共查询到20条相似文献,搜索用时 31 毫秒
1.
On the basis of the spatial conformation of gonadotropin-releasing hormone (GnRH), we have predicted that aromatic amino acids and at least one carboxyl group are involved in the recognition site of the receptor. Therefore, various specific reagents were examined for their ability to interfere with the binding of GnRH to its receptor. Pretreatment of pituitary membrane preparations with sodium periodate decreased the specific binding in a dose-dependent manner (IC50 = 0.5 mM) due to a decrease in receptor affinity. This indicated the presence of a sugar moiety in the binding site. Tryptophan is another constituent that participates in the GnRH binding site, as pretreatment of pituitary membranes with 2-methoxy-5-nitrobenzyl bromide inhibited the binding (IC50 = 0.22 mM) by decreasing receptor affinity. In addition, the native hormone conferred on the binding site a protective effect against inactivation by 2-methoxy-5-nitrobenzyl bromide. Pretreatment of membranes with p-diazobenzenesulfonic acid also inhibited the binding of 125I-Buserelin (IC50 = 0.1 mM), indicating the presence of tyrosine within or near the binding site. Pretreatment of pituitary membrane preparations with dithiothreitol also inhibited the binding due to a decrease in the binding affinity, which was accompanied by an increase in receptor number. These data suggest that there are disulfide bonds within or near the binding region. Treatment with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and glycine ethyl ester also prevented binding in a dose-dependent manner and implies that free carboxylic groups are involved in the binding site.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
J Pirrwitz G Lassmann H Rein G R J?nig S Pecar K Ruckpaul 《Acta biologica et medica Germanica》1979,38(2-3):235-247
For the characterization of the substrate binding site optical and EPR measurements with spin labelled substrates on solubilized and pure cytochrome P-450 were performed. Analogously to the unlabelled derivatives spin labelled n-alkylamines and isocyanides with different chain lengths are type II substrates. The Ks-values evaluated from optical (P-450 = 1.98 . 10(-6) M) and ESR (P-450 = 1.98 . 10(-4) M) measurements are very similar indicating no concentration dependences. Contrary to the unlabelled n-alkylamines the spin labelled compounds show an affinity almost independent of the chain lengths. The SL-substrates with a short distance between the functional group and the NO-group bound to P-450 induce pronounced changes of the ligand field of the heme iron and a large broadening of the signal of the immobilized nitroxide indicating intensive interactions between the unpaired electron of the nitroxide group and the paramagnetic heme iron. Elongation of the alkyl chains results in spectra of the Fe3+ complexes with only slight modification and a remained unbroadened signal of the immobilized nitroxide. The binding of the substrate through their functional groups together with a 1:1 stoichiometry of the P-450 SL-IC-complex give evidence for the same binding site in the near vicinity of the heme iron. 相似文献
3.
The binding site topography of progesterone-binding globulin (PBG) purified from pregnant guinea pig serum was examined using synthesized spin-labeled ligands and electron spin resonance (ESR) spectroscopy. A series of deoxycorticosterone-nitroxide (DOC-NO) derivatives were prepared, bearing the free radical on the side chain at increasing distance (d) from the steroid nucleus. The ability of the spin-labeled steroids to specifically bind to PBG was assessed by measurement of their relative binding affinity as compared to progesterone. ESR spectra of the bound steroid nitroxide radical were used to calculate the rotational correlation times tau c for the nitroxides as a function of their distance d to the protein-bound steroid nucleus. The data showed that the side chain nitroxide exhibited an unrestrained rotation in a water-like environment when d reached about 18 A. This would correspond to a PBG steroid binding site depth of about 28 A and suggests that the bound steroid in the PBG site is oriented with the side chain at C-17 directed toward the outside of the protein binding crevice. 相似文献
4.
The site of substrate and fructose 2,6-bisphosphate binding to rabbit liver fructose-1,6-bisphosphatase 总被引:1,自引:0,他引:1
The binding site(s) in rabbit liver fructose-1,6-bisphosphatase for the active site binding ligand, fructose 6-phosphate, and the inhibitor, fructose 2,6-bisphosphate, have been investigated by using nuclear magnetic resonance spectroscopy. The distance from a nitroxide spin label to the bound ligands and the distance from the structural metal site to the bound ligands are about the same within experimental error. These data indicate that the two ligands probably bind at the active site in the rabbit liver enzyme. 相似文献
5.
Gingras AR Vogel KP Steinhoff HJ Ziegler WH Patel B Emsley J Critchley DR Roberts GC Barsukov IL 《Biochemistry》2006,45(6):1805-1817
Talin is a key protein involved in linking integrins to the actin cytoskeleton. The long flexible talin rod domain contains a number of binding sites for vinculin, a cytoskeletal protein important in stabilizing integrin-mediated cell-matrix junctions. Here we report the solution structure of a talin rod polypeptide (residues 1843-1973) which contains a single vinculin binding site (VBS; residues 1944-1969). Like other talin rod polypeptides, it consists of a helical bundle, in this case a four-helix bundle with a right-handed topology. The residues in the VBS important for vinculin binding were identified by studying the binding of a series of VBS-related peptides to the vinculin Vd1 domain. The key binding determinants are buried in the interior of the helical bundle, suggesting that a substantial structural change in the talin polypeptide is required for vinculin binding. Direct evidence for this was obtained by NMR and EPR spectroscopy. [1H,15N]-HSQC spectra of the talin fragment indicate that vinculin binding caused approximately two-thirds of the protein to adopt a flexible random coil. For EPR spectroscopy, nitroxide spin labels were attached to the talin polypeptide via appropriately located cysteine residues. Measurements of inter-nitroxide distances in doubly spin-labeled protein showed clearly that the helical bundle is disrupted and the mobility of the helices, except for the VBS helix, is markedly increased. Binding of vinculin to talin is thus a clear example of the unusual phenomenon of protein unfolding being required for protein/protein interaction. 相似文献
6.
A combined fluorescence-photochrome approach was used for investigation of the molecular dynamics antiDNP antibody binding site and its cavity. A 4-(N-2,4-dinitrophenylamino)-4'-(N,N'-dimethylamino)stilbene (StDNP) fluorescence DNP analog was incorporated into the antibody binding site. This was followed by measurements of fluorescence and photochrome parameters such as the StDNP excitation and emission spectra, fluorescence lifetime, steady-state and time-resolved fluorescence polarization, kinetics of trans-cis and cis-trans photoisomerization, and fluorescence quenching by nitroxide radicals freely diffused in solution. In parallel, computational modeling studies on the location and dynamics of DNP/TEMPO spin-label (NslDNP) and StDNP guests within a model of the binding site were performed. When all the experimental evidence is considered (including data from the antibody X-ray study), one can conclude that wobbling of the Trp 91 L/Trp 96 H binding-site.bound-hapten moiety (StDNP), can be responsible for the label's nanosecond dynamics monitored by fluorescence polarization techniques. A similar conclusion may be reached as a result of data analysis on NslDNP mobility within the antibody binding site. The mobility of Trp 91 L and Trp 96 H moieties provides the induced fit needed for effective stacking and release of the DNP epitope. Analysis of the above-mentioned data allows one to explore the mechanism of the probe's movement within the binding site and enables one to discuss the local dynamics of the binding site region. The combined fluorescence-photochrome approach can be used for investigation of local medium molecular dynamics in the immediate vicinity of specific sites of proteins and nucleic acids, as well as for other biologically important structures and synthetic analogues. 相似文献
7.
Christoph von Ballmoos Yvonne Appoldt Josef Brunner Thierry Granier Andrea Vasella Peter Dimroth 《The Journal of biological chemistry》2002,277(5):3504-3510
A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na(+)-translocating F(1)F(0) ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na(+) ions but not in its presence, indicating the specific reaction with the Na(+) binding c-Glu(65) residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu(65) was therefore in close contact with phospholipid but does not contact subunit a. Na(+)in/(22)Na(+)out exchange activity of the ATP synthase was not affected by modifying the c-Glu(65) sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na(+) ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospholipases C and A(2) and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu(65) is located within the core of the membrane. 相似文献
8.
P Chaussepied 《Biochemistry》1989,28(23):9123-9128
Using a complementary sequence or antipeptide to selectively neutralize the stretch of residues 633-642 of skeletal myosin heavy chain, we recently demonstrated that this segment is an actin binding site operating in the absence as in the presence of nucleotide and that this stretch 633-642 is not part of the nucleotide binding site [Chaussepied & Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471-7475]. In the present study, we determined that the covalent cross-linking of the antipeptide to the stretch 633-642 [induced by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] does not alter the overall polypeptide conformation since no changes were observed on the far-ultraviolet CD spectra and thiol reactivity measurements. The presence of the antipeptide did not influence significantly the enhancement of tryptophan fluorescence induced by ATP.Mg2+ or ADP.Mg2+ binding to the myosin head (S1) nor did it on the ATP.Mg2+-induced tryptic proteolysis of S1 heavy chain. Moreover, fluorescence quenching studies, using acrylamide and the analogue, 1,N6-ethenoadenosine 5'-triphosphate, indicated that the nucleotide bound to antipeptide-S1 complex has an accessibility to the solute quencher close to that observed when it is bound to native S1. Additionally, neutralization of the stretch 633-642 of the S1 heavy chain by the antipeptide did not influence the stabilization of the Mg2+.ADP.sodium vanadate-S1 complex. On the other hand, experiments using antipeptide-induced protection against the cleavage of the S1 heavy chain by Arg-C protease demonstrated that the presence of Mg2+.ADP.sodium vanadate in the S1 nucleotide site did not affect the interaction of the antipeptide with the stretch of residues 633-642.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
Shift of binding site at the interface between actin and myosin 总被引:2,自引:0,他引:2
K Yamamoto 《Biochemistry》1990,29(3):844-848
The molar ratio dependent change in the binding manner between actin and the lysine-rich sequence at the junction between 50K and 20K domains of subfragment 1 was studied by both protease digestion and cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The tryptic cleavage site at the function between 50K and 20K was found to be located between the third and fourth lysine residues in the lysine-rich sequence -KKGGKKK-. This site was not protected by actin when the molar ratio of actin to subfragment 1 was 1:1 but was protected at 2:1 and 3:1. The V8 protease cleavage site of chicken subfragment 1 and the elastase cleavage site of rabbit subfragment 1 were found to be located four residues away from the N-terminus of the lysine-rich sequence. Unlike the tryptic cleavage site, this site was protected by actin more when the molar ratio of actin to subfragment 1 was 1:1 than when it was 2:1 and 3:1. To understand the reason for the opposite effect of the molar ratio observed at the middle of and at four residues away from the lysine-rich sequence, actual cross-linked residue(s) was (were) determined by subjecting cross-linked product to a protein sequencer. It was found that the cross-linked sites were mainly at the first and second lysine residues of the lysine-rich sequence when the molar ratio of actin to subfragment 1 was 1:1.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
Localization of the binding site on fibrin for the secondary binding site of thrombin 总被引:8,自引:0,他引:8
Affinity chromatography of active site inhibited thrombin on immobilized fragments derived from the central (desAB-NDSK) and terminal (D1) globular domains of fibrinogen revealed that the site responsible for the binding of thrombin at its secondary fibrin binding site is located in the central domain. Chromatography of various domains of the central nodule (desAB-NDSK, fibrinogen E, and fibrin E) having nonidentical amino acid sequences showed that all of these fragments are capable of binding to PMSF-thrombin-Sepharose, suggesting that the thrombin binding site resides within the peptide regions common to all of these fragments: alpha(Gly17-Met51), beta(Val55-Met118), and gamma(Tyr1-Lys53). Competitive affinity chromatography of the same binding domains revealed that there is no detectable difference in their binding constants to PMSF-thrombin-Sepharose, indicating that the alpha(Lys52-Lys78), beta(Gly15-Lys54)/(Tyr119-Lys122), and gamma(Thr54-Met78) peptide segments do not contribute significantly to the binding of thrombin. Chromatography of the isolated chains of fibrinogen E showed that the alpha(Gly17-Lys78) peptide region itself contains a strong binding site for PMSF-thrombin-Sepharose. The location of the binding site suggests that the secondary site interaction may play an important role in determining the cleavage specificity of thrombin on fibrinogen and can affect the rate of release of the fibrinopeptides. Affinity chromatography of fragments prepared from polymerized fibrin showed that cross-linked DD (D x D) itself does not bind to thrombin, whereas the D x DE complex remained attached to the column, suggesting that the binding site on fragment E for thrombin is distinct from its binding site for D x D.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
J Stonehuerner P O'Brien L Geren F Millett J Steidl L Yu C A Yu 《The Journal of biological chemistry》1985,260(9):5392-5398
The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c. 相似文献
12.
The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by YZ. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[-Mn]) membranes. The rate of Mn(II) oxidation saturates at > or = 10 microM in PSII(-Mn) membranes and > or = 500 microM in EDC-treated PSII (-Mn) samples. Intact PSII(-Mn) membranes have only one site for Mn(II) oxidation via YZ (dissociation constant, Kd = 0.64 microM), while EDC-treated PSII(-Mn) samples have two sites (Kd = 1.52 and 22 microM; the latter is the low-affinity site). When PSII(-Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (Kd = 2.3 microM) for exogenous Mn(II) oxidation by Y(Z)() radical. In this case, the rate of electron donation via YZ saturated at a Mn(II) concentration > or = 15 microM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HAZ site is protected from the action of the modifier by the iron cations blocking the HAZ site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HAZ site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site. 相似文献
13.
Erika Aloi Jin-Hua Guo Rita Guzzi Ren-Wang Jiang Lucy Kate Ladefoged Derek Marsh Mikael Esmann Rosa Bartucci Natalya U. Fedosova 《Biophysical journal》2021,120(13):2679-2690
Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D2O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled. 相似文献
14.
Sentjurc M Pecar S Stojan J Marchot P Radić Z Grubic Z 《Biochimica et biophysica acta》1999,1430(2):349-358
Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6, 6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound fasciculin at its rim. 相似文献
15.
Proteolytic dissection of a hapten binding site 总被引:1,自引:0,他引:1
IgG Gar, a human myeloma protein that binds riboflavin with a high affinity, was used to derive variable region fragments from the heavy chain and the light chain. Riboflavin binding ability of the active site generated by V(H) and light chain and the active site generated by V(H) and V(L) was compared to riboflavin binding by the F(ab) fragment. The riboflavin binding ability of the F(ab) fragment is the same as the intact molecule, while the binding ability of the active site formed by V(H) and light chain is lowered by two to three orders of magnitude, indicating that the removal of C(H1) domain decreases the interaction between riboflavin and the amino acids that is important in tight binding of riboflavin. Removal of the third hypervariable region and the constant region domain from the light chain further lowers the binding constant by one order of magnitude. The results indicate that the V(H) and V(L) segments of IgG Gar can reconstitute a riboflavin binding site. The decrease in affinity probably reflects a decrease in the rigidity with which the hypervariable loops are held together to place the contact amino acid residues in optimal contact with the hapten. 相似文献
16.
1. Chemical modification of carboxyl groups on the chloroplast membrane with a water-soluble carbodiimide plus a nucleophile caused inhibition of Ca-2plus binding. 2. Both binding sites were affected and showed a decrease in the number of binding sites and an increase in the dissociation constant. 3. Cation-induced changes in chlorophyll a fluorescence and structural changes (deltaA540) were inhibited at the same carbodiimide concentrations as Ca-2plus binding, emphasizing the relationship between these processes. 4. Chloroplasts that were illuminated with high intensity light for short time periods showed a decrease in the carbodiimide-mediated inhibition of Ca-2plus binding. 相似文献
17.
Spin-labeling study on the depth of the active site of papain 总被引:1,自引:0,他引:1
S Nakayama M Hoshino K Takahashi T Watanabe M Yoshida 《Biochemical and biophysical research communications》1981,98(2):471-475
Papain is alkylated with a series of haloacetamide spin labels with varying distance between the haloacetamide residue and the nitroxide portion. The dependence of τc on the distance and temperature revealed that the active site has a narrow neck and one auxiliary binding site around the neck. The depth of the active site is estimated about 10 A. 相似文献
18.
A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1 -yl )]aminophenyl pyrrolidine (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand. The equilibrium dissociation constant (K(D)) is about 23 microM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS. It is concluded that NN binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label. 相似文献
19.
Electron-spin resonance (ESR) spectra of a nitroxide spin-label attached to residue i6A-37 of yeast tRNATyr were measured in complexes of deacylated tRNATyr with Escherichia coli ribosomes. A Scatchard plot, obtained in the absence of mRNA, indicated strong binding with an association constant of 1 X 10(7) l X mol-1, suggesting the P-site binding. The ESR spectrum of free tRNATyr, characteristic for a rapidly tumbling nitroxide, changes to a spectrum with extensively broadened lines in the ribosome-tRNA complex. The original spectrum can be restored upon long incubations of the complex with an excess of extraneous tRNA. ESR spectra suggest that the spin-label motion is drastically perturbed though not completely blocked in the ribosome-tRNATyr complex. Since ESR spectra of a spin-label attached to the opposite, i.e. 5', side of the anticodon loop are only slightly perturbed by the messenger-free binding to ribosomes [Rodriguez et al. (1980) J. Biol. Chem. 255, 8116-8120], it is concluded that the two sides of the anticodon loop face entirely different environments when bound to the P site, the 3' side being oriented towards the surface of the ribosome, and the other side towards its environment or a large cavity. 相似文献
20.
ABSTRACT: BACKGROUND: The ability to predict protein-protein binding sites has a wide range of applications,including signal transduction studies, de novo drug design, structure identification andcomparison of functional sites. The interface in a complex involves two structurallymatched protein subunits, and the binding sites can be predicted by identifying structuralmatches at protein surfaces. RESULTS: We propose a method which enumerates "all" the configurations (or poses) between twoproteins (3D coordinates of the two subunits in a complex) and evaluates eachconfiguration by the interaction between its components using the Atomic Contact Energyfunction. The enumeration is achieved efficiently by exploring a set of rigidtransformations. Our approach incorporates a surface identification technique and amethod for avoiding clashes of two subunits when computing rigid transformations. Whenthe optimal transformations according to the Atomic Contact Energy function areidentified, the corresponding binding sites are given as predictions. Our results show thatthis approach consistently performs better than other methods in binding siteidentification. CONCLUSIONS: Our method achieved a success rate higher than other methods, with the prediction qualityimproved in terms of both accuracy and coverage. Moreover, our method is being able topredict the configurations of two binding proteins, where most of other methods predictonly the binding sites. The software package is available athttp://sites.google.com/site/guofeics/dobi for non-commercial use. 相似文献