genetic algorithm - Abstract
Due to the inherent problems with their structural elucidation in the laboratory, the computational prediction of membrane protein structure is an essential step toward understanding the function of these leading targets for drug discovery. In this work, the development of a genetic algorithm technique is described that is able to generate predictive 3D structures of membrane proteins in an ab initio fashion that possess high stability and similarity to the native structure. This is accomplished through optimisation of the distances between TM regions and the end-on rotation of each TM helix. The starting point for the genetic algorithm is from the model of general TM region arrangement predicted using the TMRelate program. From these approximate starting coordinates, the TMBuilder program is used to generate the helical backbone 3D coordinates. The amino acid side chains are constructed using the MaxSprout algorithm. The genetic algorithm is designed to represent a TM protein structure by encoding each alpha carbon atom starting position, the starting atom of the initial residue of each helix, and operates by manipulating these starting positions. To evaluate each predicted structure, the SwissPDBViewer software (incorporating the GROMOS force field software) is employed to calculate the free potential energy. For the first time, a GA has been successfully applied to the problem of predicting membrane protein structure. Comparison between newly predicted structures (tests) and the native structure (control) indicate that the developed GA approach represents an efficient and fast method for refinement of predicted TM protein structures. Further enhancement of the performance of the GA allows the TMGA system to generate predictive structures with comparable energetic stability and reasonable structural similarity to the native structure.
- Published
- 2007
118. Membrane Chemistry Tunes the Structure of a Peptide Transporter.
- Author
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Lasitza‐Male, Tanya, Bartels, Kim, Jungwirth, Jakub, Wiggers, Felix, Rosenblum, Gabriel, Hofmann, Hagen, and Löw, Christian
- Subjects
- *
MEMBRANE proteins , *CHEMISTRY , *MEMBRANE lipids , *PROTEIN structure , *LIPIDS - Abstract
Membrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities, high‐resolution structures are usually obtained in artificial detergents. To understand whether and how lipids guide membrane protein function, we use single‐molecule FRET to probe the dynamics of DtpA, a member of the proton‐coupled oligopeptide transporter (POT) family, in various lipid environments. We show that detergents trap DtpA in a dynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity, allowing an opening to the extracellular side and a sampling of all relevant states. Bilayer compositions tune the abundance of these states. A novel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups. Hence, chemical diversity of membranes translates into structural diversity, with the current POT structures only sampling a portion of the full structural space. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
119. Caught in the Hinact: Crystal Structure and Spectroscopy Reveal a Sulfur Bound to the Active Site of an O2‐stable State of [FeFe] Hydrogenase.
- Author
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Rodríguez‐Maciá, Patricia, Galle, Lisa M., Bjornsson, Ragnar, Lorent, Christian, Zebger, Ingo, Yoda, Yoshitaka, Cramer, Stephen P., DeBeer, Serena, Span, Ingrid, and Birrell, James A.
- Subjects
- *
HYDROGENASE , *INFRARED spectroscopy , *CRYSTAL structure , *X-ray spectroscopy , *SPECTROMETRY - Abstract
[FeFe] hydrogenases are the most active H2 converting catalysts in nature, but their extreme oxygen sensitivity limits their use in technological applications. The [FeFe] hydrogenases from sulfate reducing bacteria can be purified in an O2‐stable state called Hinact. To date, the structure and mechanism of formation of Hinact remain unknown. Our 1.65 Å crystal structure of this state reveals a sulfur ligand bound to the open coordination site. Furthermore, in‐depth spectroscopic characterization by X‐ray absorption spectroscopy (XAS), nuclear resonance vibrational spectroscopy (NRVS), resonance Raman (RR) spectroscopy and infrared (IR) spectroscopy, together with hybrid quantum mechanical and molecular mechanical (QM/MM) calculations, provide detailed chemical insight into the Hinact state and its mechanism of formation. This may facilitate the design of O2‐stable hydrogenases and molecular catalysts. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
120. Cellular Synthesis and X‐ray Crystal Structure of a Designed Protein Heterocatenane.
- Author
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Liu, Yajie, Duan, Zelin, Fang, Jing, Zhang, Fan, Xiao, Junyu, and Zhang, Wen‐Bin
- Subjects
- *
PROTEIN structure , *CRYSTAL structure , *GEL permeation chromatography , *X-rays , *PROTEIN synthesis , *LIQUID chromatography-mass spectrometry - Abstract
Herein, we report the biosynthesis of protein heterocatenanes using a programmed sequence of multiple post‐translational processing events including intramolecular chain entanglement, in situ backbone cleavage, and spontaneous cyclization. The approach is general, autonomous, and can obviate the need for any additional enzymes. The catenane topology was convincingly proven using a combination of SDS‐PAGE, LC‐MS, size exclusion chromatography, controlled proteolytic digestion, and protein crystallography. The X‐ray crystal structure clearly shows two mechanically interlocked protein rings with intact folded domains. It opens new avenues in the nascent field of protein‐topology engineering. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
121. Functional Characterization and Crystal Structure of the Type II Peptidyl Carrier Protein ColA1a in Collismycins Biosynthesis†.
- Author
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Ma, Xueyang, Wang, Guiyang, Liu, Tan, Chi, Changbiao, Zhang, Zhongyi, Yang, Donghui, Liu, Wen, and Ma, Ming
- Subjects
- *
CARRIER proteins , *CRYSTAL structure , *PICOLINIC acid , *AMINO acids , *PROTEINS - Abstract
Summary of main observation and conclusion: Collismycins (COLs) are antibiotics characterized by a 2,2′‐bipyridine (2,2′‐BP) core composed of a trisubstituted ring A and an unmodified ring B. The 2,2′‐BP core, which possesses metal‐chelating ability and plays key roles in various biological activities of COLs, is biosynthesized by a nonribosomal peptide synthetase (NRPS)‐polyketide synthase (PKS) hybrid machinery. The starter module of the NRPS‐PKS hybrid machinery consists of a type II peptidyl carrier protein (PCP) ColA1a and an adenylation protein ColA1b. We here report the functional characterization of ColA1a and ColA1b in vitro, confirming their functions in selection and loading of picolinic acid (PA), instead of normal amino acid substrates, as the origin of ring B in COLs. The 2.1 Å crystal structure of ColA1a was solved, revealing structural features including the additional helices α1a, α1b and missing helix α3, which may reflect unique interactions of ColA1a with other NRPS‐PKS proteins/domains or substrate. Primary and tertiary structural comparison of ColA1a with other PCPs revealed the structural basis for their typical α‐helical bundle, providing a better understanding of the structural flexibility of PCPs. These results facilitate the starter module engineering for the generation of COL derivatives with ring B modifications in the future. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
122. Functional Characterization and Crystal Structure of the Type II Peptidyl Carrier Protein ColA1a in Collismycins Biosynthesis†.
- Author
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Ma, Xueyang, Wang, Guiyang, Liu, Tan, Chi, Changbiao, Zhang, Zhongyi, Yang, Donghui, Liu, Wen, and Ma, Ming
- Subjects
CARRIER proteins ,CRYSTAL structure ,PICOLINIC acid ,AMINO acids ,PROTEINS - Abstract
Summary of main observation and conclusion: Collismycins (COLs) are antibiotics characterized by a 2,2′‐bipyridine (2,2′‐BP) core composed of a trisubstituted ring A and an unmodified ring B. The 2,2′‐BP core, which possesses metal‐chelating ability and plays key roles in various biological activities of COLs, is biosynthesized by a nonribosomal peptide synthetase (NRPS)‐polyketide synthase (PKS) hybrid machinery. The starter module of the NRPS‐PKS hybrid machinery consists of a type II peptidyl carrier protein (PCP) ColA1a and an adenylation protein ColA1b. We here report the functional characterization of ColA1a and ColA1b in vitro, confirming their functions in selection and loading of picolinic acid (PA), instead of normal amino acid substrates, as the origin of ring B in COLs. The 2.1 Å crystal structure of ColA1a was solved, revealing structural features including the additional helices α1a, α1b and missing helix α3, which may reflect unique interactions of ColA1a with other NRPS‐PKS proteins/domains or substrate. Primary and tertiary structural comparison of ColA1a with other PCPs revealed the structural basis for their typical α‐helical bundle, providing a better understanding of the structural flexibility of PCPs. These results facilitate the starter module engineering for the generation of COL derivatives with ring B modifications in the future. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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123. Haruspex: A Neural Network for the Automatic Identification of Oligonucleotides and Protein Secondary Structure in Cryo‐Electron Microscopy Maps.
- Author
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Mostosi, Philipp, Schindelin, Hermann, Kollmannsberger, Philip, and Thorn, Andrea
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PROTEIN structure , *AUTOMATIC identification , *CONVOLUTIONAL neural networks , *PROTEOMICS - Abstract
In recent years, three‐dimensional density maps reconstructed from single particle images obtained by electron cryo‐microscopy (cryo‐EM) have reached unprecedented resolution. However, map interpretation can be challenging, in particular if the constituting structures require de‐novo model building or are very mobile. Herein, we demonstrate the potential of convolutional neural networks for the annotation of cryo‐EM maps: our network Haruspex has been trained on a carefully curated set of 293 experimentally derived reconstruction maps to automatically annotate RNA/DNA as well as protein secondary structure elements. It can be straightforwardly applied to newly reconstructed maps in order to support domain placement or as a starting point for main‐chain placement. Due to its high recall and precision rates of 95.1 % and 80.3 %, respectively, on an independent test set of 122 maps, it can also be used for validation during model building. The trained network will be available as part of the CCP‐EM suite. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
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124. Variation in RNA‐editing sites of chloroplast protein‐coding genes in early‐maturity mutant induced by carbon‐ion beam in Sweet Sorghum.
- Author
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Liu, Ruiyuan, Li, Wenjian, Lu, Dong, Li, Jiangang, Qu, Ying, Jin, Wenjie, Dong, Xicun, and Prasad, Manoj
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SORGO , *CHLOROPLASTS , *PROTEIN structure , *GENES , *CHLOROPLAST DNA , *RNA editing - Abstract
Using prediction and experimental determination, we compared the RNA‐editing sites in chloroplast protein‐coding genes in an early‐maturity mutant (KFJT‐1) induced by carbon‐ion beam irradiation in sweet sorghum and its wild type (KFJT‐CK). Twenty‐two and 20 editing sites were identified in the KFJT‐CK and KFJT‐1 chloroplast, respectively, and the differential editing sites between KFJT‐CK and KFJT‐1 were mainly in mat, ndh and rpo genes. Furthermore, mutations in RNA‐editing sites in KFJT‐1 affected the structure of the encoded protein, which implying that RNA‐editing events might influence the function of essential genes. Finally, a comparative analysis of the RNA‐editing events between KFJT‐1 and other 10 Poaceae species revealed that the RNA‐editing sites in matK‐1002, rpoC1‐1163 and rpoC2‐242 were unique to KFJT‐1. This suggested that RNA‐editing events due to carbon‐ion beam irradiation affected the structure of the encode proteins, thereby influenced their biological functions. This study is the first to report on the variation in chloroplast RNA‐editing patterns induced by carbon‐ion beams irradiation, and its effect on the chloroplast in KFJT‐1 at the post‐transcriptional level. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
125. Molecular Dynamics Simulations of the Elusive Matrix‐Open State of Mitochondrial ADP/ATP Carrier.
- Author
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Škulj, Sanja, Brkljača, Zlatko, and Vazdar, Mario
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MOLECULAR dynamics , *PROTEIN analysis , *MEMBRANE proteins , *PROTEIN structure - Abstract
We studied and compared in detail an elusive matrix open (m‐state) and cytoplasmic open (c‐state) state of ADP/ATP carrier (AAC) protein embedded in the DOPC bilayer by microsecond molecular dynamics (MD) simulations. We analyzed both states with and without cardiolipin (CDL) molecules, with a special emphasis on the recently obtained crystallographic structure of the AAC m‐state. The obtained results show that both states of the protein are stable in the DOPC bilayer and impermeable to water. In comparison with the c‐state of AAC, the m‐state is more dynamic, but at the same time possesses a larger occluded area in the protein cavity. Both states of the protein are less flexible in simulations when CDL molecules are present, which is especially visible for the m‐state. Finally, the analysis of the protein conformational changes during MD simulations shows that protein parts at the protein/lipid boundaries are prone to larger conformational changes in contrast to central region of the protein embedded in the bilayer core, thus further supporting the cycling mechanism suggested for ADP/ATP exchange by AAC. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
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126. Bovine Serum Albumin Catalysed Hydrogen and Deuterium Evolution at Mercury Electrodes.
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Dorčák, Vlastimil, Černocká, Hana, and Paleček, Emil
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MERCURY electrodes , *KINETIC isotope effects , *HYDROGEN evolution reactions , *PROTEIN structure , *BLOOD proteins , *SERUM albumin , *DEUTERIUM , *PROTON transfer reactions - Abstract
The hydrogen evolution reaction (HER), catalysed by proteins at mercury electrodes and reflected in chronopotentiometric stripping peak H, provides a label-free and reagentless analytical technique that is sensitive to protein structure. Here we show how the kinetic isotope effect affected the HER catalysed by the protein bovine serum albumin (BSA). We found that the deuteron bond, which is stronger than that of a proton, contributed to less effective transport of deuterons mediated by BSA at the HgjD2O interface, and enhanced structural stability of the surface-attached native BSA in D2O solution. A structural transition was also observed in the surface-attached urea-denatured BSA, and is probably due to the destabilisation of some secondary structural remnants retained by the 17 SSbonds. Because the catalytically active groups involved in proton or deuteron transfer in native proteins are often exposed towards solutions and their protons exchange almost instantly, no signs of H/D exchange were observed in native BSA using peak H under the given conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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127. The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl‐Acyl Carrier Protein Binding Site of FabI.
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Fage, Christopher D., Lathouwers, Thomas, Vanmeert, Michiel, Gao, Ling‐Jie, Vrancken, Kristof, Lammens, Eveline‐Marie, Weir, Angus N. M., Degroote, Ruben, Cuppens, Harry, Kosol, Simone, Simpson, Thomas J., Crump, Matthew P., Willis, Christine L., Herdewijn, Piet, Lescrinier, Eveline, Lavigne, Rob, Anné, Jozef, and Masschelein, Joleen
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CARRIER proteins , *BINDING sites , *BIOSYNTHESIS , *ANTIBIOTICS , *ESSENTIAL fatty acids , *POLYKETIDE synthases , *MUPIROCIN - Abstract
The enoyl‐acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti‐staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically‐relevant activity against multidrug‐resistant S. aureus. By combining X‐ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti‐staphylococcal drug development. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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128. Cationic Carbosilane Dendritic Systems as Promising Anti‐Amyloid Agents in Type 2 Diabetes.
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Lozano‐Cruz, Tania, Alcarraz‐Vizán, Gema, Mata, F. Javier, Pablo, Sara, Ortega, Paula, Duarte, Yorley, Bravo‐Moraga, Felipe, González‐Nilo, Fernando D., Novials, Anna, and Gómez, Rafael
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TYPE 2 diabetes , *AMYLIN , *PROTEIN folding , *POLYAMIDOAMINE dendrimers , *ISLANDS of Langerhans , *AMYLOID beta-protein , *TRANSGENIC mice - Abstract
The most common denominator of many of the neurodegenerative diseases is badly folded protein accumulation, which results in the formation of insoluble protein deposits located in different parts of the organism, causing cell death and tissue degeneration. Dendritic systems have turned out to be a promising new therapeutic approach for the treatment of these diseases due to their ability to modulate the folding of these proteins. With this perspective, and focused on type 2 diabetes (T2D), characterized by the presence of deposits containing the amyloidogenic islet amyloid polypeptide (IAPP), we demonstrate how different topologies of cationic carbosilane dendrimers inhibit the formation of insoluble protein deposits in pancreatic islets isolated from transgenic Tg‐hIAPP mice. Also, the results obtained by the modification of dendritic carbosilane wedges with the chemical chaperone 4‐phenylbutyric acid (4‐PBA) at the focal point confirmed their potential as anti‐amyloid agents with a concentration efficiency in their therapeutic action five orders of magnitude lower than that observed for free 4‐PBA. Computational studies, which determined the main interaction between IAPP and dendrimers at the atomic level, support the experimental work. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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129. Effect of Phosphorylation on the Structural Behaviour of Peptides Derived from the Intrinsically Disordered C‐Terminal Domain of Histone H1.0.
- Author
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Chaves‐Arquero, Belén, Pérez‐Cañadillas, José M., and Jiménez, M. Angeles
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PEPTIDES , *CHARGE-charge interactions , *PROTEIN domains , *PHOSPHORYLATION , *HISTONES - Abstract
To investigate the structural impact of phosphorylation on the human histone H1.0 C‐terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T118‐H1.0 (T118PKK motif) and T140‐H1.0 (T140PVK motif). Both model peptides are mainly disordered in aqueous solution in their non‐phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T118‐H1.0 and pT118‐H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104–115 and a short α/310 helix (residues 119–123), that are almost perpendicular in T118‐H1.0 but have a poorly defined orientation in pT118‐H1.0. Peptides T140‐H1.0 and pT140‐H1.0 form very similar α helices between residues 141–147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side‐chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT118‐H1.0 and pT140‐H1.0 has pKa values below the intrinsic values, which can be explained by non‐specific charge–charge interactions with nearby Lys. The non‐polar Val in the TPVK motif accounts for the pT140 pKa being closer to the intrinsic pKa value than the pT118 pKa. Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short‐lived intrinsically disordered proteins and domains. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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130. Crystals in Minutes: Instant On‐Site Microcrystallisation of Various Flavours of the CYP102A1 (P450BM3) Haem Domain.
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Stanfield, Joshua Kyle, Omura, Keita, Matsumoto, Ayaka, Kasai, Chie, Sugimoto, Hiroshi, Shiro, Yoshitsugu, Watanabe, Yoshihito, and Shoji, Osami
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HEME , *CRYSTALS , *CRYSTAL structure , *METALLOPORPHYRINS , *ENZYMES , *LIGAND binding (Biochemistry) - Abstract
Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are indispensable for the efficient structure‐based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N‐abietoyl‐l‐tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild‐type P450BM3 haem domain, with visible crystals forming within 2 hours and diffracting to a near‐atomic resolution of 1.22 Å. Using these crystals as seeds in a cross‐microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem‐domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
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131. A Hydrolase‐Catalyzed Cyclization Forms the Fused Bicyclic β‐Lactone in Vibralactone.
- Author
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Feng, Ke‐Na, Yang, Yan‐Long, Xu, Yu‐Xing, Zhang, Yue, Feng, Tao, Huang, Sheng‐Xiong, Liu, Ji‐Kai, and Zeng, Ying
- Subjects
- *
CYTOCHEMISTRY , *SYNTHETIC enzymes , *SYNTHETIC biology , *NATURAL products , *GLYCOSIDASE inhibitors , *PERICYCLIC reactions , *LIPASE inhibitors - Abstract
Vibralactone is isolated from the basidiomycete fungus Boreostereum vibrans as one of the strongest lipase inhibitors. Its unusual β‐lactone‐fused bicycle is derived from an aryl ring moiety by an oxidative ring‐expansion prior to an intramolecular cyclization. Herein, we report the discovery of the cyclase VibC which belongs to the α/β‐hydrolase superfamily and is involved in the vibralactone biosynthesis. Biochemical and crystal studies suggest that VibC may catalyze an aldol or an electrocyclic reaction initiated by the Ser‐His‐Asp catalytic triad. For the aldol and pericyclic chemistry in living cells, VibC is a unique hydrolase performing the carbocycle formation of an oxepinone to a fused bicyclic β‐lactone. This presents a naturally occurring, new enzymatic reaction in both aldol and hydrolase (bio)chemistry that will guide future exploitation of these enzymes in synthetic biology for chemical‐diversity expansion of natural products. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
132. IMRPS: Inserted and Modified Residues in Protein Structures. A database.
- Author
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Santhosh, R., Bankoti, Namrata, Gurudarshan, M., Jeyakanthan, J., and Sekar, K.
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PROTEIN structure , *INTERNET servers , *ONLINE databases , *GRAPHICAL user interfaces , *POST-translational modification , *DATABASES - Abstract
Modified residues present in proteins are the result of post‐translational modifications (PTMs). These PTMs increase the functional diversity of the proteome and influence various biological processes and diseased conditions. Therefore, identification and understanding of PTMs in various protein structures is of great significance. In view of this, an online database, Inserted and Modified Residues in Protein Structures (IMRPS), has been developed. IMRPS is a derived database that furnishes information on the residues modified and inserted in the protein structures available in the Protein Data Bank (PDB). The database is equipped with a graphical user interface and has an option to view the data for non‐redundant protein structures (25 and 90%) as well. A quality criteria cutoff has been incorporated to assist in displaying the specific set of PDB codes. The entire protein structure along with the inserted or modified residues can be visualized in JSmol. This database will be updated regularly (presently, every three months) and can be accessed through the URL http://cluster.physics.iisc.ac.in/imrps/. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
133. Mobility of water and of protein atoms at the protein-water interface, monitored by anisotropic atomic displacement parameters, are largely uncorrelated.
- Author
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Carugo, Oliviero
- Subjects
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ATOMS , *CRYSTALLOIDS (Botany) , *PROTEIN structure , *WATER of crystallization , *DATABASES , *ATOMIC displacements - Abstract
A non-redundant set of 231 protein crystal structures refined at a resolution better than (or equal to) 1 Å was extracted from the Protein Data Bank and the degree of conformational rigidity at the protein-water interface was examined by means of the Hirshfeld test and by comparing the orientations of the anisotropic Us for contacting protein and water atoms. Contacts between protein and water atoms are more rigid that contacts between water atoms and the degree of rigidity increases for shorter contacts and for more hydrogen-bonded atoms. Nevertheless, water and protein atoms are not rigidly held together. On the contrary, they seem to have little influence on their mobility to such an extent that hydration water, different from the protein atoms, cannot be considered to be properly in the solid state [ABSTRACT FROM AUTHOR]
- Published
- 2020
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134. Synthesis of C12‐Keto Saxitoxin Derivatives with Unusual Inhibitory Activity Against Voltage‐Gated Sodium Channels.
- Author
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Adachi, Kanna, Yamada, Tomoshi, Ishizuka, Hayate, Oki, Mana, Tsunogae, Shunsuke, Shimada, Noriko, Chiba, Osamu, Orihara, Tatsuya, Hidaka, Masafumi, Hirokawa, Takatsugu, Odagi, Minami, Konoki, Keiichi, Yotsu‐Yamashita, Mari, and Nagasawa, Kazuo
- Subjects
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SODIUM channels , *SAXITOXIN , *CONOTOXINS , *ION channels , *MEMBRANE proteins , *KETONES , *INDIUM gallium zinc oxide - Abstract
A novel series of C12‐keto‐type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV‐inhibitory activity has been evaluated in a cell‐based assay as well as whole‐cell patch‐clamp recording. Among these compounds, 11‐benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell‐based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7 nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin‐resistant subtype of NaV1.5, with an IC50 value of 94.1 nm. Derivatives 3 a–d and 3 f showed low recovery rates from NaV1.2 subtype (ca 45–79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12‐keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
135. Structure‐Based Design of a Macrocyclic PROTAC.
- Author
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Testa, Andrea, Hughes, Scott J., Lucas, Xavier, Wright, Jane E., and Ciulli, Alessio
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MACROCYCLIC compounds , *CHEMICAL inhibitors , *ENZYME inhibitors , *PROTEIN structure - Abstract
Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Herein, we report the design and synthesis of a macrocyclic PROTAC by adding a cyclizing linker to the BET degrader MZ1. A co‐crystal structure of macroPROTAC‐1 bound in a ternary complex with VHL and the second bromodomain of Brd4 validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12‐fold loss of binary binding affinity for Brd4, macroPROTAC‐1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
136. Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis.
- Author
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Wheeler, Ryan, Yu, Xia, Hou, Caixia, Mitra, Prithiba, Chen, Jhong‐Min, Herkules, Frank, Ivanov, Dmitri N., Tsodikov, Oleg V., and Rohr, Jürgen
- Subjects
- *
ISOMERIZATION , *NICOTINAMIDE adenine dinucleotide phosphate , *CRYSTAL structure , *BIOSYNTHESIS , *PROTEIN structure , *IONS , *ION channels - Abstract
MtmOIV and MtmW catalyze the final two reactions in the mithramycin (MTM) biosynthetic pathway, the Baeyer–Villiger opening of the fourth ring of premithramycin B (PMB), creating the C3 pentyl side chain, strictly followed by reduction of the distal keto group on the new side chain. Unexpectedly this results in a C2 stereoisomer of mithramycin, iso‐mithramycin (iso‐MTM). Iso‐MTM undergoes a non‐enzymatic isomerization to MTM catalyzed by Mg2+ ions. Crystal structures of MtmW and its complexes with co‐substrate NADPH and PEG, suggest a catalytic mechanism of MtmW. The structures also show that a tetrameric assembly of this enzyme strikingly resembles the ring‐shaped β subunit of a vertebrate ion channel. We show that MtmW and MtmOIV form a complex in the presence of PMB and NADPH, presumably to hand over the unstable MtmOIV product to MtmW, yielding iso‐MTM, as a potential self‐resistance mechanism against MTM toxicity. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
137. A 3.3 Å‐Resolution Structure of Hyperthermophilic Respiratory Complex III Reveals the Mechanism of Its Thermal Stability.
- Author
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Zhu, Guoliang, Zeng, Hui, Zhang, Shuangbo, Juli, Jana, Pang, Xiaoyun, Hoffmann, Jan, Zhang, Yan, Morgner, Nina, Zhu, Yun, Peng, Guohong, Michel, Hartmut, and Sun, Fei
- Subjects
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THERMAL stability , *THERMOPHILIC bacteria , *MATHEMATICAL complexes , *ATOMIC structure , *EXTREME environments , *CYTOCHROME c - Abstract
Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc1 complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N‐terminus of cyt. c1 was found to greatly enhance the interaction between cyt. b and cyt. c1, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc1 complex in an extreme thermal environment. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
138. In Silico Prediction of Target-Inhibitor Interaction
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Reichelt, Joachim and Böldicke, Thomas, editor
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- 2016
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139. Nutlin-3a-aa: Improving the Bioactivity of a p53/MDM2 Interaction Inhibitor by Introducing a Solvent-Exposed Methylene Group
- Author
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Química orgánica I, Kimika organikoa I, Nietzold, Florian, Rubner, Stefan, Labuzek, Beata, Golik, Przemyslaw, Surmiak, Ewa, Del Corte Solaguren-Beascoa, Xabier, Kitel, Radoslaw, Protzel, Christoph, Reppich-Sacher, Regina, Stichel, Jan, Magiera-Mularz, Katarzyna, Holak, Tad A. A., Berg, Thorsten, Química orgánica I, Kimika organikoa I, Nietzold, Florian, Rubner, Stefan, Labuzek, Beata, Golik, Przemyslaw, Surmiak, Ewa, Del Corte Solaguren-Beascoa, Xabier, Kitel, Radoslaw, Protzel, Christoph, Reppich-Sacher, Regina, Stichel, Jan, Magiera-Mularz, Katarzyna, Holak, Tad A. A., and Berg, Thorsten
- Abstract
Nutlin-3a is a reversible inhibitor of the p53/MDM2 interaction. We have synthesized the derivative Nutlin-3a-aa bearing an additional exocyclic methylene group in the piperazinone moiety. Nutlin-3a-aa is more active than Nutlin-3a against purified wild-type MDM2, and is more effective at increasing p53 levels and releasing transcription of p53 target genes from MDM2-induced repression. X-ray analysis of wild-type MDM2-bound Nutlin-3a-aa indicated that the orientation of its modified piperazinone ring was altered in comparison to the piperazinone ring of MDM2-bound Nutlin-3a, with the exocyclic methylene group of Nutlin-3a-aa pointing away from the protein surface. Our data point to the introduction of exocyclic methylene groups as a useful approach by which to tailor the conformation of bioactive molecules for improved biological activity.
- Published
- 2023
140. Beyond sequence : Structure-based machine learning
- Author
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Durairaj, Janani, de Ridder, Dick, van Dijk, Aalt D.J., Durairaj, Janani, de Ridder, Dick, and van Dijk, Aalt D.J.
- Abstract
Recent breakthroughs in protein structure prediction demarcate the start of a new era in structural bioinformatics. Combined with various advances in experimental structure determination and the uninterrupted pace at which new structures are published, this promises an age in which protein structure information is as prevalent and ubiquitous as sequence. Machine learning in protein bioinformatics has been dominated by sequence-based methods, but this is now changing to make use of the deluge of rich structural information as input. Machine learning methods making use of structures are scattered across literature and cover a number of different applications and scopes; while some try to address questions and tasks within a single protein family, others aim to capture characteristics across all available proteins. In this review, we look at the variety of structure-based machine learning approaches, how structures can be used as input, and typical applications of these approaches in protein biology. We also discuss current challenges and opportunities in this all-important and increasingly popular field.
- Published
- 2023
141. New graph learning approaches for exploring gene and protein function
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Muzio, Giulia; id_orcid 0000-0001-5999-2030
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- Proteins, Machine Learning, genome wide association studies (GWAS), Deep Learning, Graph Neural Networks (GNNs), Genetic data, complex traits, Graphs, Graph Learning, Protein structures, Life sciences, Data processing, computer science
- Published
- 2024
142. MOLECULAR UNDERSTANDING AND DESIGN OF DEEP EUTECTIC SOLVENTS AND PROTEINS USING COMPUTER SIMULATIONS AND MACHINE LEARNING
- Author
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Abbas, Usman Lame
- Subjects
- non-ionic deep eutectic solvents, molecular dynamics simulation, AlphaFold, protein structures, protein language models, machine learning, Biochemical and Biomolecular Engineering, Bioinformatics, Computational Chemistry, Data Science, Other Chemical Engineering
- Abstract
Hydrophobic deep eutectic solvents (DESs) have emerged as excellent extractants. A major challenge is the lack of an efficient tool to discover DES candidates. Currently, the search relies heavily on the researchers’ intuition or a trial-and-error process, which leads to a low success rate or bypassing of promising candidates. DES performance depends on the heterogeneous hydrogen bond environment formed by multiple hydrogen bond donors and acceptors. Understanding this heterogeneous hydrogen bond environment can help develop principles for designing high performance DESs for extraction and other separation applications. This work investigates the structure and dynamics of hydrogen bonds in hydrophobic DESs using molecular dynamics (MD) simulations. The results show the diversity of hydrogen bonds in the eight DESs and their impact on diffusivity and molecular association. The dominating hydrogen bonds determine whether the DESs are governed by intra- or inter-component associations. The DES-aqueous liquid–liquid interface also plays a vital role in the extraction ability of DESs. One question is how the DES compositions affect the structural features of the interface. This work also investigates the density profile, dipole moment, and hydrogen bonds of hydrophobic DES-aqueous interfaces using MD simulations. The results show the variations of dipole moment and hydrogen bond structure and dynamics at the interfaces. These variations could influence the extraction ability of DES through adjusting the partition and kinetics of organic substrates in the DES-aqueous biphasic systems. Recognizing the central role that hydrogen bonds play in the DES formation, this work aims to decipher the hydrogen bond features for DESs and develop machine learning models to predict the potential of a system to be DES based on the hydrogen bond-based descriptors. Based on our MD simulation results, we developed 30 machine learning models using ten algorithms and three types of hydrogen bond-based descriptors. The model performance is first benchmarked using their average and minimal ROC-AUC values. We also analyze the importance of individual features in the models and the results are consistent with the simulation-based statistical analysis. Finally, we validate the prediction ability of the models using the experimental results of 34 systems. Our work iterates the importance of hydrogen bond in DES formation and shows the potential of machine learning in discovering new DESs. Large protein language models (PLMs) have presented excellent potential to reshape protein research. The trained PLMs encode the amino acid sequence of a protein to a mathematical embedding that can be used for protein design or property prediction. It is recognized that protein 3D structure plays an important role in protein properties and functions. However, most PLMs are trained only on sequence data and lack protein 3D structure information. The lack of such crucial 3D structure information hampers the prediction capacity of PLMs in various applications. We utilize contrastive learning to develop a 3D structure-aware protein language model (S-PLM). The model encodes the sequence and 3D structure of proteins separately and deploys a multi-view contrastive loss function to enable the information exchange between the sequence and 3D structure embeddings. Our analysis shows that contrastive learning effectively incorporates 3D structure information into sequence-based embeddings.
- Published
- 2024
143. SARS-CoV-2 Virus−Host Interaction: Currently Available Structures and Implications of Variant Emergence on Infectivity and Immune Response
- Author
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Luís Queirós-Reis, Priscilla Gomes da Silva, José Gonçalves, Andrea Brancale, Marcella Bassetto, and João R. Mesquita
- Subjects
SARS-CoV-2 ,Spike protein ,hACE2 ,protein structures ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Coronavirus disease 19, or COVID-19, is an infection associated with an unprecedented worldwide pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which has led to more than 215 million infected people and more than 4.5 million deaths worldwide. SARS-CoV-2 cell infection is initiated by a densely glycosylated spike (S) protein, a fusion protein, binding human angiotensin converting enzyme 2 (hACE2), that acts as the functional receptor through the receptor binding domain (RBD). In this article, the interaction of hACE2 with the RBD and how fusion is initiated after recognition are explored, as well as how mutations influence infectivity and immune response. Thus, we focused on all structures available in the Protein Data Bank for the interaction between SARS-CoV-2 S protein and hACE2. Specifically, the Delta variant carries particular mutations associated with increased viral fitness through decreased antibody binding, increased RBD affinity and altered protein dynamics. Combining both existing mutations and mutagenesis studies, new potential SARS-CoV-2 variants, harboring advantageous S protein mutations, may be predicted. These include mutations S13I and W152C, decreasing antibody binding, N460K, increasing RDB affinity, or Q498R, positively affecting both properties.
- Published
- 2021
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144. Structural Characterization and Extended Substrate Scope Analysis of Two Mg2+-Dependent O-Methyltransferases from Bacteria**
- Author
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Nika Sokolova, Lili Zhang, Sadaf Deravi, Rick Oerlemans, Matthew R. Groves, Kristina Haslinger, Chemical and Pharmaceutical Biology, Drug Design, Medicinal Chemistry and Bioanalysis (MCB), and Biopharmaceuticals, Discovery, Design and Delivery (BDDD)
- Subjects
biocatalysis ,natural products ,methyltransferases ,Organic Chemistry ,protein structures ,Molecular Medicine ,methylation ,Molecular Biology ,Biochemistry ,phenylpropanoids - Abstract
Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by 15 O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for 16 sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we 17 characterized two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis in 18 terms of their enzymatic properties and substrate scope and determined their crystal structures. Both 19 OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, 20 hydroxybenzoic acids and their respective aldehydes, an anthraquinone and an indole. One enzyme also 21 accepted a steroid. The product range included pharmaceutically relevant compounds such as 22 (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether and 2-methoxyestradiol. Interestingly, 23 certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the 24 knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way 25 for their use in (combinatorial) pathway engineering.
- Published
- 2023
145. 17O NMR Spectroscopy: A Novel Probe for Characterizing Protein Structure and Folding
- Author
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Srinivasan Muniyappan, Yuxi Lin, Young-Ho Lee, and Jin Hae Kim
- Subjects
17O NMR spectroscopy ,protein structures ,protein folding ,oxygen-17 ,Biology (General) ,QH301-705.5 - Abstract
Oxygen is a key atom that maintains biomolecular structures, regulates various physiological processes, and mediates various biomolecular interactions. Oxygen-17 (17O), therefore, has been proposed as a useful probe that can provide detailed information about various physicochemical features of proteins. This is attributed to the facts that (1) 17O is an active isotope for nuclear magnetic resonance (NMR) spectroscopic approaches; (2) NMR spectroscopy is one of the most suitable tools for characterizing the structural and dynamical features of biomolecules under native-like conditions; and (3) oxygen atoms are frequently involved in essential hydrogen bonds for the structural and functional integrity of proteins or related biomolecules. Although 17O NMR spectroscopic investigations of biomolecules have been considerably hampered due to low natural abundance and the quadruple characteristics of the 17O nucleus, recent theoretical and technical developments have revolutionized this methodology to be optimally poised as a unique and widely applicable tool for determining protein structure and dynamics. In this review, we recapitulate recent developments in 17O NMR spectroscopy to characterize protein structure and folding. In addition, we discuss the highly promising advantages of this methodology over other techniques and explain why further technical and experimental advancements are highly desired.
- Published
- 2021
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146. Machine Learning Models to Interrogate Proteomewide Covalent Ligandabilities Directed at Cysteines.
- Author
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Liu R, Clayton J, Shen M, Bhatnagar S, and Shen J
- Abstract
Machine learning (ML) identification of covalently ligandable sites may accelerate targeted covalent inhibitor design and help expand the druggable proteome space. Here we report the rigorous development and validation of the tree-based models and convolutional neural networks (CNNs) trained on a newly curated database (LigCys3D) of over 1,000 liganded cysteines in nearly 800 proteins represented by over 10,000 three-dimensional structures in the protein data bank. The unseen tests yielded 94% and 93% AUCs (area under the receiver operating characteristic curve) for the tree models and CNNs, respectively. Based on the AlphaFold2 predicted structures, the ML models recapitulated the newly liganded cysteines in the PDB with over 90% recall values. To assist the community of covalent drug discoveries, we report the predicted ligandable cysteines in 392 human kinases and their locations in the sequence-aligned kinase structure including the PH and SH2 domains. Furthermore, we disseminate a searchable online database LigCys3D (https://ligcys.computchem.org/) and a web prediction server DeepCys (https://deepcys.computchem.org/), both of which will be continuously updated and improved by including newly published experimental data. The present work represents a first step towards the ML-led integration of big genome data and structure models to annotate the human proteome space for the next-generation covalent drug discoveries.
- Published
- 2024
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147. Ice Isn't the Only Crystal in Town: Structure Determination of Ice-Binding Proteins via X-Ray Crystallography.
- Author
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Vance TDR
- Subjects
- Crystallography, X-Ray, Caspase 1, Microscopy, Electron, Transmission, Carrier Proteins, Ice
- Abstract
Ice-binding proteins (IBPs) are proteins that have the remarkable ability to bind to ice, and their study has intrigued researchers for decades. This chapter explores the importance of structural biology in understanding IBPs and highlights the significant contributions of IBPs to the field of structural biology. The structures of various IBPs from different organisms have been elucidated, revealing key elements involved in ice binding. Structural biology techniques, including nuclear magnetic resonance (NMR) spectroscopy, transmission electron cryo-microscopy (cryo-EM), and X-ray crystallography, play crucial roles in solving protein structures. This article focuses on X-ray crystallography as a tool for investigating IBP structures, providing insights into its theoretical and practical aspects, experimental workflows, and common pitfalls to avoid., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2024
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148. A Mechanistic Understanding of the Distinct Regio- and Chemoselectivity of Multifunctional P450s by Structural Comparison of IkaD and CftA Complexed with Common Substrates.
- Author
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Jiang P, Jin H, Zhang G, Zhang W, Liu W, Zhu Y, Zhang C, and Zhang L
- Subjects
- Oxidation-Reduction, Catalysis, Substrate Specificity, Cytochrome P-450 Enzyme System metabolism, Lactams
- Abstract
Regio- and chemoselective C-H activation at multi-positions of a single molecule is fascinating but chemically challenging. The homologous cytochrome P450 enzymes IkaD and CftA catalyze multiple C-H oxidations on the same polycyclic tetramate macrolactam (PoTeM) ikarugamycin, with distinct regio- and chemoselectivity. Herein we provide mechanistic understanding of their functional differences by solving crystal structures of IkaD and CftA in complex with ikarugamycin and unnatural substrates. Distinct conformations of the F/G region in IkaD and CftA are found to differentiate the orientation of PoTeM substrates, by causing different binding patterns with polar moieties to determine site selection, oxidation order, and chemoselectivity. Fine-tuning the polar subpocket altered the regioselectivity of IkaD, indicating that substrate re-orientation by mutating residues distal to the oxidation site could serve as an important method in future engineering of P450 enzymes., (© 2023 Wiley-VCH GmbH.)
- Published
- 2023
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149. Fluorination Influences the Bioisostery of Myo-Inositol Pyrophosphate Analogs.
- Author
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Hostachy S, Wang H, Zong G, Franke K, Riley AM, Schmieder P, Potter BVL, Shears SB, and Fiedler D
- Subjects
- Humans, Halogenation, Phosphorylation, Diphosphates, Inositol Phosphates chemistry
- Abstract
Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP
5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)- Published
- 2023
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150. Evaluation of Transmembrane Protein Structural Models Using HPMScore
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Stéphane Téletchéa, Jérémy Esque, Aurélie Urbain, Catherine Etchebest, and Alexandre G. de Brevern
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structural models ,protein structures ,membrane bilayer ,DOPE ,Modeller ,AlphaFold2 ,Ocean Engineering - Abstract
Transmembrane proteins (TMPs) are a class of essential proteins for biological and therapeutic purposes. Despite an increasing number of structures, the gap with the number of available sequences remains impressive. The choice of a dedicated function to select the most probable/relevant model among hundreds is a specific problem of TMPs. Indeed, the majority of approaches are mostly focused on globular proteins. We developed an alternative methodology to evaluate the quality of TMP structural models. HPMScore took into account sequence and local structural information using the unsupervised learning approach called hybrid protein model. The methodology was extensively evaluated on very different TMP all-α proteins. Structural models with different qualities were generated, from good to bad quality. HPMScore performed better than DOPE in recognizing good comparative models over more degenerated models, with a Top 1 of 46.9% against DOPE 40.1%, both giving the same result in 13.0%. When the alignments used are higher than 35%, HPM is the best for 52%, against 36% for DOPE (12% for both). These encouraging results need further improvement particularly when the sequence identity falls below 35%. An area of enhancement would be to train on a larger training set. A dedicated web server has been implemented and provided to the scientific community. It can be used with structural models generated from comparative modeling to deep learning approaches.
- Published
- 2023
- Full Text
- View/download PDF
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