首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 0 毫秒
1.
    
The conformational changes and aggregation process of beta-lactoglobulin (beta-LG) subjected to gamma irradiation are presented. Beta-LG in solutions of different protein concentrations (3 and 10 mg/ml) and in solid state with different water activities (a(w)) (0.22; 0.53; 0.74) was irradiated using a Cobalt-60 radiation source at dose level of 1-50 kGy. Small-angle X-ray scattering (SAXS) was used to study the conformational changes of beta-LG due to the irradiation treatment. The irradiated protein was also examined by high performance size exclusion chromatography (HPSEC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing and reducing conditions and fluorescence. SAXS analysis showed that the structural conformation of irradiated beta-LG in solid state at different a(w) and dose level was essentially the same as the nonirradiated beta-LG. The scattering data also showed that the irradiation of beta-LG in solution promoted the formation of oligomers. Interestingly, from the data analysis and model building, it could be shown that the formed oligomers are linear molecules, built by linear combinations of beta-LG dimers (tetramers, hexamers, etc). The formation of oligomers was also evidenced by SDS-PAGE analysis and HPSEC chromatograms, in which products with higher molecular mass than that of the dimeric beta-LG were detected. Formation of intermolecular cross-linking between tyrosyl radicals are proposed to be at least partially responsible for this occurrence. From the results it could be shown that the samples irradiated in solution presented some conformational changes under gamma irradiation, resulting in well ordered oligomers and aggregates formed by cross-linking of beta-LG dimers subunits, while the samples irradiated in the solid state were not modified.  相似文献   

2.
    
Structural biology provides essential information for elucidating molecular mechanisms that underlie biological function. Advances in hardware, sample preparation, experimental methods, and computational approaches now enable structural analysis of protein complexes with increasing complexity that more closely represent biologically entities in the cellular environment. Integrated multidisciplinary approaches are required to overcome limitations of individual methods and take advantage of complementary aspects provided by different structural biology techniques. Although X‐ray crystallography remains the method of choice for structural analysis of large complexes, crystallization of flexible systems is often difficult and does typically not provide insights into conformational dynamics present in solution. Nuclear magnetic resonance spectroscopy (NMR) is well‐suited to study dynamics at picosecond to second time scales, and to map binding interfaces even of large systems at residue resolution but suffers from poor sensitivity with increasing molecular weight. Small angle scattering (SAS) methods provide low resolution information in solution and can characterize dynamics and conformational equilibria complementary to crystallography and NMR. The combination of NMR, crystallography, and SAS is, thus, very useful for analysis of the structure and conformational dynamics of (large) protein complexes in solution. In high molecular weight systems, where NMR data are often sparse, SAS provides additional structural information and can differentiate between NMR‐derived models. Scattering data can also validate the solution conformation of a crystal structure and indicate the presence of conformational equilibria. Here, we review current state‐of‐the‐art approaches for combining NMR, crystallography, and SAS data to characterize protein complexes in solution.  相似文献   

3.
    
The X‐ray crystal structure of sheep liver sorbitol dehydrogenase (slSDH) has been determined using the crystal structure of human sorbitol dehydrogenase (hSDH) as a molecular‐replacement model. slSDH crystallized in space group I222 with one monomer in the asymmetric unit. A conserved tetramer that superposes well with that seen in hSDH (despite belonging to a different space group) and obeying the 222 crystal symmetry is seen in slSDH. An acetate molecule is bound in the active site, coordinating to the active‐site zinc through a water molecule. Glycerol, a substrate of slSDH, also occupies the substrate‐binding pocket together with the acetate designed by nature to fit large polyol substrates. The substrate‐binding pocket is seen to be in close proximity to the tetramer interface, which explains the need for the structural integrity of the tetramer for enzyme activity. Small‐angle X‐ray scattering was also used to identify the quaternary structure of the tetramer of slSDH in solution.  相似文献   

4.
    
The solution structure of the full‐length DNA helicase minichromosome maintenance protein from Methanothermobacter thermautotrophicus was determined by small‐angle neutron scattering (SANS) data together with all‐atom molecular modeling. The data were fit best with a dodecamer (dimer of hexamers). The 12 monomers were linked together by the B/C domains, and the adenosine triphosphatase (AAA+) catalytic regions were found to be freely movable in the full‐length dodecamer both in the presence and absence of Mg2+ and 50‐meric single‐stranded DNA (ssDNA). In particular, the SANS data and molecular modeling indicate that all 12 AAA+ domains in the dodecamer lie approximately the same distance from the axis of the molecule, but the positions of the helix–turn–helix region at the C‐terminus of each monomer differ. In addition, the A domain at the N‐terminus of each monomer is tucked up next to the AAA+ domain for all 12 monomers of the dodecamer. Finally, binding of ssDNA does not lock the AAA+ domains in any specific position, which leaves them with the flexibility to move both for helicase function and for binding along the ssDNA. Proteins 2014; 82:2364–2374. © 2014 Wiley Periodicals, Inc.  相似文献   

5.
    
Histidine‐rich, unstructured peptides adsorb to charged interfaces such as mineral surfaces and microbial cell membranes. At a molecular level, we investigate the adsorption mechanism as a function of pH, salt, and multivalent ions showing that (1) proton charge fluctuations are—in contrast to the majority of proteins—optimal at neutral pH, promoting electrostatic interactions with anionic surfaces through charge regulation and (2) specific zinc(II)‐histidine binding competes with protons and ensures an unusually constant charge distribution over a broad pH interval. In turn, this further enhances surface adsorption. Our analysis is based on atomistic molecular dynamics simulations, coarse grained Metropolis Monte Carlo, and classical polymer density functional theory. This multiscale modeling provides a consistent picture in good agreement with experimental data on Histatin 5, an antimicrobial salivary peptide. Biological function is discussed and we suggest that charge regulation is a significant driving force for the remarkably robust activity of histidine‐rich antimicrobial peptides. Proteins 2014; 82:657–667. © 2013 Wiley Periodicals, Inc.  相似文献   

6.
    
Infection by the four serotypes of Dengue virus (DENV‐1 to DENV‐4) causes an important arthropod‐borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA‐dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all four Dengue virus serotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full‐length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV‐1 to DENV‐4 NS5 forms are elongated and flexible in solution, with DENV‐4 NS5 being more compact relative to NS5 from DENV‐1, DENV‐2 and DENV‐3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten‐residue linker region to the flexibility of the entire NS5. Swapping the ten‐residue linker between DENV‐4 NS5FL and DENV‐3 NS5FL demonstrated its importance in MTase–RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.  相似文献   

7.
    
Dengue virus (DENV) nonstructural protein 5 (NS5) consists of a methyltransferase (MTase) domain and an RNA‐dependent RNA polymerase (RdRp) domain. The cross‐talk between these domains occurs via a ten‐residue linker. Recent solution studies of DENV NS5 from all four serotypes (DENV‐1 to DENV‐4) showed that NS5 adopts multiple conformations owing to its flexible linker and that DENV‐4 NS5 is more compact and less flexible compared with NS5 from DENV‐1 to DENV‐3 [Saw et al. (2015), Acta Cryst. D 71 , 2309–2327]. Here, using a variety of single, double, triple and quadruple mutants of DENV‐4 NS5 combined with solution X‐ray scattering studies, insight into the critical residues responsible for the differential flexibility of DENV‐4 NS5 is presented. The DENV‐4 NS5 mutants K271T and S266N/T267A as well as the deletion mutant ΔS266T267 showed enlarged dimensions and flexibility similar to those of DENV‐3 NS5. The data indicate that the residues Lys271, Ser266 and Thr267 are important for the compactness of DENV‐4 NS5 and therefore may be critical for the regulation of virus replication. Furthermore, quantitative characterization of the flexibility of these DENV‐4 NS5 linker mutants using the ensemble‐optimization method revealed that these mutants possess a similar conformational distribution to DENV‐3 NS5, confirming that these residues in the linker region cause the higher compactness of DENV‐4 NS5.  相似文献   

8.
    
Tear lipocalin and β‐lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non‐native helical structures are formed during the early stage of β‐lactoglobulin folding. To address whether the non‐native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped‐flow methods measuring the time‐dependent changes in circular dichroism (CD) spectrum and small‐angle X‐ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst‐phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst‐phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non‐native helix formation is not general for folding of all lipocalin family members. The non‐native helix content in the burst‐phase folding appears to depend on helical propensities of the amino acid sequence. Proteins 2009. © 2008 Wiley‐Liss, Inc.  相似文献   

9.
    
Small‐angle X‐ray scattering (SAXS) is useful for determining the oligomeric states and quaternary structures of proteins in solution. The average molecular mass in solution can be calculated directly from a single SAXS curve collected on an arbitrary scale from a sample of unknown protein concentration without the need for beamline calibration or protein standards. The quaternary structure in solution can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from proposed models of the oligomer. This approach is especially robust when the crystal structure of the target protein is known, and the candidate oligomer models are derived from the crystal lattice. When SAXS data are obtained at multiple protein concentrations, this analysis can provide insight into dynamic self‐association equilibria. Herein, we summarize the computational methods that are used to determine protein molecular mass and quaternary structure from SAXS data. These methods are organized into a workflow and demonstrated with four case studies using experimental SAXS data from the published literature.  相似文献   

10.
    
By simulations on the distance distribution function (DDF) derived from small angle X‐ray scattering (SAXS) theoretical data of a dense monodisperse system, we found a quantitative mathematical correlation between the apparent size of a spherically symmetric (or nearly spherically symmetric) homogenous particle and the concentration of the solution. SAXS experiments on protein solutions of human hemoglobin and horse myoglobin validated the correlation. This gives a new method to determine, from the SAXS DDF, the size of spherically symmetric (or nearly spherically symmetric) particles of a dense monodisperse system, specifically for protein solutions with interference effects.  相似文献   

11.
    
Small‐angle X‐ray scattering can be used to determine the molecular shape of macromolecules in solution which are otherwise refractory to conventional high‐resolution studies. DAMMIN and GASBOR are applications that utilize ab initio methods to build models of proteins using simulated annealing; both DAMMIN and GASBOR have to be run numerous times on the same input data to generate the most likely protein shape. Condor is a specialized workload‐management system for PC computation‐intensive tasks. Using Condor, DAMMIN and GASBOR can be run a number of times simultaneously on the same input data, allowing the shape of proteins to be determined in a fraction of the time it would have taken to have run DAMMIN and GASBOR sequentially. The main advantage of this approach is that it allows quicker data processing; therefore, results are obtained promptly and without undue delay. Tissue transglutaminase is a multidomain enzyme that catalyses the formation of isopeptide bonds between polypeptide chains. This reaction requires the enzyme to undergo a series of conformational changes that are not well understood in order to allow the sequential interaction with the two substrate proteins and their subsequent release when cross‐linked. Condor was applied to determine the solution shape of tissue transglutaminase in a rapid fashion. Eventually, the next step will be to move towards online analysis at synchrotron sources by developing a graphical user interface that will enable remote access, allowing users to submit jobs to Condor whilst at synchrotrons.  相似文献   

12.
    
Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil‐globule transition)? To answer this question, we measured small‐angle X‐ray scattering for a series of β‐lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil‐globule transitions. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 651–658, 2014.  相似文献   

13.
    
Small angle solution X‐ray and neutron scattering recently resurfaced as powerful tools to address an array of biological problems including folding, intrinsic disorder, conformational transitions, macromolecular crowding, and self or hetero‐assembling of biomacromolecules. In addition, small angle solution scattering complements crystallography, nuclear magnetic resonance spectroscopy, and other structural methods to aid in the structure determinations of multidomain or multicomponent proteins or nucleoprotein assemblies. Neutron scattering with hydrogen/deuterium contrast variation, or X‐ray scattering with sucrose contrast variation to a certain extent, is a convenient tool for characterizing the organizations of two‐component systems such as a nucleoprotein or a lipid‐protein assembly. Time‐resolved small and wide‐angle solution scattering to study biological processes in real time, and the use of localized heavy‐atom labeling and anomalous solution scattering for applications as FRET‐like molecular rulers, are amongst promising newer developments. Despite the challenges in data analysis and interpretation, these X‐ray/neutron solution scattering based approaches hold great promise for understanding a wide variety of complex processes prevalent in the biological milieu.  相似文献   

14.
    
The radius of gyration is a fundamental structural parameter that is particularly useful for describing polymers. It has been known since Flory's seminal work in the mid‐20th century that polymers show a power‐law dependence, where the radius of gyration is proportional to the number of residues raised to a power. The power‐law exponent has been measured experimentally for denatured proteins and derived empirically for folded monomeric proteins using crystal structures. Here, the biological assemblies in the Protein Data Bank are surveyed to derive the power‐law parameters for protein oligomers having degrees of oligomerization of 2–6 and 8. The power‐law exponents for oligomers span a narrow range of 0.38–0.41, which is close to the value of 0.40 obtained for monomers. This result shows that protein oligomers exhibit essentially the same power‐law behavior as monomers. A simple power‐law formula is provided for estimating the oligomeric state from an experimental measurement of the radius of gyration. Several proteins in the Protein Data Bank are found to deviate substantially from power‐law behavior by having an atypically large radius of gyration. Some of the outliers have highly elongated structures, such as coiled coils. For coiled coils, the radius of gyration does not follow a power law and instead scales linearly with the number of residues in the oligomer. Other outliers are proteins whose oligomeric state or quaternary structure is incorrectly annotated in the Protein Data Bank. The power laws could be used to identify such errors and help prevent them in future depositions.  相似文献   

15.
    
Mycoplasma genitalium is one of the smallest organisms capable of self‐replication and its sequence is considered a starting point for understanding the minimal genome required for life. MG289, a putative phosphonate substrate binding protein, is considered to be one of these essential genes. The crystal structure of MG289 has been solved at 1.95 Å resolution. The structurally identified thiamine binding region reveals possible mechanisms for ligand promiscuity. MG289 was determined to be an extracytoplasmic thiamine binding lipoprotein. Computational analysis, size exclusion chromatography, and small angle X‐ray scattering indicates that MG289 homodimerizes in a concentration‐dependant manner. Comparisons to the thiamine pyrophosphate binding homolog Cypl reveal insights into the metabolic differences between mycoplasmal species including identifying possible kinases for cofactor phosphorylation and describing the mechanism of thiamine transport into the cell. These results provide a baseline to build our understanding of the minimal metabolic requirements of a living organism. Proteins 2011. © 2010 Wiley‐Liss, Inc.  相似文献   

16.
    
The conformational flexibility of linkage‐specific polyubiquitin chains enables ubiquitylated proteins and their receptors to be involved in a variety of cellular processes. Linear or Met1‐linked polyubiquitin chains, associated with nondegradational cellular signalling pathways, have been known to adopt multiple conformations from compact to extended conformations. However, the extent of such conformational flexibility remains open. Here, the crystal structure of linear Ub2 was determined in a more compact conformation than that of the previously known structure (PDB entry 3axc ). The two structures differ significantly from each other, as shown by an r.m.s.d. between Cα atoms of 3.1 Å. The compactness of the linear Ub2 structure in comparison with PDB entry 3axc is supported by smaller values of the radius of gyration (Rg; 18 versus 18.9 Å) and the maximum interatomic distance (Dmax; 55.5 versus 57.8 Å). Extra intramolecular hydrogen bonds formed among polar residues between the distal and proximal ubiquitin moieties seem to contribute to stabilization of the compact conformation of linear Ub2. An ensemble of three semi‐extended and extended conformations of linear Ub2 was also observed by small‐angle X‐ray scattering (SAXS) analysis in solution. In addition, the conformational heterogeneity in linear polyubiquitin chains is clearly manifested by SAXS analyses of linear Ub3 and Ub4: at least three distinct solution conformations are observed in each chain, with the linear Ub3 conformations being compact. The results expand the extent of conformational space of linear polyubiquitin chains and suggest that changes in the conformational ensemble may be pivotal in mediating multiple signalling pathways.  相似文献   

17.
  相似文献   

18.
    
Brome mosaic virus (BMV) is a small icosahedral plant virus of mean diameter 268 Å. Interactions between BMV particles in solution were studied by means of small‐angle X‐ray scattering in order to find crystallization conditions. The interactions between biomacromolecules as large as these viruses have not yet been systematically studied by this method. As it is known that usually proteins crystallize in, or close to, attractive regimes, the interactions between BMV particles in solution were studied as a function of pH, type of salt and size and concentration of polyethylene glycol. An unexpected result of these studies is that the precipitates obtained upon addition of PEG alone or PEG combined with salt were in fact made of microcrystals, which were all characterized by the same series of diffraction peaks, with positions close to those of a centered cubic space group. A phase diagram of the virus as a function of PEG concentration was established by means of microbatch experiments. From the precipitation zones, conditions for crystallization were tested from 5 to 40 mg ml−1 virus with 3−10%(w/v) PEG 8000 or PEG 20 000. Small crystals were obtained in several conditions after a few days and continued growing for several weeks.  相似文献   

19.
    
Enteropathogenic Yersinia expresses several invasins that are fundamental virulence factors required for adherence and colonization of tissues in the host. Within the invasin‐family of Yersinia adhesins, to date only Invasin has been extensively studied at both structural and functional levels. In this work, we structurally characterize the recently identified inverse autotransporter InvasinE from Yersinia pseudotuberculosis (formerly InvasinD from Yersinia pseudotuberculosis strain IP31758) that belongs to the invasin‐family of proteins. The sequence of the C‐terminal adhesion domain of InvasinE differs significantly from that of other members of the Yersinia invasin‐family and its detailed cellular and molecular function remains elusive. In this work, we present the 1.7 Å crystal structure of the adhesion domain of InvasinE along with two Immunoglobulin‐like domains. The structure reveals a rod shaped architecture, confirmed by small angle X‐ray scattering in solution. The adhesion domain exhibits strong structural similarities to the C‐type lectin‐like domain of Yersinia pseudotuberculosis Invasin and enteropathogenic/enterohemorrhagic E. coli Intimin. However, despite the overall structural similarity, the C‐type lectin‐like domain in InvasinE lacks motifs required for Ca2+/carbohydrate binding as well as sequence or structural features critical for Tir binding in Intimin and β1‐integrin binding in Invasin, suggesting that InvasinE targets a distinct, yet unidentified molecule on the host‐cell surface. Although the biological role and target molecule of InvasinE remain to be elucidated, our structural data provide novel insights into the architecture of invasin‐family proteins and a platform for further studies towards unraveling the function of InvasinE in the context of infection and host colonization.  相似文献   

20.
    
The pathogenic bacteria Legionella pneumophila is known to cause Legionnaires' Disease, a severe pneumonia that can be fatal to immunocompromised individuals and the elderly. Shohdy et al. identified the L. pneumophila vacuole sorting inhibitory protein VipF as a putative N‐acetyltransferase based on sequence homology. We have characterized the basic structural and functional properties of VipF to confirm this original functional assignment. Sequence conservation analysis indicates two putative CoA‐binding regions within VipF. Homology modeling and small angle X‐ray scattering suggest a monomeric, dual‐domain structure joined by a flexible linker. Each domain contains the characteristic beta‐splay motif found in many acetyltransferases, suggesting that VipF may contain two active sites. Docking experiments suggest reasonable acetyl‐CoA binding locations within each beta‐splay motif. Broad substrate screening indicated that VipF is capable of acetylating chloramphenicol and both domains are catalytically active. Given that chloramphenicol is not known to be N‐acetylated, this is a surprising finding suggesting that VipF is capable of O‐acetyltransferase activity. Proteins 2016; 84:1422–1430. © 2016 Wiley Periodicals, Inc.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号