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1.
A novel correlation for protein diffusion coefficients based on molecular weight and radius of gyration 总被引:2,自引:0,他引:2
A new correlation is proposed for the prediction of protein diffusion coefficients in free solution. Molecular weight and radius of gyration of proteins are employed as correlation parameters in this method. Both parameters can be easily found in the literature. The correlation works well for diverse proteins with different shapes and extensive molecular weight. Furthermore, this method does not require a preassumption regarding the protein shape while it offers a rapid and convenient calculation with a high accuracy. Also, the proposed correlation can elucidate the estimation deviation of previous correlation methods in the literature. 相似文献
2.
The transporter associated with antigen processing (TAP) plays a key role in the class I major histocompatibility complex (MHC) mediated immune surveillance. It translocates peptides generated by the proteasome complex into the endoplasmic reticulum (ER) for loading onto MHC class I molecules. At the cell surface these MHC complexes are monitored for their antigenic cargo by cytotoxic T-lymphocytes. Peptide binding to TAP is the essential step for peptide selection and for subsequent ATP-dependent translocation into the ER lumen. To examine the pathway of substrate recognition by TAP, we employed peptide epitopes, which were labeled with an environmentally sensitive fluorophore. Upon binding to TAP, a drastic fluorescence quenching of the fluorescent substrate was detected. This allowed us to analyze TAP function in real-time by using a homogeneous assay. Formation of the peptide-TAP complex is composed of a fast association step followed by a slow isomerization of the transport complex. Proton donor groups moving in proximity to the fluorescence label cause fluorescence quenching. Taken together, this peptide-induced structural reorganization may reflect the crosstalk of structural information between the peptide binding site and both nucleotide-binding domains within the TAP complex. 相似文献
3.
HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities. 相似文献
4.
5.
Peptide inhibitors, specifically labeled at the alpha-amino terminus by dansylation, have been prepared by utilizing solid-phase peptide synthesis. Changes in fluorescence have been observed upon mixing these peptides with porcine pepsin that can be attributed to the formation of at least two complexes. Energy transfer between tryptophan residues of the protein and the dansyl group of the inhibitors has been detected by the unique excitation spectra generated. The kinetics of formation of the second complex can be correlated with inhibition of the catalytic activity of pepsin. Evidence for complex formation has also been obtained from gel filtration experiments using the fluorescent peptides. 相似文献
6.
Group-specific polysaccharides isolated by means of a cetavlon procedure are immunogenic in man and induce protective immunity against meningococcal meningitis. Minute quantities of the polymers in solution can act as vaccines. We now report the first characterization of a fractionated (C-1) group C polysaccharide in 0.4KM KCl and 0.05M sodium acetate by means of light-scattering spectroscopy. Independent measurements of refractive index increments, absolute scattered intensities, angular scattering intensities and line widths as a function of scattering angles and delay times at different concentrations using incident wavelengths of 632.8 nm from a He–Ne laser and of 488 nm from an argon–ion laser yield information on aggregation properties, molecular weight (Mr), radius of gyration 〈r0g〉1/2z, translational diffusion coefficient 〈D〉0z, and second virial coefficients A2 and B2 of C-1 polysaccharide. At relatively high ionic strength (0.04M KCl + 0.05M sodium acetate), we obtain for the C-1 polysaccharide in solution Mr = 5.15 × 105, 〈r2g〉1/2z = 345 Å, A2 = 1.25 × 10?4 ml/g, 〈D〉 = 1.16 × 10?7 cm2/sec with a corresponding Stokes radius of 240 Å and B2 = 4.4 ml/g. A2 and B2 are the second virial coefficients from intensity- and diffusion-coefficient measurements. The C-1 polysaccharide aggregates in solution and behaves hydrodynamically like random coils. Viscosity and sedimentation studies further confirm our conclusions that the fractioned C-1 polysaccharide aggregates in solution and EDTA can partially break up those aggregates. However, the system remains polydisperse even after adding an excess amount of EDTA. The weight-average molecular weight of the C-1 polysaccharide in solution depends upon ionic strength and exhibits a minimum at ~0.2M KCl. Finally, viscosity, light-scattering, and sedimentation results all show that the aggregated macromolecular system behaves like random-coiled polymers with no measurable shape factors. 相似文献
7.
Zarutskie JA Sato AK Rushe MM Chan IC Lomakin A Benedek GB Stern LJ 《Biochemistry》1999,38(18):5878-5887
To investigate a conformational change accompanying peptide binding to class II MHC proteins, we probed the structure of a soluble version of the human class II MHC protein HLA-DR1 in empty and peptide-loaded forms. Peptide binding induced a large decrease in the effective radius of the protein as determined by gel filtration, dynamic light scattering, and analytical ultracentrifugation. It caused a substantial increase in the cooperativity of thermal denaturation and induced alterations in MHC polypeptide backbone structure as determined by circular dichroism. These changes suggest a condensation of the protein around the bound peptide. An antibody specific for beta58-69 preferentially bound the empty protein, indicating that the peptide-induced conformational change involves the beta-subunit helical region. The conformational change may have important implications for the mechanisms of intracellular antigen presentation pathways. 相似文献
8.
The grafting of l-phenylalanine onto low molecular weight chitosan is accomplished by using carbodiimide as a coupling agent. As increase in the amount of phenylalanine in feed, the grafting chain length increases, while a number of grafting chains hardly change. The obtained product, LMWCts-g-Phe, performs sphere with an average size of ~80 nm when the % grafting is less than 123. The complexes of the LMWCts-g-Phe and DNA (LMWCts-g-Phe/DNA) prepared by a complex coacervation method possess various shapes with an average size of ~50–150 nm and a negatively charged surface. The LMWCts-g-Phe and its complex show very reduced toxicity to fibroblast cells. The release of DNA from the complex is very fast in high pH media (tris buffer, pH 8.0 and carbonate buffer, pH 9.5), and relatively slow or more sustainable in neutral and low pH ones (PBS, pH 7.4 and citric acid/trisodium citrate buffer, pH 3.0). The results suggest that the LMWCts-g-Phe be an alternative promising carrier for negatively charged active molecules. 相似文献
9.
Molecular dynamic simulation is a practical and powerful technique for analysis of protein structure. Several programs have been
developed to facilitate the mentioned investigation, under them the visual molecular dynamic or VMD is the most frequently used
programs. One of the beneficial properties of the VMD is its ability to be extendable by designing new plug-in. We introduce here a
new facility of the VMD for distance analysis and radius of gyration of biopolymers such as protein and DNA.
Availability
The database is available for free at http://trc.ajums.ac.ir/HomePage.aspx/?TabID/=12618/&Site/=trc.ajums.ac/&Lang/=fa-IR 相似文献10.
K Nelson J Baranowska-Kortylewicz J R van Nagell H H Gallion E S Donaldson D E Kenady W P McRoberts E J Pavlik 《Biochemistry》1989,28(25):9741-9749
After exposure to ligand at 0-4 degrees C, estrogen receptors from mouse uteri characteristically eluted between thyroglobulin (Mr 669,000) and ferritin (Mr 443,000) during size-exclusion HPLC. However, when preparations were warmed with ligand under mild activating conditions, most or all of the receptor was observed as a much larger complex, which eluted between dextran blue 2000 and thyroglobulin. Formation of the large complex required ligand, was inhibited by molybdate, and occurred even in 0.4 M KCl. Slower ligand dissociation characterized the large complex, indicating that activated receptors were included preferentially. This large complex did not form when charged cytosols were aged, concentrated, or precipitated, indicating that formation was not the result of random aggregation. After exposure to conditions commonly used for activation (25 degrees C, 60 min), most receptor existed as a very large, monodisperse complex of finite size, predicting an ordered structure for these large complexes that should be useful for defining the types of proteins which can interact with estrogen receptors. Formation of the large complex was not impeded or disrupted by EDTA, RNase, DNase I, thiourea, or mercaptoethanol; however, the capacity to form this large complex was not demonstrated by preparations that had been exposed to trypsin or by the small receptor forms obtained after salt extraction. Proteolytic sensitivity and lack of sensitivity to RNase or DNase indicate that interactions between receptors and other proteins are involved in peak A formation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
Calcium-induced increase in the radius of gyration and maximum dimension of calmodulin measured by small-angle X-ray scattering 总被引:6,自引:0,他引:6
We have used solution small-angle X-ray scattering to characterize bovine brain calmodulin in the presence and absence of calcium. In the presence of calcium, calmodulin exists in solution as an elongated molecule with a radius of gyration of 21.5 A and a maximum vector length of approximately 62 A. These values are consistent with the dimensions recently determined for the crystal form of rat testis calmodulin. In the absence of calcium, the calmodulin molecule is shorter, the radius of gyration decreases to 20.6 A, and the maximum vector length decreases to approximately 58 A. This change in dimensions is consistent with an overall contraction of the protein through movement of the two lobes closer to each other upon removal of calcium from calmodulin. 相似文献
12.
A procedure is described for the rapid preparation of a partial cyanogen bromide digest of horse heart cytochrome c. This digest serves as a calibration mixture for estimation of peptide molecular weight in the range 500 to 20,000 by exelusion chromatography in denaturing solvents. The elution profile of the calibration mixture from Sephadex G-50 equilibrated and developed with 10% formic acid is described by the relationship log M = 4.79 ? 0.42 (). 相似文献
13.
Demicheli C Frézard F Pereira FA Santos DM Mangrum JB Farrell NP 《Journal of inorganic biochemistry》2011,105(12):1753-1758
The interaction of arsenite with a Cys3His (CCHC) zinc finger model (34-51) HIV-1 nucleocapsid protein p7 (NCp7) peptide in the absence and presence of ZnII was studied using fluorescence spectroscopy, CD (circular dichroism) and ESI-MS (Electrospray Ionization Mass Spectrometry). We found that arsenic forms different complexes with the free peptide and the zinc finger peptide. In the former case the peptide conformation differed greatly from that of the zinc finger, whereas in the second case a mixed As-Zn-peptide complex was formed with partial preservation of zinc finger conformation. An apparent stability constant was estimated for the mixed As-Zn-peptide complex (K = 2083 M− 1 and 442 M− 1 at 25 °C and pHs 6 and 7, respectively). Our study also shows that the interaction of arsenic with the CCHC motif is facilitated by glutathione (GSH), through formation of a GS-As-peptide conjugate. 相似文献
14.
Two GTPases in the signal recognition particle (SRP) and SRP receptor (SR) interact with one another to mediate the cotranslational protein targeting pathway. Previous studies have shown that a universally conserved SRP RNA facilitates an efficient SRP–SR interaction in the presence of a signal sequence bound to SRP. However, a remarkable exception has been found in chloroplast SRP (cpSRP) pathway, in which the SRP RNA is missing. Based on biochemical and structural analyses, it is proposed that free cpSRP receptor (cpFtsY) has already been preorganized into a closed state for efficient cpSRP–cpFtsY association. However, no direct evidence has been reported to support this postulation thus far. In this study, we characterized the structural dynamics of cpFtsY and its conformational rearrangements induced by GTP binding using molecular dynamics (MD) simulations. Our results showed that the GTP-binding event triggered substantial conformational changes in free cpFtsY, including the relative orientation of N–G domain and several conserved motifs that are critical in complex formation. These rearrangements enabled the cpFtsY to relax into a preorganized ‘closed’ state that favored the formation of a stable complex with cpSRP54. Interestingly, the intrinsic flexibility of αN1 helix facilitated these rearrangements. In addition, GTP binding in cpFtsY was mediated by conserved residues that have been shown in other SRP GTPases. These findings suggested that GTP-bound cpFtsY could fluctuate into conformations that are favorable to form the stable complex, providing explanation of why SRP–SR interaction bypasses the requirement of the SRP RNA at a molecular level. 相似文献
15.
Frataxin is an iron-binding mitochondrial matrix protein that has been shown to mediate iron delivery during iron–sulfur cluster
and heme biosynthesis. Mitochondrial processing peptidase (MPP) yields a form of human frataxin corresponding to residues
56–210. However, structural and functional studies have focused on a core structure that results from an ill-defined cleavage
event at the N-terminus. Herein we show that the N-terminus of MPP-processed frataxin shows a unique high-affinity iron site
and that this iron center appears to mediate a self-cleavage reaction. Moreover, the N-terminus appears to block previously
defined iron-binding sites located on the carboxylate-rich surface defined by the helix (α1) and the β-sheet (β1), most likely
through electrostatic contact with the carboxylate-rich surface on the core protein, as well as inhibiting iron-promoted binding
of the iron–sulfur cluster assembly scaffold partner protein, ISU. The physiological significance of iron-mediated release
of the N-terminal residues from this anionic surface is discussed. 相似文献
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17.
Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K(d)=1.5(+/-0.1)mM and an off-rate on the order of 10(4)s(-1). 1H and 15N NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. 15N relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory precision were extracted for the monomer, enabling model-free analyses of backbone fluctuations on pico- to nanosecond time scales. The dimer relaxation data are of lower quality due to extrapolation errors and the possible presence of higher-order oligomers at higher concentrations. A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric (target bound) states. The exchange rate compares rather well with that of the catalyzed reaction step, supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer. 相似文献
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19.
La Gruta NL Liu H Dilioglou S Rhodes M Wiest DL Vignali DA 《Journal of immunology (Baltimore, Md. : 1950)》2004,172(6):3662-3669
A hallmark of T cell activation is the ligation-induced down-modulation of the TCR:CD3 complex. However, little is known about the molecular events that drive this process. The CD3 zeta-chain has been shown to play a unique role in regulating the assembly, transport, and cell surface expression of the TCR:CD3 complex. In this study we have investigated the relationship between CD3zeta and the TCRalphabetaCD3epsilondeltagamma complex after ligation by MHC:peptide complexes. Our results show that there is a significant increase in free surface CD3zeta, which is not associated with the TCR:CD3 complex, after T cell stimulation. This may reflect dissociation of CD3zeta from the TCRalphabetaCD3epsilondeltagamma complex or transport of intracellular CD3zeta directly to the cell surface. We also show that MHC:peptide ligation also results in exposure of the TCR-associated CD3zeta NH2 terminus, which is ordinarily buried in the complex. These observations appears to be dependent on Src family protein tyrosine kinases, which are known to be critical for efficient T cell activation. These data suggest a mechanism by which ligated TCR may be differentiated from unligated TCR and selectively down-modulated. 相似文献
20.
Rabbit histidine-rich glycoprotein (HRG, 94 kDa) binds heparin with high affinity (apparent Kd 60-110 nM). Eosin Y (1 equiv) bound to HRG was used as a reporter group to monitor associations of HRG with heparins of molecular mass 10, 17.5, and 30 kDa. The stoichiometries of the heparin-HRG complexes were determined by fluorescence and absorbance measurements as well as by analytical ultracentrifugation. Two types of complex form: complexes of 1 heparin:1 HRG and of 1 heparin:2 HRG. The 1:2 complex formation requires a minimum heparin chain length since 17.5-kDa but not 10-kDa heparin binds two HRG molecules. The formation of the 1:2 complexes of the larger heparin fractions is enhanced by divalent copper or zinc (1-10 equiv) bound to HRG. However, metal is not required for complex formation since all sizes of heparin examined interact tightly with HRG in the presence of ethylenediaminetetraacetic acid. Between 0.1 and 0.3 M ionic strength, both 1:1 and 1:2 complexes of heparin with HRG are progressively destabilized. No heparin-HRG complex is found at ionic strengths of 0.5 M. Between pH 8.5 and pH 6.5 both 1:2 and 1:1 complexes are found with 17.5-kDa heparin, but at pH 5.5 only 1:1 complexes are formed. The heparin-HRG interaction is progressively decreased by modification of the histidine residues of HRG, whereas modification of 22 of the 33 lysine residues of HRG has little effect.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献