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1.
The crystal structure of the pentapeptide p-toluene-sulfonyl-(α-aminoisobutyryl)5-methyl ester (Tosyl-(Aib)5-OMe) has been determined in the space group PI. Pentapeptide molecules are folded in the 310 helical conformation and packed together, so as to yield a hydrophobic channel with a minimim diameter of 5.2 Å. The channel contains crystallographically disordered material. This structure provides a model for channel formation by hydrophobic peptide aggregates and should prove useful in studies of alamethicin, suzukacillin and related Aib containing membrane channels. Triclinic (PI) crystals of the pentapeptide are obtained in the presence of LiClO4 in aqueous methanol, whereas crystallization from methanol alone yields crystals in the space group Pbca. The conformations of the peptide in the two crystal forms are very similar and only the molecular packing is dramatically different.  相似文献   

2.
Peptidoglycan monomer (GlcNAc-MurNac-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala), labeled with 14C both in the disaccharide and pentapeptide portions, was incubated with slices of mouse liver, kidney or spleen as well as with mouse and human blood cells, plasma and serum. Peptidoglycan monomer was isolated unchanged after incubations with mouse organs and blood cells. However, upon incubation with mouse or human blood, 10–50% of the peptidoglycan monomer underwent hydrolysis to the corresponding disaccharide and pentapeptide. After incubations with plasma and serum more than 90% of the [14C]peptidoglycan monomer was metabolized: about 50% of the administered radioactive dose was recovered in the disaccharide unit and about 35% in the pentapeptide part. These results suggest that in blood, plasma and serum of mouse and man, an N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) exists which splits the amide bond between the lactyl carboxyl group of the muramyl residue and the amino group of the peptide moiety in the peptidoglycan molecule.  相似文献   

3.
Abstract

The crystal-state preferred conformations of two tripeptides, one tetrapeptide, and one pen- tapeptide, each containing a single residue of the chiral, Cα,α-disubstituted glycine Cα-methyl, Cα-benzylglycine [(αMe)Phe], have been determined by X-ray diffraction. The tripeptides are Z-L-(αMe)Phe-(Aib)2-OH dihydrate and Z-Aib-D-(αMe)Phe-Aib-OtBu, the tetrapeptide is Z-(Aib)2-D-(αMe)Phe-Aib-OtBu, and the pentapeptide is pBrBz-(Aib)2-DL-(αMe)Phe-(Aib)2-OtBu. While the two tripeptides are folded in a β-bend conformation, two such conformations are consecutively formed by the tetrapeptide. The pentapeptide adopts a regular 310-helix promoted by three consecutive β-bends. This study confirms the strong propensity of short peptides containing Cα-methylated α-aminoacids to fold into β-bends and 310-helical structures. Since Aib is achiral, the handedness of the observed bends and helices is dictated by the presence of the (αMe)Phe residue. In general, we have found that the relationship between (αMe)Phe chirality and helix handedness is opposite to that exhibited by protein aminoacids. A comparison with the preferred conformation of other extensively investigated Cα-methylated aminoacids is made.  相似文献   

4.
The crystal-state preferred conformations of six Nα-blocked pentapeptide esters, each containing four helicogenic, achiral α-aminoisobutyric acid (Aib) residues followed by one chiral L -valine (L -Val) or Cα-methyl-L -valine [(αMe)Val] residue at the C-terminus, have been assessed by x-ray diffraction analysis. In all of the compounds the  (Aib)4 sequence is folded in a regular 310-helical conformation. In the four pentapeptides characterized by the L -(αMe)Val residue two conformationally distinct molecules occur in the asymmetric unit. Conversely, only one molecule is observed in the asymmetric unit of two pentapeptides with the C-terminal L -Val residue. In the L -Val based peptides the helical screw sense of the  (Aib)4 sequence is right-handed, whereas in the L  (αMe)Val analogues both right- and left-handed helical screw senses concomitantly occur in the two crystallographically independent molecules. © 1998 John Wiley & Sons, Inc. Biopoly 46: 433–443, 1998  相似文献   

5.
Synthesis of diphtheria toxin in E. coli cell-free lysate   总被引:7,自引:0,他引:7  
An E. coli cell-free lysate was used to translate C. diphtheriae RNA from nontoxinogenic C7(?), C7 infected with β tox+ corynebacteriophage, and C. diphtheriae strain PW8. De novo synthesis of toxin was detected by immune precipitation with antitoxin, ADP-ribosylation of mammalian elongation factor 2 and rabbit skin test. The results indicated that toxin is produced in the E. coli protein synthesizing system primed with RNA from cells infected with tox+ bacteriophage and is absent in systems primed with RNA from C7(?) cells.  相似文献   

6.
A comparative study of four peptidomimetics of the sequence Phe-Met-Arg-Phe-amide (FMRFa) was performed to compare the conformational bias caused by trans-2,3-methanomethionine and α-methylmethionine stereoisomers. The specific compounds studied were F[(2S,3S)-cyclo-M] RFa, F[(2R,3R)-cyclo-M]RFa, F[(S)-α-MeM]RFa, and F[(R)-α-MeM]RFa. Molecular simulations based on CHARMm 22 indicate that γ-turn, inverse γ-turn, and α-helical conformations about the cyclo-M residue are accessible to the two F[cyclo-M]RFa stereoisomers. Similar calculations for F[(S)-α-MeM]RFa, and F[(R)-α-MeM]RFa indicate that the α-methylamino acids tend to favor α-helical conformations. The nmr data is presented for the four peptidomimetics. Most informative were the rotating frame nuclear Overhauser effect cross peaks between the NH protons proximal to the methionine surrogates, and the Cβ hydrogens. Overall, these nmr data indicate F[(2S,3S)-cyclo-M]RFa and F[(2R,3R)-cyclo-M]RFa preferentially adopt inverse γ-turn and γ-turn conformations, respectively, whereas F[(S)-α-MeM]RFa and F[(R)-α-MeM]RFa tend to form partial left- and right-handed helical structures (although energy differences between the two turn structures, and between the two helical structures are likely to be small). It is suggested that the wider NH-Cα-CO angle of cyclopropane amino acids and their more severe steric requirements around the Cβ carbons force the peptidomimetic N- and C-termini into the same region of conformational space. This favors C7 turns in the cyclopropane amino acid series relative to the less constrained α-methyl derivatives. © 1997 John Wiley & Sons, Inc. Biopoly 42: 439–453, 1997  相似文献   

7.
The preferred conformations of N-acetyl-N′-methyl amides of some dialkylglycines have been determined by empirical conformational-energy calculations; minimum-energy conformations were located by minimizing the energy with respect to all the dihedral angles of the molecules. The conformational space of these compounds is sterically restricted, and low-energy conformations are found only in the regions of fully extended and helical structures. Increasing the bulkiness of the substituents on the Cα, the fully extended conformation becomes gradually more stable than the helical structure preferred in the cases of dimethylglycine. This trend is, however, strongly dependent on the bond angles between the substituents on the Cα atom: In particular, helical structures are favored by standard values (111°) of the N-Cα-C′ angle, while fully extended conformations are favored by smaller values of the same angle, as experimentally observed, for instance, in the case of α,α-di-n-propylglycine.  相似文献   

8.
Binding of the structural protein soc to the head shell of bacteriophage T4   总被引:5,自引:0,他引:5  
Qβ plus strands with a 70 S ribosome bound to the coat cistron initiation site were used as template for Qβ replicase. Minus strand synthesis proceeded until the replicase reached the ribosome. The ribosome was removed and elongation was continued in a substrate-controlled, stepwise fashion. The nucleotide analog N4-hydroxyCMP was introduced into the positions complementary to the third and fourth nucleotides of the coat cistron. The minus strands were elongated to completion, purified and used as template for Qβ replicase. The final plus strand preparation consisted of four species, with the sequences -A-U-G-G- (wild type), -A-U-A-G- (mutant C3), -A-U-G-A- (mutant C4) and -A-U-A-A- (mutant C3C4) at the coat initiation site. The ribosome binding capacity of the mutant RNAs relative to wild type was <0.1 (C3), 3.2 (C4) and 0.3 (C3C4). The finding that mutant C3 no longer formed an initiation complex suggests that the interaction of the ribosome binding site with fMet-tRNA plays an essential role in the formation of the 70 S initiation complex. The fact that mutant C4 RNA bound more efficiently than wild type, and that mutant C3C4 RNA showed substantial ribosome binding capacity whereas the single mutant C3 did not, can be explained by assuming that an A residue following the A-U-G triplet interacts with a complementary U residue in the anticodon loop sequence. In the case of C3C4 this additional base-pair may offset the reduced codon-anticodon interaction resulting from the modification of the A-U-G codon.  相似文献   

9.
Intramolecularly hydrogen bonded conformations of (Aib-Pro)n sequences have been analysed theoretically. Both 4→1 (C10 and 3→1 (C7 hydrogen bonded regular structures are shown to be stereochemically feasible. Conformational energies for the helical structures have been estimated using classical potential energy methods. Both C10 and C7 conformations have very similar energies. Pyrrolidine ring puckering has a pronounced effect on the energies, and only -endo puckered Pro residues can be accommodated. The theoretical calculations using spectroscopic data suggest that the recently proposed novel 310 helical conformation for benzyloxycarbonyl(Aib-Pro)4-methyl ester is in solution, is indeed energetically and stereochemically favourable.  相似文献   

10.
Sun-Shine Yuan 《Steroids》1982,39(3):279-289
A-ring enollactones 1a, 1b or 9 derived from 4-cholesten-3-one, testosterone benzoate or 3-oxo-4-estren-17β-yl benzoate were condensed with [1,2-13C2]acetyl chloride to give intermediates 2a, 2b or 10. 2a and 2b were cyclized by acid or base to give 3,4-13C2-labeled 4-cholesten-3-one and testosterone, respectively. [3,4-13C2]4-Cholesten-3-one was converted via reduction of its trimethylsilyl enol ether to [3,4-13C2]cholesterol. Acetyl enollactone 10 was cyclized in acetic acid to [3,4-13C2]3-oxo-4-estren-17β-yl benzoate followed by aromatization and hydrolysis to produce [3,4-13C2]estradiol-17β. Alternatively, cyclization of 10 with base afforded [3,4-13C2]3-oxo-4-estren-17β-ol directly, which was then oxidized and aromatized to yield [3,4-13C2]estrone. Ozonolysis of progesterone, conversion to the diketal ester 16 and acylation followed by acid hydrolysis furnished [3,4-13C2]progesterone.  相似文献   

11.
One chiral L ‐valine (L ‐Val) was inserted into the C‐terminal position of achiral peptide segments constructed from α‐aminoisobutyric acid (Aib) and α,β‐dehydrophenylalanine (ΔZPhe) residues. The IR, 1H NMR and CD spectra indicated that the dominant conformations of the pentapeptide Boc‐Aib‐ΔPhe‐(Aib)2‐L ‐Val‐NH‐Bn (3) and the hexapeptide Boc‐Aib‐ΔPhe‐(Aib)3‐L ‐Val‐NH‐Bn (4) in solution were both right‐handed (P) 310‐helical structures. X‐ray crystallographic analyses of 3 and 4 revealed that only a right‐handed (P) 310‐helical structure was present in their crystalline states. The conformation of 4 was also studied by molecular‐mechanics calculations. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

12.
The X-ray diffraction and i.r. absorption conformational analysis of N-tert-butyloxycarbonyl-l-phenylalanine has showed the absence of intramolecularly hydrogen-bonded peptide conformations in the solid state. The molecules are held together in rows of ‘cyclic dimer’ motifs through intermolecular NHOC (acid) and OHOC {urethane} hydrogen bonds, the secondary amide-like group of the urethane moiety being in the unusual cis conformation, whereas the carboxylic acid group in the common syn conformation. The two molecules in the unit cell present a centrosymmetric set of ?, ψ1, and ψ2 values. In polar solvents solvated species largely predominate. In saturated hydrocarbon solution non-associated and associated (mostly involving the carboxylic acid CO as the proton acceptor) species simultaneously occur. The extent of association decreases with dilution. The amount of intramolecularly hydrogen-bonded oxy-C7 and C5 forms if any, should be extremely small. The type of association at saturation seems to differ from that found in the crystalline compound obtained by precipitation with saturated aliphatic hydrocarbons (from a diethyl ether solution).  相似文献   

13.
We have studied the receptor binding activities of C-terminal free and amidated enkephalins with and without the dehydrophenylalanine4 residue. For the selective labeling of so-called δ and μ opiate receptors, specific tracers were used at low concentrations in rat brain membranes and neuroblastoma cells containing pure δ receptors. C-Terminal free enkephalins are five to eight times more potent in the assay for δ receptors than in μ assay, while the amides are almost equipotent in both assays. The presence of a C-terminal carboxyl group is a determining factor for selective activity. [D-Ala2, ΔPhe4, Met5]-enkephalin amide is very potent in all of the binding assays examined, and, in particular, twice as active as the saturated amide and the C-terminal free enkephalin in the δ assay. We suggest that the steric arrangement of the dehydrophenylalanine residue in position 4 is very important to the enhanced interaction with the δ receptors.  相似文献   

14.
A pentapeptide, Z-Gly-Gly-Phe-Phe-Ala · OH (1b) and the corresponding unsaturated pentapeptide, Z-Gly-Gly-Phe-ΔZPhe-Ala · OH (1a), have been synthesized. The saturated compound (1b) was rapidly hydrolyzed by both chymotrypsin and thermolysin to the expected products, but the dehydropeptide was completely unhydrolyzed by either enzyme even after thirty hours. A new method of peptide stabilization to enzymolysis is made available.  相似文献   

15.
Recently, it was suggested that the measured rate of reduction of ferricyto chrome C by O?2 below pH 8, was too high in the presence of high concentrations of formate (Koppenol, W.H., Van Buuren, K.J.H., Butler J. and Braams, R. (1976) Biochim. Biophys. Acta 449, 157–168).The high values were attributed to the presence of impurities of copper, which compete for O?2. This assumption is consistent with either a decrease in the reduction yield of ferricytochrome C in the presence of copper, or with a very fast reaction of Cu(I) with ferricytochrome C.It was previously shown by us and by others that the reduction yield of ferricytochrome C by O?2 is 100%. We measured the rate of reduction of ferricytochrome C by Cu(I), and found that this reaction is slow: k = (1.5±0.5) · 103M?1) · s?1.Therefore, our results rule out the possibility that below pH 8 copper impurities affect the measured rate constant of the reduction of ferricytochrome C by O?2.  相似文献   

16.
Quercetin inhibited a dog kidney (Na+ + K+)-ATPase preparation without affecting Km for ATP or K0.5 for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the (Na+ + K+)-ATPase activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the Km for ATP and the K0.5 for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on Vmax, Km for substrate, and K0.5 for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations.  相似文献   

17.
Crystals of alkaline phosphatase (EC 3.1.3.1; Mr 94,000) grown at pH 9.5 from 2.25 m-(NH4)2SO4 with 5 × 10?5 m-Zn and 10?2 m-Mg present were analyzed by X-ray diffraction at pH 7.5 in 2.66 m-(NH4)2SO4 with 10?2 m-Zn and 10?2 m-Mg present. The crystals are orthorhombic with a = 195.5 A?, b = 168.3 A?and c = 76.33 A?, and the space group is I222. X-ray phases were determined by the multiple isomorphous replacement and anomalous dispersion method using K2PtCl4, KAu(CN)2 and K2OsO4 derivatives. The electron density maps and analysis of metal binding sites reveal one molecule per asymmetric unit with an internal, non-crystallographic, 2-fold rotation axis relating the subunits. Each subunit contains a major αβ domain with a seven-stranded β-sheet flanked by helices. The sheets are roughly coplanar but the general direction of the strands in each is at 20 ° to the rotation axis and thus 40 ° from each other. The helical content of the αβ domain is approximately 27% of the 459 residues in the monomer and the β content is approximately 7%. The chains in a smaller domain are more convoluted and less easily characterized than in the αβ domain. In both there is extensive monomer-monomer contact.Removal of the zinc and magnesium from the parent crystal produces a stable apoenzyme crystal and addition of cobalt at 10?2 m or cadmium at 10?2 or 5 × 10?2 m reveals seven metal binding sites per dimer. The active centers are 32 Å apart and each is shown by anomalous dispersion data to contain two metal binding sites, A and B. The cadmium derivative refinement determined the A-B separation to be 4.9 Å. Comparison of the parent and apo structures by means of difference maps reveals the double metal site with Zn at A and probably Mg at B. A prominent, partially resolved peak centered 7 Å away is interpreted as a stabilization of the backbone in this position by the metal ion co-ordination to a side-chain. Several negative peaks within 10 Å of the metals indicate local differences between apo and native structures but no significant differences are seen in the other parts of the molecule. At 5 × 10?2 m-Cd two metal sites (D and D′) are found 25.5 Å from the active center, on the surface of the minor domain. They are related to each other by the molecular 2-fold axis with a D-D′ distance of 25 Å. The seventh Cd site, E, is 20 Å from the active center, on the major domain, near a crystalline contact region, and devoid of any molecular symmetry mate.The apparent dissociation constants for cadmium at the A, B and D sites (and A′, B′, D′) are 3 × 10?3 m, 1.5 × 10?1 m and 1.3 × 10?2 m, respectively. Thus in these conditions cadmium is seen to distribute between A and B sites when the combined stoichiometry is two metal ions per dimer.  相似文献   

18.
tRNACUGLeu from E. Coli was purified by column chromatography on benzoylated DEAE-cellulose, followed by hydroxyapatite prepared by an improved method. Crystals obtained by vapour diffusion gave X-ray diffraction out to 7 Å in the hk0 projection and 10 Å in h0?. The space group was P42212 with a = b = 133 A?, c = 66 A? and 8 molecules in the unit cell. Birefringence showed preferred orientation of RNA helical regions in the ab plane.  相似文献   

19.
The peptide t-butyloxycarbonyl-α-aminoisobutyryl-L-prolyl-L-prolyl-N-methylamide has been shown to adopt an extended structure in the solid state. The Pro-Pro segment occurs in the poly-proline II conformation. On dissolution of single crystals at ~ 233°K, a single species corresponding to the all trans peptide backbone is observed by 270 MHz 1H NMR. On warming, trans to cis isomerization about the Pro-Pro bond is facilitated. Both cis' (ψ ~?50°) and trans' (ψ ~ 130°) rotamers about the Pro3 CαCO bond are detectable in the Pro-Pro cis conformer, at low temperature. These observations demonstrate unambiguously the large differences in the solid state and solution conformations of a Pro-Pro sequence.  相似文献   

20.
RNA (guanine-7) methyltransferase, partially purified from N.crassa mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both N.crassa poly A(+) RNA and reovirus unmethylated mRNA. RNase T2 digestion of the invitro methylated poly A(+) RNA from N.crassa yielded the “cap” structures m 7G(5′)pppAp and m 7G(5′)pppGp in a ratio of 2:1 respectively. RNase T2 digestion of the invitro methylated reovirus mRNA yielded m 7G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in N.crassa mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. 187, 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate.  相似文献   

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