共查询到20条相似文献，搜索用时 0 毫秒
1.
2.
Our recently developed off-lattice bead model capable of simulating protein structures with mixed alpha/beta content has been extended to model the folding of a ubiquitin-like protein and provides a means for examining the more complex kinetics involved in the folding of larger proteins. Using trajectories generated from constant-temperature Langevin dynamics simulations and sampling with the multiple multi-histogram method over five-order parameters, we are able to characterize the free energy landscape for folding and find evidence for folding through compact intermediates. Our model reproduces the observation that the C-terminus loop structure in ubiquitin is the last to fold in the folding process and most likely plays a spectator role in the folding kinetics. The possibility of a productive metastable intermediate along the folding pathway consisting of collapsed states with no secondary structure, and of intermediates or transition structures involving secondary structural elements occurring early in the sequence, is also supported by our model. The kinetics of folding remain multi-exponential below the folding temperature, with glass-like kinetics appearing at T/T(f) approximately 0.86. This new physicochemical model, designed to be predictive, helps validate the value of modeling protein folding at this level of detail for genomic-scale studies, and motivates further studies of other protein topologies and the impact of more complex energy functions, such as the addition of solvation forces. 相似文献
3.
Fredrik Ekstrm Gunter Stier Uwe H. Sauer 《Acta Crystallographica. Section D, Structural Biology》2003,59(4):724-726
α‐Actinin forms antiparallel homodimers that cross‐link actin filaments from adjacent sarcomeres within the Z‐discs of striated muscle. The N‐terminal actin‐binding domain (ABD) is composed of two calponin homology (CH) domains followed by four spectrin‐like repeats and a calmodulin‐like EF‐hand domain at the C‐terminus. The ABD of human α‐actinin crystallizes in space group P21, with unit‐cell parameters a = 101.9, b = 38.4, c = 154.9 Å, β = 109.2°. A complete native data set from a native crystal was collected extending to 2.0 Å resolution and a single‐wavelength anomalous dispersion (SAD) data set to 2.9 Å resolution was collected from a selenomethionine‐labelled microcrystal using the microfocusing beamline ID‐13 at the ESRF. Analysis of the anomalous contribution shows a rapid decrease in the σnormal/σanomal ratio owing to radiation damage. 相似文献
4.
Armin Wagner Michel Pieren Clemens Schulze‐Briese Kurt Ballmer‐Hofer Andrea E. Prota 《Acta Crystallographica. Section D, Structural Biology》2006,62(11):1430-1434
The crystal structure of VEGF‐E was solved by the sulfur single‐wavelength anomalous dispersion method (S‐SAD) using highly redundant low‐resolution data collected at a wavelength of λ≃ 1.7 Å with an estimated anomalous signal of 1.5%. 11 sulfur sites, nine out of 16 disulfide bonds and two out of 12 methionines could be located in the asymmetric unit using data truncated at a resolution of 4.1 Å; however, none of the common diffraction data‐quality indicators for SAD allowed clear discrimination between successful and unsuccessful resolution cutoffs. The high solvent content of 75% allowed efficient density modification to be performed and an unbiased electron‐density map of good quality to be generated. This study demonstrates the strength of S‐SAD for phasing using accurate highly redundant data at low resolution. 相似文献
5.
D. E. Brodersen E. De La Fortelle C. Vonrhein G. Bricogne J. Nyborg M. Kjeldgaard 《Acta Crystallographica. Section D, Structural Biology》2000,56(4):431-441
Two examples of the application of single‐wavelength anomalous dispersion (SAD) in macromolecular structure determination are described, both using the statistical phasing program SHARP. For the holmium‐substituted calcium‐binding protein psoriasin (22.7 kDa), a set of accurate phases has been obtained to a resolution of 1.05 Å without recourse to an atomic model of the molecule. The accuracy of the phases resulted in an electron‐density map of a quality comparable to σA‐weighted 2mFo−DFc maps derived from the final model refined with SHELX97. Comparison of the refined and SAD electron‐density maps showed significant discrepancies resulting from the iterative refinement in reciprocal space. Additionally, it is shown that the structure of psoriasin can be determined from native data extending to 2.0 Å alone by exploiting the minute anomalous signal from a bound zinc ion. 相似文献
6.
Tobias Beck Andrius Krasauskas Tim Gruene George M. Sheldrick 《Acta Crystallographica. Section D, Structural Biology》2008,64(11):1179-1182
Obtaining phase information for the solution of macromolecular structures is still one of the bottlenecks in X‐ray crystallography. 5‐Amino‐2,4,6‐triiodoisophthalic acid (I3C), in which three covalently bound iodines form an equilateral triangle, was incorporated into proteins in order to obtain phases by single‐wavelength anomalous dispersion (SAD). An improved binding capability compared with simple heavy‐metal ions, ready availability, improved recognition of potential heavy‐atom sites and low toxicity make I3C particularly suitable for experimental phasing. 相似文献
7.
Recently, we have shown that a modified energy model based on the param99 force field with the generalized Born (GB) solvation model produces reliable free energy landscapes of mini-proteins with a betabetaalpha motif (BBA5, 1FSD, and 1PSV), with the native structures of the mini-proteins located in their lowest free energy minimum states. One of the main features in the modified energy model is a significant improvement for more balanced treatments of alpha and beta strands in proteins. In this study, using the replica exchange molecular dynamics (REMD) simulation method with this new force field, we have carried out extensive ab initio folding studies of several well-known peptides with alpha or beta strands (C-peptide, EK-peptide, le0q, and gbl). Starting from fully extended conformations as the initial conditions, all of the native-like structures of the target peptides were successfully identified by REMD, with reasonable representations of free energy surfaces. The present simulation results with the modified energy model are consistent with experiments, demonstrating an extended applicability of the energy model to folding studies of a variety of alpha-helices, beta-strands, and alpha/beta proteins. 相似文献
8.
Rezaei-Ghaleh N Zweckstetter M Morshedi D Ebrahim-Habibi A Nemat-Gorgani M 《Biopolymers》2009,91(1):28-36
Amyloid fibril formation is widely believed to be a generic property of polypeptide chains. In the present study, alpha-chymotrypsin, a well-known serine protease has been driven toward these structures by the use of two different conditions involving (I) high temperature, pH 2.5, and (II) low concentration of trifluoroethanol (TFE), pH 2.5. A variety of experimental methods, including fluorescence emission, dynamic quenching, steady-state fluorescence anisotropy, far-UV circular dichroism, nuclear magnetic resonance spectroscopy, and dynamic light scattering were employed to characterize the conformational states of alpha-chymotrypsin that precede formation of amyloid fibrils. The structure formed under Condition I was an unfolded monomer, whereas an alpha-helical rich oligomer was induced in Condition II. Both the amyloid aggregation-prone species manifested a higher solvent exposure of hydrophobic and aromatic residues compared with the native state. Upon incubation of the protein in these conditions for 48 h, amyloid-like fibrils were formed with diameters of about 10-12 nm. In contrast, at neutral pH and low concentration of TFE, a significant degree of amorphous aggregation was observed, suggesting that charge neutralization of acidic residues in the amyloid core region has a positive influence on amyloid fibril formation. In summary, results presented in this communication suggest that amyloid fibrils of alpha-chymotrypsin may be obtained from a variety of structurally distinct conformational ensembles highlighting the critical importance of protein evolution mechanisms related to prevention of protein misfolding. 相似文献
9.
The restriction endonuclease (REase) R. HphI is a Type IIS enzyme that recognizes the asymmetric target DNA sequence 5'-GGTGA-3' and in the presence of Mg(2+) hydrolyzes phosphodiester bonds in both strands of the DNA at a distance of 8 nucleotides towards the 3' side of the target, producing a 1 nucleotide 3'-staggered cut in an unspecified sequence at this position. REases are typically ORFans that exhibit little similarity to each other and to any proteins in the database. However, bioinformatics analyses revealed that R.HphI is a member of a relatively big sequence family with a conserved C-terminal domain and a variable N-terminal domain. We predict that the C-terminal domains of proteins from this family correspond to the nuclease domain of the HNH superfamily rather than to the most common PD-(D/E)XK superfamily of nucleases. We constructed a three-dimensional model of the R.HphI catalytic domain and validated our predictions by site-directed mutagenesis and studies of DNA-binding and catalytic activities of the mutant proteins. We also analyzed the genomic neighborhood of R.HphI homologs and found that putative nucleases accompanied by a DNA methyltransferase (i.e. predicted REases) do not form a single group on a phylogenetic tree, but are dispersed among free-standing putative nucleases. This suggests that nucleases from the HNH superfamily were independently recruited to become REases in the context of RM systems multiple times in the evolution and that members of the HNH superfamily may be much more frequent among the so far unassigned REase sequences than previously thought. 相似文献
10.
Vigna radiata plant defensin 1 (VrD1) is the first reported plant defensin exhibiting insecticidal activity. We report herein the nuclear magnetic resonance solution structure of VrD1 and the implication on its insecticidal activity. The root-mean-square deviation values are 0.51 +/- 0.35 and 1.23 +/- 0.29 A for backbone and all heavy atoms, respectively. The VrD1 structure comprises a triple-stranded antiparallel beta-sheet, an alpha-helix, and a 3(10) helix stabilized by four disulfide bonds, forming a typical cysteine-stabilized alphabeta motif. Among plant defensins of known structure, VrD1 is the first to contain a 3(10) helix. Glu26 is highly conserved among defensins; VrD1 contains an arginine at this position, which may induce a shift in the orientation of Trp10, thereby promoting the formation of this 3(10) helix. Moreover, VrD1 inhibits Tenebrio molitor alpha-amylase. Alpha-amylase has an essential role in the digestion of plant starch in the insect gut, and expression of the common bean alpha-amylase inhibitor 1 in transgenic pea imparts complete resistance against bruchids. These results imply that VrD1 insecticidal activity has its basis in the inhibition of a polysaccharide hydrolase. Sequence and structural comparisons between two groups of plant defensins having different specificity toward insect alpha-amylase reveal that the loop between beta2 and beta3 is the probable binding site for the alpha-amylase. Computational docking experiments were used to study VrD1-alpha-amylase interactions, and these results provide information that may be used to improve the insecticidal activity of VrD1. 相似文献
11.
12.
A soft-sensor for monitoring solubility of native-like alpha-lactalbumin (alpha-LA) and beta-lactoglobulin (beta-LG) and their aggregation behavior following heat treatment of mixtures under different treatment conditions was developed using fluorescence spectroscopy data regressed with a multivariate Partial Least Squares (PLS) regression algorithm. PLS regression was used to correlate the concentrations of alpha-LA and beta-LG to the fluorescence spectra obtained for their mixtures. Data for the calibration and validation of the soft sensor was derived from fluorescence spectra. The process of thermal induced aggregation of beta-LG and alpha-LA protein in mixtures, which involves the disappearance of native-like proteins, was studied under various treatment conditions including different temperatures, pH, total initial protein concentration and proportions of alpha-LA and beta-LG. It was demonstrated that the multivariate regression models used could effectively deconvolute multi-wavelength fluorescence spectra collected under a variety of process conditions and provide a fairly accurate quantification of respective native-like proteins despite the significant overlapping between their emission profiles. It was also demonstrated that a PLS model can be used as a black-box prediction tool for estimating protein aggregation when combined with simple mass balances. 相似文献
13.
The beta-propeller fold is a phylogenetically widespread, common protein architecture able to support a range of different functions such as catalysis, ligand binding and transport, regulation and protein binding. Interestingly, it appears that the beta-propeller topology is also compatible with strikingly diverse sequences. Amongst this diversity, there are three large groups of proteins with related sequences and very important cellular and intercellular regulatory functions: WD, kelch, and YWTD proteins. A common characteristic between these protein families is that their sequences, while distinct, all contain internal repeats 40-45 residues long. Through a pangenomic analysis using internal repeat profiles derived from the structurally known propeller modules of the eukaryotic protein RCC1 and the related prokaryotic protein BLIP-II, we have defined a new superfamily of propeller repeats, the RCC1-like repeats (RLRs). These sequences turn out to be more phylogenetically widespread than other large groups of propeller proteins, occurring in both prokaryotic and eukaryotic genomes. Interestingly, our research showed that RLR domains with different numbers of repeats exist, ranging from 3 to 7, and possibly more. A novel, intriguing finding is the discovery of sequences with 3 repeats, as well as proteins with 10 modular units, though in the latter case it is not clear whether these are made of two 5-bladed domains or a single, novel 10-bladed propeller. In addition, the results indicate that circular permutation events may have taken place in the evolution of these proteins. It is now established that the group of RLR proteins is extremely numerous and is characterized by unique, remarkable features which place it in a position of special interest as an important superfamily of proteins in nature. 相似文献
14.
Langnaese K Richter K Smalla KH Krauss M Thomas U Wolf G Laube G 《Developmental neurobiology》2007,67(4):422-437
Knock out mice deficient for the splice-isoform alphaalpha of neuronal nitric oxide synthase (nNOSalphaalpha) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, betabeta and gammagamma, we generated isoform-specific anti-peptide antibodies against the nNOSalphaalpha specific betabeta-finger motif involved in PDZ domain scaffolding and the nNOSbetabeta specific N-terminus. The nNOSalphaalpha betabeta-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOSalphaalpha on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the betabeta-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOSalphaalpha betabeta-finger antibody in pull-down assays. By contrast, nNOSalphaalpha betabeta-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOSalphaalpha knock out mice, nNOSalphaalpha was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting betabeta/gammagamma-isoforms in these cells. The nNOSbetabeta antibody clearly detected bacterial expressed nNOSbetabeta fusion protein and nNOSbetabeta in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOSbetabeta in nNOSalphaalpha deficient animals. 相似文献
15.
Reddy BV Nagarajaram HA Blundell TL 《Protein science : a publication of the Protein Society》1999,8(3):573-586
An alpha-helix and a beta-strand are said to be interactively packed if at least one residue in each of the secondary structural elements loses 10% of its solvent accessible contact area on association with the other secondary structural element. An analysis of all such 5,975 nonidentical alpha/beta units in protein structures, defined at < or = 2.5 A resolution, shows that the interaxial distance between the alpha-helix and the beta-strand is linearly correlated with the residue-dependent function, log[(V/nda)/n-int], where V is the volume of amino acid residues in the packing interface, nda is the normalized difference in solvent accessible contact area of the residues in packed and unpacked secondary structural elements, and n-int is the number of residues in the packing interface. The beta-sheet unit (beta u), defined as a pair of adjacent parallel or antiparallel hydrogen-bonded beta-strands, packing with an alpha-helix shows a better correlation between the interaxial distance and log(V/nda) for the residues in the packing interface. This packing relationship is shown to be useful in the prediction of interaxial distances in alpha/beta units using the interacting residue information of equivalent alpha/beta units of homologous proteins. It is, therefore, of value in comparative modeling of protein structures. 相似文献
16.
It has previously been shown that nicotine-evoked dopamine release from rat striatal synaptosomes and nicotine-evoked norepinephrine release from hippocampal synaptosomes are mediated by distinct nicotinic acetylcholine receptor (nAChR) subtypes. In the present study, the functional association of these nicotinic receptors with specific subtypes of voltage-gated calcium channels was examined. Cd(2+) (200 microM), as well as omega-conotoxin MVIIC (5 microM), blocks approximately 85% of nicotine-evoked dopamine release from striatal synaptosomes, indicating a major involvement of calcium channels. Furthermore, the toxin-susceptibility suggests that these calcium channels contain alpha(1A) and/or alpha(1B) subunits. Inhibition of nicotine-evoked dopamine release by conotoxins alpha-MII and omega-GVIA is additive and indicates that presynaptic alpha3beta2 nAChRs are functionally coupled to alpha(1A), but not alpha(1B), calcium channel subtypes. Conversely, insensitivity to alpha-AuIB and sensitivity to omega-MVIIC indicate that non-alpha3beta2/alpha3beta4-containing nAChRs are functionally coupled to alpha(1B)-containing calcium channels. In contrast, Cd(2+) blocks only 65% of nicotine-evoked norepinephrine release from hippocampal synaptosomes, indicating that a substantial fraction of this release occurs through mechanisms not involving calcium channels. This Cd(2+)-insensitive component of release is blocked by alpha-AuIB and therefore appears to be triggered by Ca(2+) flowing directly through the channels of presynaptic alpha3beta4 nAChRs. Thus, these data indicate that different presynaptic termini can have distinctive functional associations of specific nAChRs and voltage-gated calcium channels. 相似文献
17.
The class A beta-lactamase TEM-1 is a key bacterial resistance enzyme against beta-lactam antibiotics, but little is known about the energetic bases for complementarity between TEM-1 and its inhibitors. Most inhibitors form a covalent adduct with the catalytic Ser70, making the measurement of equilibrium constants, and hence interaction energies, technically difficult. This study evaluates noncovalent interactions within covalent complexes by examining the differential stability of TEM-1 and its inhibitor adducts. The thermal denaturation of TEM-1 follows a two-state, reversible model with a melting temperature (T(m)) of 51.6C and a van't Hoff enthalpy of unfolding (DeltaH(VH)) of 146.2 kcal/mol at pH 7.0. The stability of the enzyme changes on forming an inhibitor adduct. As expected, some inhibitors stabilize TEM-1; transition-state analogues increase the T(m) by up to 3.7C (1.7 kcal/mol). Surprisingly, all beta-lactam covalent acyl--enzyme complexes tested destabilize TEM-1 significantly relative to the apo-enzyme. For instance, the clinically used inhibitor clavulanic acid and the beta-lactamase-resistant beta-lactams moxalactam and imipenem destabilize TEM-1 by over 2.6C (1.2 kcal/mol) in their covalent adducts. Based on the structure of the TEM-1/imipenem complex (Maveyraud et al., J Am Chem Soc 1998;120:9748--52), destabilization by moxalactam and imipenem is thought to be caused by a steric clash between the side-chain of Asn132 and the 6(7)-alpha group of these beta-lactams. To test this hypothesis, the mutant enzyme N132A was made. In contrast with wild-type, the covalent complexes between N132A and both imipenem and moxalactam stabilize the enzyme, consistent with the hypothesis. To investigate the structural bases of this dramatic change in stability, the structure of N132A/imipenem was determined by X-ray crystallography. In the complex with N132A, imipenem adopts a very different conformation from that observed in the wild-type complex, and the putative destabilizing interaction with residue 132 is relieved. Studies of several enzymes suggest that beta-lactams, and covalent inhibitors in general, can have either net favorable or net unfavorable noncovalent interaction energies within the covalent complex. In the case of TEM-1, such unfavorable interactions convert substrate analogues into very effective inhibitors. 相似文献
18.
J P Scheerlinck I Lasters M Claessens M De Maeyer F Pio P Delhaise S J Wodak 《Proteins》1992,12(4):299-313
A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed. 相似文献
19.
Heterotrimeric G proteins mediate cell growth and differentiation by coupling cell surface receptors to intracellular effector enzymes. The G-protein alpha subunit, Galpha(16), and its murine homologue Galpha(15), are expressed specifically in hematopoietic cells and their expression is highly regulated during differentiation of normal and leukemic cells. In this study, we examined the phosphorylation of Galpha(15)/Galpha(16) and its role in receptor and effector coupling. We observed a PMA-stimulated intact cell phosphorylation of Galpha(15) in COS7 cells transfected with Galpha(15) and protein kinase Calpha (PKCalpha), and phosphorylation of endogenous Galpha(16) in HL60 cells. We also showed that peptides derived from the two G-proteins were phosphorylated in vitro using purified brain PKC. Furthermore, we identified the putative phosphorylation site and showed that mutation or deletion of this PKC phosphorylation site inhibited phospholipase C (PLC) activation. The behavior of double mutants with the constitutively active G-protein mutation (QL-mutant) and mutation in the putative phosphorylation site suggests that the phosphorylation site of Galpha(15/16) is essential for receptor-coupled activation of PLC, but not for direct interaction of the G-protein with PLC-beta. 相似文献
20.
Klaus Fütterer Raimond B. G. Ravelli Scott A. White Andrew J. Nicoll Rudolf K. Allemann 《Acta Crystallographica. Section D, Structural Biology》2008,64(3):264-272
The high photon flux at third‐generation synchrotron sources can inflict significant primary radiation damage upon macromolecular crystals, even when the crystals are cryocooled. However, specific radiation‐induced structural changes can be exploited for de novo phasing by an approach known as radiation damage‐induced phasing (RIP). Here, RIP and single‐wavelength anomalous dispersion (SAD) phasing were alternatively used to derive experimental phases to 1.2 Å resolution for crystals of an α‐helical 18‐residue peptide, MINTS, which was derived from the neurotoxin apamin and the palladium‐bound structure of which is now reported. Helix formation is induced by the binding of palladium (or copper) to two histidines spaced four residues apart, while two disulfide bonds tether the N‐terminal helix to the C‐terminal loop‐like part of the peptide. Either RIP or SAD phasing of the palladium‐bound and copper‐bound forms of MINTS, which crystallized in different space groups, resulted in density maps of superb quality. Surprisingly, RIP phasing of the metal‐bound complex structures of MINTS was a consequence of differential radiation damage, resting primarily on the reduction of the disulfide bonds in Pd‐MINTS and on depletion of the metal sites in Cu‐MINTS. Its miniprotein‐like characteristics, versatile metal‐binding properties and ease of crystallization suggest MINTS to be a convenient test specimen for methods development in crystallographic phasing based on either synchrotron or in‐house X‐ray diffraction data. 相似文献