Large-scale evaluation of protein reductive methylation for improving protein crystallization.
Ontology highlight
ABSTRACT:
SUBMITTER: Kim Y
PROVIDER: S-EPMC2678869 | biostudies-literature | 2008 Oct
REPOSITORIES: biostudies-literature
Publications
Large-scale evaluation of protein reductive methylation for improving protein crystallization.
Kim Youngchang Y Quartey Pearl P Li Hui H Volkart Lour L Hatzos Catherine C Chang Changsoo C Nocek Boguslaw B Cuff Marianne M Osipiuk Jerzy J Tan Kemin K Fan Yao Y Bigelow Lance L Maltseva Natalia N Wu Ruiying R Borovilos Maria M Duggan Erika E Zhou Min M Binkowski T Andrew TA Zhang Rong-guang RG Joachimiak Andrzej A
Nature methods 20081001 10
Similar Datasets
Project description:Surface lysine methylation (SLM) is a technique for improving the rate of success of protein crystallization by chemically methylating lysine residues. The exact mechanism by which SLM enhances crystallization is still not clear. To study these mechanisms, and to analyze the conditions where SLM will provide the optimal benefits for rescuing failed crystallization experiments, we compared 40 protein structures containing N,N-dimethyl-lysine (dmLys) to a nonredundant set of 18,972 nonmethylated structures from the PDB. By measuring the relative frequency of intermolecular contacts (where contacts are defined as interactions between the residues in proximity with a distance of 3.5 A or less) of basic residues in the methylated versus nonmethylated sets, dmLys-Glu contacts are seen more frequently than Lys-Glu contacts. Based on observation of the 10 proteins with both native and methylated structures, we propose that the increased rate of contact for dmLys-Glu is due to both a slight increase in the number of amine-carboxyl H-bonds and to the formation of methyl C--H...O interactions. By comparing the relative contact frequencies of dmLys with other residues, the mechanism by which methylation of lysines improves the formation of crystal contacts appears to be similar to that of Lys to Arg mutation. Moreover, analysis of methylated structures with the surface entropy reduction (SER) prediction server suggests that in many cases SLM of predicted SER sites may contribute to improved crystallization. Thus, tools that analyze protein sequences and mark residues for SER mutation may identify proteins with good candidate sites for SLM.
| S-EPMC2974831 | biostudies-literature
Project description:Develop a novel de-glyco-assisted methylation site identification (DOMAIN) strategy which enables straightforward, fast, and reproducible analysis of protein methylation in a proteome-wide manner. Combining multidimensional fractionation and multiprotease digestion, our method enabled the identification of 573 methylated forms in 270 proteins, including 311 new methylation forms, in A549 cells. Combining this technique with stable isotope labeling quantitative proteomics and RNA interference, we determined the differential regulation of several putative methylated sites that are related to the protein arginine N-methyltransferase 3 (PRMT3). Collectively, our integrated proteomics workflow for comprehensive mapping of methylation sites enables a better understanding of protein methylation, while providing a rapid and effective approach for global protein methylation analysis in biomedical research.
2019-07-18 | PXD014574 | Pride
Project description:Methylation can occur on histidine, lysine and arginine residues in proteins and often serves a regulatory function. Histidine methylation has recently attracted attention through the discovery of the human histidine methyltransferase enzymes SETD3 and METTL9. There are currently no methods to enrich histidine methylated peptides for mass spectrometry analysis and large-scale studies of the modification are hitherto absent. Here, we query ultra-comprehensive human proteome datasets to generate a resource of histidine methylation sites. In HeLa cells alone, we report 299 histidine methylation sites as well as 895 lysine methylation events. We use this resource to explore the frequency, localization, targeted domains, protein types and sequence requirements of histidine methylation and benchmark all analyses to methylation events on lysine and arginine. Our results demonstrate that histidine methylation is widespread in human cells and tissues and that the modification is over-represented in regions of mono-spaced histidine repeats. We also report colocalization of the modification with functionally important phosphorylation sites and disease associated mutations to identify regions of likely regulatory and functional importance. Taken together, we here report a system level analysis of human histidine methylation and our results represent a comprehensive resource enabling targeted studies of individual histidine methylation events.
| S-EPMC8140740 | biostudies-literature
Project description:The lack of methods for proteome-scale detection of arginine methylation restricts our knowledge of its relevance in physiological and pathological processes. Here we show that most tryptic peptides containing methylated arginine(s) are highly basic and hydrophilic. Consequently, they could be considerably enriched from total cell extracts by simple protocols using either one of strong cation exchange chromatography, isoelectric focusing, or hydrophilic interaction liquid chromatography, the latter being by far the most effective of all. These methods, coupled with heavy methyl-stable isotope labeling by amino acids in cell culture and mass spectrometry, enabled in T cells the identification of 249 arginine methylation sites in 131 proteins, including 190 new sites and 93 proteins not previously known to be arginine methylated. By extending considerably the number of known arginine methylation sites, our data reveal a novel proline-rich consensus motif and identify for the first time arginine methylation in proteins involved in cytoskeleton rearrangement at the immunological synapse and in endosomal trafficking.
| S-EPMC3494207 | biostudies-literature
Project description:Lysine methylation mediated by methyltransferase enzymes is present on multiple proteins throughout the cell; however, methods to uncover and characterize global protein lysine methylation patterns do not readily exist. Here we developed pan-specific methyl lysine antibodies that we utilized in immunoprecipitation experiments coupled with mass spectrometry to yield one of the first large-scale surveys of protein lysine methylation in vivo. In total, 552 different lysine methylation sites were determined, making this one of the most comprehensive global studies published to date. The large majority of these sites have not been yet reported. These sites showed significantly enriched sequence motifs and resided in proteins that are involved in diverse biological processes, particularly in chromatin organization. Our data provide a comprehensive view of lysine methylation in human cells and a powerful resource to facilitate investigations into the function of lysine methylation on non-histone proteins.
| S-EPMC3741217 | biostudies-literature
Project description:Rv0765c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and crystallized. The native protein crystallized in a hexagonal crystal form which diffracted to 7 A resolution. In an attempt to improve the quality of the Rv0765c crystals, the protein was modified by reductive methylation using dimethylaminoborane and formaldehyde. The modified protein crystallized under different conditions in a tetragonal crystal form, from which diffraction data could be collected to a resolution of 3.2 A. In both crystal forms of Rv0765c, the asymmetric unit contained two copies of the protein molecule.
| S-EPMC2335070 | biostudies-literature
Project description:Automated function prediction (AFP) of proteins is of great significance in biology. AFP can be regarded as a problem of the large-scale multi-label classification where a protein can be associated with multiple gene ontology terms as its labels. Based on our GOLabeler-a state-of-the-art method for the third critical assessment of functional annotation (CAFA3), in this paper we propose NetGO, a web server that is able to further improve the performance of the large-scale AFP by incorporating massive protein-protein network information. Specifically, the advantages of NetGO are threefold in using network information: (i) NetGO relies on a powerful learning to rank framework from machine learning to effectively integrate both sequence and network information of proteins; (ii) NetGO uses the massive network information of all species (>2000) in STRING (other than only some specific species) and (iii) NetGO still can use network information to annotate a protein by homology transfer, even if it is not contained in STRING. Separating training and testing data with the same time-delayed settings of CAFA, we comprehensively examined the performance of NetGO. Experimental results have clearly demonstrated that NetGO significantly outperforms GOLabeler and other competing methods. The NetGO web server is freely available at http://issubmission.sjtu.edu.cn/netgo/.
| S-EPMC6602452 | biostudies-literature
Project description:Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be high. Here we report the results from the first large-scale community-based critical assessment of protein function annotation (CAFA) experiment. Fifty-four methods representing the state of the art for protein function prediction were evaluated on a target set of 866 proteins from 11 organisms. Two findings stand out: (i) today's best protein function prediction algorithms substantially outperform widely used first-generation methods, with large gains on all types of targets; and (ii) although the top methods perform well enough to guide experiments, there is considerable need for improvement of currently available tools.
| S-EPMC3584181 | biostudies-literature
Project description:Crystallization is the most serious bottleneck in high-throughput protein-structure determination by diffraction methods. We have used data mining of the large-scale experimental results of the Northeast Structural Genomics Consortium and experimental folding studies to characterize the biophysical properties that control protein crystallization. This analysis leads to the conclusion that crystallization propensity depends primarily on the prevalence of well-ordered surface epitopes capable of mediating interprotein interactions and is not strongly influenced by overall thermodynamic stability. We identify specific sequence features that correlate with crystallization propensity and that can be used to estimate the crystallization probability of a given construct. Analyses of entire predicted proteomes demonstrate substantial differences in the amino acid-sequence properties of human versus eubacterial proteins, which likely reflect differences in biophysical properties, including crystallization propensity. Our thermodynamic measurements do not generally support previous claims regarding correlations between sequence properties and protein stability.
| S-EPMC2746436 | biostudies-literature
Project description:The mammalian genome depends on patterns of methylated cytosines for normal function, but the relationship between genomic methylation patterns and the underlying sequence is unclear. We have characterized the methylation landscape of the human genome by global analysis of patterns of CpG depletion and by direct sequencing of 3073 unmethylated domains and 2565 methylated domains from human brain DNA. The genome was found to consist of short (<4 kb) unmethylated domains embedded in a matrix of long methylated domains. Unmethylated domains were enriched in promoters, CpG islands, and first exons, while methylated domains comprised interspersed and tandem-repeated sequences, exons other than first exons, and non-annotated single-copy sequences that are depleted in the CpG dinucleotide. The enrichment of regulatory sequences in the relatively small unmethylated compartment suggests that cytosine methylation constrains the effective size of the genome through the selective exposure of regulatory sequences. This buffers regulatory networks against changes in total genome size and provides an explanation for the C value paradox, which concerns the wide variations in genome size that scale independently of gene number. This suggestion is compatible with the finding that cytosine methylation is universal among large-genome eukaryotes, while many eukaryotes with genome sizes <5 x 10(8) bp do not methylate their DNA.
| S-EPMC1361710 | biostudies-literature