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1.
Dokudovskaya S Williams R Devos D Sali A Chait BT Rout MP 《Structure (London, England : 1993)》2006,14(4):653-660
Limited proteolysis is widely used in biochemical and crystallographic studies to determine domain organization, folding properties, and ligand binding activities of proteins. The method has limitations, however, due to the difficulties in obtaining sufficient amounts of correctly folded proteins and in interpreting the results of the proteolysis. A new limited proteolysis method, named protease accessibility laddering (PAL), avoids these complications. In PAL, tagged proteins are purified on magnetic beads in their natively folded state. While attached to the beads, proteins are probed with proteases. Proteolytic fragments are eluted and detected by immunoblotting with antibodies against the tag (e.g., Protein A, GFP, and 6xHis). PAL readily detects domain boundaries and flexible loops within proteins. A combination of PAL and comparative protein structure modeling allows characterization of previously unknown structures (e.g., Sec31, a component of the COPII coated vesicle). PAL's high throughput should greatly facilitate structural genomic and proteomic studies. 相似文献
2.
Niccolai N Ciutti A Spiga O Scarselli M Bernini A Bracci L Di Maro D Dalvit C Molinari H Esposito G Temussi PA 《The Journal of biological chemistry》2001,276(45):42455-42461
Characterization of protein surface accessibility represents a new frontier of structural biology. A surface accessibility investigation for two structurally well-defined proteins, tendamistat and bovine pancreatic trypsin inhibitor, is performed here by a combined analysis of water-protein Overhauser effects and paramagnetic perturbation profiles induced by the soluble spin-label 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl on NMR spectra. This approach seems to be reliable not only for distinguishing between buried and exposed residues but also for finding molecular locations where a network of more ordered waters covers the protein surface. From the presented set of data, an overall picture of the surface accessibility of the two proteins can be inferred. Detailed knowledge of protein accessibility can form the basis for successful design of mutants with increased activity and/or greater specificity. 相似文献
3.
4.
Competition between solution and cell surface receptors for ligand. Dissociation of hapten bound to surface antibody in the presence of solution antibody. 总被引:6,自引:5,他引:1
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B Goldstein R G Posner D C Torney J Erickson D Holowka B Baird 《Biophysical journal》1989,56(5):955-966
We present a joint theoretical and experimental study on the effects of competition for ligand between receptors in solution and receptors on cell surfaces. We focus on the following experiment. After ligand and cell surface receptors equilibrate, solution receptors are introduced, and the dissociation of surface bound ligand is monitored. We derive theoretical expressions for the dissociation rate and compare with experiment. In a standard dissociation experiment (no solution receptors present) dissociation may be slowed by rebinding, i.e., at high receptor densities a ligand that dissociates from one receptor may rebind to other receptors before separating from the cell. Our theory predicts that rebinding will be prevented when S much greater than N2Kon/(16 pi 2D a4), where S is the free receptor site concentration in solution, N the number of free surface receptor sites per cell, Kon the forward rate constant for ligand-receptor binding in solution, D the diffusion coefficient of the ligand, and a the cell radius. The predicted concentration of solution receptors needed to prevent rebinding is proportional to the square of the cell surface receptor density. The experimental system used in these studies consists of a monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), that reversibly binds to a monoclonal anti-DNP immunoglobulin E (IgE). This IgE is both a solution receptor and, when anchored to its high affinity Fc epsilon receptor on rat basophilic leukemia (RBL) cells, a surface receptor. For RBL cells with 6 x 10(5) binding sites per cell, our theory predicts that to prevent DCT rebinding to cell surface IgE during dissociation requires S much greater than 2,400 nM. We show that for S = 200-1,700 nM, the dissociation rate of DCT from surface IgE is substantially slower than from solution IgE where no rebinding occurs. Other predictions are also tested and shown to be consistent with experiment. 相似文献
5.
Recombinant antibody fragments consisting of variable domains can be easily produced in various host cells, but there is no universal system that can be used to purify and detect them in the free form or complexed with their antigen. Protein L (PpL) is a cell wall protein isolated from Peptostreptococcus magnus, which has been reported to interact with the V-KAPPA chain of some, but not all, antibodies. Here we grafted the V-KAPPA framework region 1 (FR1) sequence of a high-affinity PpL-binding antibody onto single-chain antibody fragments (scFvs), which have no reactivity with PpL. This substitution made it possible to purify and detect scFvs using PpL conjugates. It did not hinder scFv folding and expression in recombinant bacteria, and it did not interfere with their antigen-binding function. We also identified residue 12 as being potentially able to alter PpL binding. This study, therefore, suggests a way of engineering a PpL-binding site on any scFv without interfering with its function. This could provide a universally applicable method both for the rapid purification of functional recombinant antibody fragments and for their detection even when complexed with their antigen without requiring fusion to an epitope Flag. 相似文献
6.
A new, simple method based on information theory is introduced to predict the solvent accessibility of amino acid residues in various states defined by their different thresholds. Prediction is achieved by the application of information obtained from a single amino acid position or pair-information for a window of seventeen amino acids around the desired residue. Results obtained by pairwise information values are better than results from single amino acids. This reinforces the effect of the local environment on the accessibility of amino acid residues. The prediction accuracy of this method in a jackknife test system for two and three states is better than 70 and 60 %, respectively. A comparison of the results with those reported by others involving the same data set also testifies to a better prediction accuracy in our case. 相似文献
7.
A conjugable analogue of the benzodiazepine 5-(2-hydroxiphenyl)-7-nitro-benzo[ e][1,4]diazepin-2(3 H)-one N 1-substituted with an aliphatic chain (CNZ acyl derivative, CAd) was synthesized. CAd inhibited FNZ binding to GABA A-R with an inhibition binding constant K i = 176 nM and expanded a model membrane packed up to 13 mN/m when penetrating from the aqueous phase. CAd exhibited surface activity with a collapse pressure pi = 18.8 mN/m and minimal molecular area A min = 49 A (2)/molecule at the closest molecular packing, resulting in full and nonideal mixing with a phospholipid in a monolayer up to a molar fraction x congruent with 0.1, decreasing its surface potential and contributing with a dipole that pointed its positive end toward the air and reoriented at the interface upon compression. These findings suggested that CAd could be stabilized at the membrane-water interface with its CNZ moiety stacked at the GABA A-R while its acyl chain can be inserted into the membrane depth. 相似文献
8.
Steric effects on multivalent ligand-receptor binding: exclusion of ligand sites by bound cell surface receptors.
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Steric effects can influence the binding of a cell surface receptor to a multivalent ligand. To account for steric effects arising from the size of a receptor and from the spacing of binding sites on a ligand, we extend a standard mathematical model for ligand-receptor interactions by introducing a steric hindrance factor. This factor gives the fraction of unbound ligand sites that are accessible to receptors, and thus available for binding, as a function of ligand site occupancy. We derive expressions for the steric hindrance factor for various cases in which the receptor covers a compact region on the ligand surface and the ligand expresses sites that are distributed regularly or randomly in one or two dimensions. These expressions are relevant for ligands such as linear polymers, proteins, and viruses. We also present numerical algorithms that can be used to calculate steric hindrance factors for other cases. These theoretical results allow us to quantify the effects of steric hindrance on ligand-receptor kinetics and equilibria. 相似文献
9.
Detection and separation of lymphocytes with specific surface receptors, by using microparticles.
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Horse anti-(human lymphocyte) globulin was immobilized together with fluorescein labelled dextran in spherical microparticles of polyacrylamide (AHLG-particles). The particles had a diameter of 1-5 micrometer and a density of 1.12g/cm3, with globulin exposed on the surface. Human lymphocytes bearing the antigen (thymus-derived lymphocytes) bound the particles, which were easily detected by fluorescence microscopy. In this way, about 58% of circulating human lymphocytes were able to bind AHLG-particles at 23 degrees C. Non-specific binding was low, only 3% when human serum albumin was present in the buffer, and only 4% when non-specific horse globulins were incorporated in the microparticles. The cell-particle complexes could be separated from cells that had not reacted by density-gradient centrifugation in Ficoll/metrizoate. The viability was not changed after the separation procedure. The number of cells binding AHLG-particles corresponded well the the relative amount of T-cells. When the cells binding AHLG-particles were separated from the lymphocytes, the number of T-cells decreased remarkably, indicating that the antibodies bind preferably to the T-cell population. Concanavalin A immobilized in microparticles was sufficiently exposed to initiate the agglutination of the lymphocytes. The agglutination was completely inhibited by preincubating the microparticles with alpha-methyl mannoside. 相似文献
10.
Grafting of triggering site onto lymphocytes; distribution of grafted dinitrophenyl groups on cell surface glycoproteins and glycolipids 总被引:1,自引:0,他引:1
Quantitative investigation of membrane-bound sialoglycoconjugates on lymphocyte surface was performed by chemical modification of the sialic acid residues with radioactive N4-dinitrophenyl-L-2,4-diaminobutyric acid hydrazide (DNP-DABH). This labeled both glycoproteins and glycolipids with concomitant preservation of the mitogenic activity by multivalent hapten binding protein (anti-DNP antibody). Under conditions where maximum stimulation of thymocytes occurred radioactive DNP-DABH labeled 1.1 X 10(7) glycolipids molecules/cell but, only 3 X 10(6) glycoproteins molecules/cell. When B lymphocytes, which do not undergo DNP-mediated stimulation were used, glycolipids labeling could not be detected. Major differences between stimulation committed and non-committed DNP-modified lymphocytes was the amount of ligand attached to the cell surface sialoglycolipids (gangliosides). 相似文献
11.
Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection 总被引:22,自引:0,他引:22
Zanata SM Lopes MH Mercadante AF Hajj GN Chiarini LB Nomizo R Freitas AR Cabral AL Lee KS Juliano MA de Oliveira E Jachieri SG Burlingame A Huang L Linden R Brentani RR Martins VR 《The EMBO journal》2002,21(13):3307-3316
Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis. 相似文献
12.
J L Finney 《Journal of molecular biology》1975,96(4):721-732
The volumes occupied by atoms or groups of atoms in ribonuclease S are calculated, using a new treatment of the surface, and considering buried and exposed atoms separately. The volumes occupied by particular atom types fall within a narrow range, with standard deviations between 10% and 15% of the mean. Summation of volumes over main and side-chain groups removes inaccuracies in the single atom data, and results in even narrower distributions. Comparison of the volume spreads with those found in model glasses suggests that the remaining variation is unlikely to be much reduced by improving further either the space-subdivision method or the surface treatment. These improved volume distributions might be used as more stringent criteria against which to test trial tertiary structures.The computer program used produces additional information that could be used to investigate quantitatively the possible freedom of movement in different parts of the molecule. It also provides complementary data on environment and surface exposure. 相似文献
13.
J H Lee T J Baker L K Mahal J Zabner C R Bertozzi D F Wiemer M J Welsh 《The Journal of biological chemistry》1999,274(31):21878-21884
The absence of viral receptors is a major barrier to efficient gene transfer in many cells. To overcome this barrier, we developed an artificial receptor based on expression of a novel sugar. We fed cells an unnatural monosaccharide, a modified mannosamine that replaced the acetyl group with a levulinate group (ManLev). ManLev was metabolized and incorporated into cell-surface glycoconjugates. The synthetic sugar decorated the cell surface with a unique ketone group that served as a foundation on which we built an adenovirus receptor by covalently binding biotin hydrazide to the ketone. The artificial receptor enhanced adenoviral vector binding and gene transfer to cells that are relatively resistant to adenovirus infection. These data are the first to suggest the feasibility of a strategy that improves the efficiency of gene transfer by using the biosynthetic machinery of the cell to engineer novel sugars on the cell surface. 相似文献
14.
J Garrido 《Experimental cell research》1975,94(1):159-175
The distribution of specific surface receptors in the course of the cell cycle has been studied on two transformed cell lines by means of ultrastructural labelling techniques employing Concanavalin A (ConA) and wheat germ agglutinin (WGA). Synchronized cultures of Cl2TSV5, an SV40-transformed hamster cell line and of CHO cells were labelled as monolayers or in suspension in the different phases of the cell cycle. In cells labelled in monolayers, a moderately discontinuous pattern of surface labelling was present during G 1, S, and G 2. On cells in mitosis, however, this pattern changes strikingly and becomes very discontinuous. These results indicate that the degree of receptor clustering is greater in mitosis than in interphase. In cells labelled in suspension, the differences in pattern between mitosis and interphase were absent. Colcemid treatment did not modify the distribution of the label, either in interphase or in mitosis. Moreover, cells in mitosis collected by Colcemid treatment and labelled at a moment in which parallel unblocked cultures had completed mitosis and passed into G 1 showed an interphase-type labelling pattern; this indicates that a certain dissociation exists between surface events and nuclear events during mitosis. These results are discussed in terms of several factors that may contribute to the production of receptor clustering, namely, direct lectin action, surface movement and membrane flow, participation of cytoplasmic structures and, finally, attachment of cells to a substratum. 相似文献
15.
The binding of fusicoccin to the microsomal preparations of maize roots in vitro is increased several-fold when segments of the tissue are washed for 2 h in distilled water before homogenization. Addition of freeze-dried wash solution to microsomal preparations of spinach leaves or fresh roots, washed roots, or coleoptiles of maize inhibited the binding of fusicoccin to particulate fractions. The freeze-dried material also blocked fusicoccin-promoted H+ extrusion from maize root segments. Roots may contain one or more water-soluble compounds competing with fusicoccin at the receptor level; such ligands might play a physiological role as modulators of the H+/K+ exchange system in higher plants.Abbreviation FC
Fusicoccin 相似文献
16.
Selectin-induced leukocyte rolling along the endothelial surface is an essential step in the immune response. Several in vitro studies showed that this cell rolling is a highly regulated adhesion phenomenon, controlled by the kinetics and forces of selectin-ligand interactions. In the flow chamber study presented here, we focused on the requirements on the ligand structure in this context. A series of neoglycolipids bearing the binding epitope Sialyl Lewis X was synthesized and used as artificial ligands. These lipids differed in their spacer structures between headgroup and membrane anchor, resulting in a gradual variation in accessibility and mobility of the binding epitope when immobilized in model membranes. Consequently, analysis of cell rolling along such membranes allowed correlation of ligand structures and functionality. All model membranes containing such ligands were further characterized by film balance measurements, epifluorescence, and atomic force microscopy. Generally, the glycolipids exhibited a high tendency for lateral aggregation, but the resulting clusters were of different morphology. This was also reflected by strong differences in the rolling experiments. Our results confirm that, in addition to a sufficient headgroup accessibility, the cell rolling process is governed by two further interdependent factors: (i) the headgroup flexibility caused by the intramolecular uncoupling between the headgroup and the hydrophobic moiety due to introduction of a spacer, and (ii) the stiffness of the molecules resulting from their supramolecular arrangement in clustered assemblies. Since both factors are influenced simultaneously by the spacer modification, we present for the first time a clear correlation between structural aspects of selectin ligands and their ability to mediate cell rolling. This might help to develop a better understanding for the function of the natural selectin ligands. 相似文献
17.
We have determined, for the first time, the enthalpic contributions to the energy change associated with ligand reorganization (LR) upon the binding of the same ligand to multiple sites within human serum albumine (HSA). Quantum mechanics based density functional theory (DFT) has been used for the LR calculations, which provides much better accuracy than previously used molecular mechanics methods (MM). Our findings show that for some ligands these enthalpic contributions can be attributed to specific structural and conformational changes. 相似文献
18.
G E Gilbert P J Sims T Wiedmer B Furie B C Furie S J Shattil 《The Journal of biological chemistry》1991,266(26):17261-17268
Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va. 相似文献
19.
Several mechanisms participate in the down-regulation of growth hormone receptor (GHR) signalling under ligand exposure. In CHO cells expressing GHR, we show that ligand stimulation induces degradation of the total cell GHR content. Experiments with 125I-hGH indicate that ligand-bound internalized receptors are not immediately replaced. Using cell surface biotinylation, we demonstrate for the first time that, concomitantly with the degradation of cell surface receptors, GHRs from the intracellular compartments are also degraded. We thus suggest that under prolonged ligand exposure, some GHRs are targeted to the cell surface, while others are routed to degradation compartments. Inhibitors of Jak2 and of the proteasome partially inhibited degradation of cell surface receptors, while these compounds completely inhibit the degradation of intracellular GHRs, resulting in their accumulation. We therefore propose that Jak2 and proteasome activities control the amount of intracellular GHRs, and thus the availability of receptors at the cell surface, during ligand exposure. 相似文献
20.