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Large Proteins (large + protein)
Terms modified by Large Proteins Selected AbstractsCollective Dynamics of Large Proteins from Mixed Coarse-Grained Elastic Network ModelMOLECULAR INFORMATICS, Issue 4 2005Ozge Kurkcuoglu Abstract Elastic network model- a coarse-grained normal mode analysis- is widely used to investigate the functionally important collective motions of proteins and their complexes. Mixed coarse-graining approach has been recently introduced to the elastic network model, so that the protein's native conformation can be modeled with regions of low and high resolution. In the mixed resolution model, each node of the elastic network may represent either a single atom (high-resolution) or a residue (low-resolution), and close-neighboring nodes are connected by harmonic springs. Here, the high-resolution parts constitute the interesting parts of the protein, such as the active site, while the rest of the structure is retained at lower resolution. By performing normal mode analysis of the resulting network, the collective dynamics (low-frequency modes) and consequent conformational changes can be analyzed even for supramolecular assemblages with reasonable computational efficiency, which may serve as a means of incorporating protein flexibility into docking algorithms and drug design. In this study, the mixed coarse-graining methodology is applied to analyze the functional motions of an important enzyme, triosephosphate isomerase. [source] Folding of epidermal growth factor-like repeats from human tenascin studied through a sequence frame-shift approachFEBS JOURNAL, Issue 21 2004Francesco Zanuttin In order to investigate the factors that determine the correct folding of epidermal growth factor-like (EGF) repeats within a multidomain protein, we prepared a series of six peptides that, taken together, span the sequence of two EGF repeats of human tenascin, a large protein from the extracellular matrix. The peptides were selected by sliding a window of the average length of tenascin EGF repeats over the sequence of EGF repeats 13 and 14. We thus obtained six peptides, EGF-f1 to EGF-f6, that are 33 residues long, contain six cysteines each, and bear a partial overlap in the sequence. While EGF-f1 corresponds to the native EGF-14 repeat, the others are frame-shifted EGF repeats. We carried out the oxidative folding of these peptides in vitro, analyzed the reaction mixtures by acid trapping followed by LC-MS, and isolated some of the resulting products. The oxidative folding of the native EGF-14 peptide is fast, produces a single three-disulfide species with an EGF-like disulfide topology and a marked difference in the RP-HPLC retention time compared with the starting product. On the contrary, frame-shifted peptides fold more slowly and give mixtures of three-disulfide species displaying RP-HPLC retention times that are closer to those of the reduced peptides. In contrast to the native EGF-14, the three-disulfide products that could be isolated are mainly unstructured, as determined by CD and NMR spectroscopy. We conclude that both kinetics and thermodynamics drive the correct pairing of cysteines, and speculate about how cysteine mispairing could trigger disulfide reshuffling in vivo. [source] Cell cycle mechanisms of sister chromatid separation; Roles of Cut1/separin and Cut2/securinGENES TO CELLS, Issue 1 2000Mitsuhiro Yanagida The correct transmission of chromosomes from mother to daughter cells is fundamental for genetic inheritance. Separation and segregation of sister chromatids in growing cells occurs in the cell cycle stage called ,anaphase'. The basic process of sister chromatid separation is similar in all eukaryotes: many gene products required are conserved. In this review, the roles of two proteins essential for the onset of anaphase in fission yeast, Cut2/securin and Cut1/separin, are discussed with regard to cell cycle regulation, and compared with the postulated roles of homologous proteins in other organisms. Securin, like mitotic cyclins, is the target of the anaphase promoting complex (APC)/cyclosome and is polyubiquitinated before destruction in a manner dependent upon the destruction sequence. The anaphase never occurs properly in the absence of securin destruction. In human cells, securin is an oncogene. Separin is a large protein (MW ,180 kDa), the C-terminus of which is conserved, and is thought to be inhibited by association with securin at the nonconserved N-terminus. In the budding yeast, Esp1/separin is thought to be a component of proteolysis against Scc1, an essential subunit of cohesin which is thought to link duplicated sister chromatids up to the anaphase. Whether fission yeast Cut1/separin is also implicated in proteolysis of cohesin is discussed. [source] Different mechanisms influencing permeation of PDGF-AA and PDGF-BB across the blood,brain barrierJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Abba J. Kastin Abstract Platelet-derived growth factor (PDGF) exerts neurotrophic and neuromodulatory effects on the CNS. To determine the permeability of the blood,brain barrier (BBB) to PDGF, we examined the blood-to-brain influx of radioactively labeled PDGF isoforms (PDGF-AA and PDGF-BB) by multiple-time regression analysis after intravenous (i.v.) injection and by in-situ perfusion, and also determined the physicochemical characteristics which affect their permeation across the BBB, including lipophilicity (measured by octanol:buffer partition coefficient), hydrogen bonding (measured by differences in octanol : buffer and isooctane : buffer partition coefficients), serum protein binding (measured by capillary electrophoresis), and stability of PDGF in blood 10 min after i.v. injection (measured by HPLC). After i.v. bolus injection, neither 125I-PDGF-AA nor 125I-PDGF-BB crossed the BBB, their influx rates being similar to that of the vascular marker 99mTc-albumin. 125I-PDGF-AA degraded significantly faster in blood than 125I-PDGF-BB. PDGF-BB, however, was completely bound to a large protein in serum whereas PDGF-AA showed no binding. Thus, degradation might explain the poor blood-to-brain influx of PDGF-AA, whereas protein binding could explain the poor influx of circulating PDGF-BB. Despite their lack of permeation in the intact mouse, both 125I-PDGF-AA and 125I-PDGF-BB entered the brain by perfusion in blood-free buffer, and the significantly faster rate of 125I-PDGF-AA than 125I-PDGF-BB may be explained by the lower hydrogen bonding potential of 125I-PDGF-AA. Thus, the lack of significant distribution of PDGF from blood to brain is not because of the intrinsic barrier function of the BBB but probably because of degradation and protein binding. Information from these studies could be useful in the design of analogues for delivery of PDGF as a therapeutic agent. [source] A network analysis of the single nucleotide polymorphisms in acute allergic diseasesALLERGY, Issue 1 2010J. Renkonen Abstract Background:, Genetics of acute allergies has focused on identifying single nucleotide polymorphisms (SNPs) within genes relevant in the pathogenesis. In this study, we begin a systems biology analysis of the interconnectivity and biological functions of these genes, their transcripts and their corresponding proteins. Methods:, The literature (Pubmed) was searched for SNPs within genes relevant in acute allergic diseases. The SNP-modified genes were converted to corresponding proteins and their protein,protein interactions were searched from six different databases. This interaction network was analysed with annotated vocabularies (ontologies), such as Gene Ontology, Reactome and Nature pathway interaction database. Time-series transcriptomics was performed with nasal epithelial cells obtained from allergic patients and their healthy control subjects. Results:, A total of 39 genes with SNPs related to acute allergic diseases were found from a literature search. The corresponding proteins were then hooked into a large protein,protein interaction network with the help of various databases. Twenty-five SNP-related proteins had more than one interacting protein and a network contained 95 proteins, and 182 connections could be generated. This network was 10-fold enriched with protein kinases and proteins involved in the host,virus interaction compared with background human proteome. Finally, eight of the 95 nodes on our network displayed nasal epithelial transcriptomal regulation in a time-series analysis collected from birch allergic patients during the spring pollen season. Conclusions:, Signal transduction with special reference to host,virus interactions dominated in the allergy-related protein interaction network. Systems level analysis of allergy-related mutation can provide new insights into pathogenetic mechanisms of the diseases. [source] Antigenic variation with a twist , the Borrelia storyMOLECULAR MICROBIOLOGY, Issue 6 2006Steven J. Norris Summary A common mechanism of immune evasion in pathogenic bacteria and protozoa is antigenic variation, in which genetic or epigenetic changes result in rapid, sequential shifts in a surface-exposed antigen. In this issue of Molecular Microbiology, Dai et al. provide the most complete description to date of the vlp/vsp antigenic variation system of the relapsing fever spirochaete, Borrelia hermsii. This elaborate, plasmid-encoded system involves an expression site that can acquire either variable large protein (vlp) or variable small protein (vsp) surface lipoprotein genes from 59 different archival copies. The archival vlp and vsp genes are arranged in clusters on at least five different plasmids. Gene conversion occurs through recombination events at upstream homology sequences (UHS) found in each gene copy, and at downstream homology sequences (DHS) found periodically among the vlp/vsp archival genes. Previous studies have shown that antigenic variation in relapsing fever Borrelia not only permits the evasion of host antibody responses, but can also result in changes in neurotropism and other pathogenic properties. The vlsE antigenic variation locus of Lyme disease spirochaetes, although similar in sequence to the relapsing fever vlp genes, has evolved a completely different antigenic variation mechanism involving segmental recombination from a contiguous array of vls silent cassettes. These two systems thus appear to represent divergence from a common precursor followed by functional convergence to create two distinct antigenic variation processes. [source] Functional analysis of the Alternaria brassicicola non-ribosomal peptide synthetase gene AbNPS2 reveals a role in conidial cell wall constructionMOLECULAR PLANT PATHOLOGY, Issue 1 2007KWANG-HYUNG KIM SUMMARY Alternaria brassicicola is a necrotrophic pathogen causing black spot disease on virtually all cultivated Brassica crops worldwide. In many plant pathosystems fungal secondary metabolites derived from non-ribosomal peptide synthetases (NPSs) are phytotoxic virulence factors or are antibiotics thought to be important for niche competition with other micro-organisms. However, many of the functions of NPS genes and their products are largely unknown. In this study, we investigated the function of one of the A. brassicicola NPS genes, AbNPS2. The predicted amino acid sequence of AbNPS2 showed high sequence similarity with A. brassicae, AbrePsy1, Cochliobolus heterostrophus, NPS4 and a Stagonospora nodorum NPS. The AbNPS2 open reading frame was predicted to be 22 kb in length and encodes a large protein (7195 amino acids) showing typical NPS modular organization. Gene expression analysis of AbNPS2 in wild-type fungus indicated that it is expressed almost exclusively in conidia and conidiophores, broadly in the reproductive developmental phase. AbNPS2 gene disruption mutants showed abnormal spore cell wall morphology and a decreased hydrophobicity phenotype. Conidia of abnps2 mutants displayed an aberrantly inflated cell wall and an increase in lipid bodies compared with wild-type. Further phenotypic analyses of abnps2 mutants showed decreased spore germination rates both in vitro and in vivo, and a marked reduction in sporulation in vivo compared with wild-type fungus. Moreover, virulence tests on Brassicas with abnps2 mutants revealed a significant reduction in lesion size compared with wild-type but only when aged spores were used in experiments. Collectively, these results indicate that AbNPS2 plays an important role in development and virulence. [source] Solid-State NMR of Matrix Metalloproteinase 12: An Approach Complementary to Solution NMRCHEMBIOCHEM, Issue 5 2007Stéphane Balayssac Dr. Data transfer. The solid-state proton-driven spin diffusion (PDSD) and J -decoupled PDSD NMR spectra of the microcrystalline catalytic domain of matrix metalloproteinase 12 (MMP-12, 17 kDa) have been recorded. It is shown that such spectra can be largely assigned in a few days by using the available liquid-state assignment and validated with an independent sequential assignment based on 3D NCACX and NCOCX PDSD experiments. This demonstrates how quickly the liquid-state assignment of comparably large protein can be transferred to the solid state. [source] TROSY effects in MAS solid-state NMRCONCEPTS IN MAGNETIC RESONANCE, Issue 2 2008Veniamin Chevelkov Abstract Use of transverse relaxation-optimized spectroscopy (TROSY) type techniques had a dramatic impact on the study of large proteins with a molecular weight >30kDa for solution-state NMR. In the solid-state, such an effect would not be expected a prior, as the investigated molecules are immobilized. However, local motions induce fluctuations of the local fields experienced by the nuclear spins and, this way, are effective for relaxation. We demonstrate that protein dynamics can significantly influence the resonance line width in ultra high resolution MAS (magic angle spinning) solid-state NMR experiments. Averaging of the 15NH,/, multiplet components as a consequence of 1H decoupling induces effective broadening of the 15N resonance. Application of TROSY type techniques that select only the narrow component of the multiplet pattern results in an increased resolution and, thus, will be of benefit for MAS solid-state NMR spectroscopy. © 2008 Wiley Periodicals, Inc. Concepts Magn Reson Part A 32A: 143,156, 2008. [source] Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4I) for capillary coatingsELECTROPHORESIS, Issue 8 2008Anisa Elhamili Abstract A monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide (M7C4I), has been evaluated as a surface derivatization reagent for CE in combination with TOF MS for the analysis of proteins, peptides, and protein digests. The M7C4I piperazine, at alkaline pH, forms a covalent bond via alkylation of the ionized silanols producing a cationic surface with a highly stable and reversed EOF. The obtained surface yields rapid separations (less than 5,min) of peptides and proteins at acidic pH with high separation efficiencies (up to 1.1×106 plates/m for peptides and up to 1.8×106 plates/m for proteins) and no observed bleeding of the coating reagent into the mass spectrometer. The simplicity of the coating procedure also enables fast (2,min) regeneration of the surface, if necessary. This is useful in the analysis of complex samples in order to prevent possible memory effects. The potential of using M7C4I-coated capillaries for MS analysis of complex samples is demonstrated by the separation of peptides, proteins, and protein digests. Even more, the spectacular thing in which large intact proteins with molecular masses over 0.5,MDa could be separated. The coating showed good ability to handle these large proteins with high efficiency and retained peak shape as demonstrated by separation of IgG1 (150,kDa) and thyroglobulin (669,kDa). [source] Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteinsELECTROPHORESIS, Issue 11 2003Chad M. Warren Abstract The electrophoretic separation of high-molecular-weight proteins (>,500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000,4000 kDa) to migrate over 10 cm in a ,13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain (,,220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes. [source] Object-oriented approach to drug design enabled by NMR SOLVE: First real-time structural tool for characterizing protein,ligand interactionsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue S37 2001Daniel S. Sem Abstract As a result of genomics efforts, the number of protein drug targets is expected to increase by an order of magnitude. Functional genomics efforts are identifying these targets, while structural genomics efforts are determining structures for many of them. However, there is a significant gap in going from structural information for a protein target to a high affinity (Kd,<,100 nM) inhibitor, and the problem is multiplied by the sheer number of new targets now available. nature frequently designs proteins in classes that are related by the reuse, through gene duplication events, of cofactor binding domains. This reuse of functional domains is an efficient way to build related proteins in that it is object-oriented. There is a growing realization that the most efficient drug design strategies for attacking the mass of targets coming from genomics efforts will be systems-based approaches that attack groups of related proteins in parallel. We propose that the most effective drug design strategy will be one that parallels the object-oriented manner by which nature designed the gene families themselves. IOPE (Integrated Object-Oriented PharmacoEngineering) is such an approach. It is a three-step technology to build focused combinatorial libraries of potential inhibitors for major families and sub-families of enzymes, using cogent NMR data derived from representatives of these protein families. The NMR SOLVE (Structurally Oriented Library Valency Engineering) data used to design these libraries are gathered in days, and data can be obtained for large proteins (>,170 kDa). Furthermore, the process is fully object-oriented in that once a given bi-ligand is identified for a target, potency is retained if different cofactor mimics are swapped. This gives the drug design process maximum flexibility, allowing for the more facile transition from in vitro potency to in vivo efficacy. J. Cell. Biochem. Suppl. 37: 99,105, 2001. © 2002 Wiley-Liss, Inc. [source] Coarse-graining of protein structures for the normal mode studiesJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 8 2007Kilho Eom Abstract The coarse-grained structural model such as Gaussian network has played a vital role in the normal mode studies for understanding protein dynamics related to biological functions. However, for the large proteins, the Gaussian network model is computationally unfavorable for diagonalization of Hessian (stiffness) matrix for the normal mode studies. In this article, we provide the coarse-graining method, referred to as "dynamic model condensation," which enables the further coarse-graining of protein structures consisting of small number of residues. It is shown that the coarser-grained structures reconstructed by dynamic model condensation exhibit the dynamic characteristics, such as low-frequency normal modes, qualitatively comparable to original structures. This sheds light on that dynamic model condensation and may enable one to study the large protein dynamics for gaining insight into biological functions of proteins. © 2007 Wiley Periodicals, Inc. J Comput Chem, 2007 [source] Binding site on human immunoglobulin G for the affinity ligand HWRGWVJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2010Haiou Yang Abstract Affinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383,Asn389 (SNGQPEN) located in the CH3 domain. Subsequent modeling has suggested a possible three-dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG. Copyright © 2009 John Wiley & Sons, Ltd. [source] Surface interaction of well-defined, concentrated poly(2-hydroxyethyl methacrylate) brushes with proteinsJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 21 2007Chiaki Yoshikawa Abstract The interaction of concentrated polymer brushes with proteins was chromatographically investigated. By the use of surface-initiated atom transfer radical polymerization, a low-polydispersity poly(2-hydroxyethyl methacrylate) (PHEMA) was densely grafted onto the inner surfaces of silica monoliths with mesopores of about 50 and 80 nm in mean size. The graft density reached 0.4,0.5 chains/nm2. The 80-nm-mesopore monolithic column with the concentrated PHEMA brush was characterized through the elution of low-polydispersity pullulans with different molecular weights, clearly showing two modes of size exclusion, that is, one by the mesopores and the other by the brush phase. The latter mode gave a sharp separation with a critical molecular weight (size-exclusion limit) of about 1000. This molecular size of pullulan was comparable to the distance between the nearest-neighbor graft points. The elution behaviors of five proteins of different sizes (bovine serum thyroglobulin, bovine serum immunoglobulin G, bovine serum albumin, horse heart myoglobin, and bovine serum aprotinin) were studied with this PHEMA-grafted column. The smallest protein, aprotinin, with a pullulan-reduced molecular weight slightly larger than the critical value of 1000, was eluted much behind the corresponding pullulan, and this indicated that it barely got into the brush layer, suffering from a strong affinity interaction within the brush. On the other hand, the other four larger proteins were eluted at the same elution volumes as the equivalent pullulans, and this meant that they were perfectly excluded from the brush layer and separated only in the size-exclusion mode by the mesopores without an affinity interaction with the brush surface. This excellent inertness of the concentrated brush in the interaction with the large proteins should afford the system long-term stability against biofouling. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4795,4803, 2007 [source] Methyl TROSY: explanation and experimental verificationMAGNETIC RESONANCE IN CHEMISTRY, Issue 10 2003Jason E. Ollerenshaw Abstract In TROSY experiments, relaxation interference effects are exploited to produce spectra with improved resolution and signal-to-noise. Such experiments cannot be explained using the standard product operator formalism, but must instead be analyzed at the level of individual density matrix elements. Herein we illustrate this point using an example from our recent work on a TROSY 1H,13C correlation experiment for methyl groups in large proteins. Methyl groups are useful spectroscopic probes of protein structure and dynamics because they are found throughout the critical core region of a folded protein and their resonances are intense and well dispersed. Additionally, it is relatively easy to produce highly deuterated protein samples that are 1H,13C labeled at selected methyl positions, facilitating studies of high molecular weight systems. Methyl groups are relaxed by a network of 1H,1H and 1H,13C dipolar interactions, and in the macromolecular limit the destructive interference of these interactions leads to unusually slow relaxation for certain density matrix elements. It is this slow relaxation that forms the basis for TROSY experiments. We present a detailed analysis of evolution and relaxation during HSQC and HMQC pulse schemes for the case of a 13C1H3 spin system attached to a macromolecule. We show that the HMQC sequence is already optimal with respect to the TROSY effect, offering a significant sensitivity enhancement over HSQC at any spectrometer field strength. The gain in sensitivity is established experimentally using samples of two large proteins, malate synthase G (81.4 kDa) and ClpP protease (305 kDa), both highly deuterated and selectively 1H,13C-labeled at isoleucine , methyl positions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Computer-aided NMR assay for detecting natively folded structural domains,PROTEIN SCIENCE, Issue 4 2006Takayuki Hondoh Abstract Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space. [source] Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEFPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2007Jukka Hellman Dr. Abstract Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20,50,ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS. [source] Use of different proteases working in acidic conditions to improve sequence coverage and resolution in hydrogen/deuterium exchange of large proteinsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2003Laetitia Cravello The combination of hydrogen exchange and mass spectrometry has been widely used in structural biology, providing views on protein structure and protein dynamics. One of the constraints is to use proteases working at low pH and low temperature to limit back-exchange during proteolysis. Although pepsin works in these conditions and is currently used in such experiments, sequence coverage is not always complete especially for large proteins, and the spatial resolution of the exchange rate is limited by the size of the resulting peptides. In this study we tried two other proteases, protease type XIII from Aspergillus saitoi and protease type XVIII from Rhizhopus species. The penicillin-binding protein X (PBP-2X*), a 77-kDa protein, was selected as a model. Like pepsin, neither of these proteases is really specific, but we found very good reproducibility in the digestion pattern. Compared with using pepsin alone, combining the results of the three independent proteolyses increased the coverage for the peptide mapping, thus avoiding missing some potentially interesting regions of the protein. Furthermore, we obtained a better spatial resolution for deuterium incorporation data, specifying accurately the deuterated regions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Going soft and SAD with manganeseACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2005Paula S. Salgado SAD phasing has been revisited recently, with experiments being carried out using previously unconventional sources of anomalous signal, particularly lighter atoms and softer X-rays. A case study is reported using the 75,kDa RNA-dependent RNA polymerase of the bacteriophase ,6, which binds a Mn atom and crystallizes with three molecules in the asymmetric unit. X-ray diffraction data were collected at a wavelength of 1.89,Å and although the calculated anomalous signal from the three Mn atoms was only 1.2%, SHELXD and SOLVE were able to locate these atoms. SOLVE/RESOLVE used this information to obtain SAD phases and automatically build a model for the core region of the protein, which possessed the characteristic features of the right-hand polymerase motif. These results demonstrate that with modern synchrotron beamlines and software, manganese phasing is a practical tool for solving the structure of large proteins. [source] Clean SEA-TROW experiments to map solvent exposed amides in large proteinsCHINESE JOURNAL OF CHEMISTRY, Issue 12 2004Dong-Hai Lin Abstract It is well known that the SEA-TROSY experiment could alleviate some of the problems of resonance overlap in 15N/2H labeled proteins as it was designed to selectively map solvent exposed amide protons. However, SEA-TROSY spectra may be contaminated with exchange-relayed NOE contributions from fast exchanged hydroxyl or amine protons and contributions from longitudinal relaxation. Also, perdeuteration of the protein sample is a prerequisite for this experiment. In this communication, a modified version, clean SEA-TROSY, was proposed to eliminate these artifacts and to allow the experiment to be applied to protonated or partially deuterated proteins and protein complexes. [source] | |||||||||||||||||||||||||