共查询到20条相似文献,搜索用时 31 毫秒
1.
Donald M. Gray A. Dean Sherry Jane Teherani Janice W. Kansy 《Journal of biomolecular structure & dynamics》2013,31(1):77-91
Abstract The ∈-amino groups of the six lysyl residues of the fd gene 5 DNA-binding protein have been modified by reductive methylation to form N∈, N∈-dimethyl lysyl derivatives containing 13C-labeled methyl groups. The α-amino terminus of the protein was not accessible to methylation. Circular dichroism studies show that the modified protein binds to fd DNA, but with a slightly reduced affinity compared with that of unmodified gene 5 protein. We also find that both the modified and unmodified proteins bind to an oligodeoxynucleotide, d(A)7, but in neither case does binding cause a decrease in the 228 nm CD band of the protein as occurs when the protein binds to long DNA polymers. 13C NMR spectra at 50.1 MHz of [13C]methylated gene 5 protein show five distinct resonances between 43.30 and 42.76 ppm originating from the six N∈, N∈-dimethyl lysyl residues. We attribute one of the resonances to two solvated lysyl residues and the other four to individual lysyl residues in different microenvironments. All four of these latter resonances are affected by the binding of d(A)7. However, since two of these resonances are similarly affected by the presence of salt in the absence of DNA, only two are uniquely affected by DNA binding. 相似文献
2.
Long-Sen Chang 《The protein journal》1996,15(5):473-480
Notexin fromNotechis scutatus scutatus snake venom was subjected to Lys modification with pyridoxal 5′-phosphate (PLP), and one major modified derivative was purified on a cation-exchanger SP-8HR column. The results of amino acid analysis and sequence determination revealed that only 2 Lys residues at positions 82 and 115 out of 11 Lys residues in notexin were modified. The incorporation of PLP into the protein was accompanied by the loss of 53% lethal toxicity, but the modified notexin showed an about 1.2-fold increase in enzymatic activity. However, the secondary structure of the toxin molecule did not significantly change after modification with PLP as revealed by the CD spectra, and the antigenicity of PLP derivative remained unchanged. The modified derivative retained its affinity for Ca2+, indicating that the modified Lys residues did not participate in Ca2+ binding. These results indicate that modification of Lys residues causes a differential effect on the enzymatic activity and lethal toxicity of notexin, and suggest that notexin might possess two functional sites, one responsible for the catalytic activity and the other associated with its lethal effect. 相似文献
3.
Long-Sen Chang 《Journal of Protein Chemistry》1996,15(5):473-480
Notexin fromNotechis scutatus scutatus snake venom was subjected to Lys modification with pyridoxal 5-phosphate (PLP), and one major modified derivative was purified on a cation-exchanger SP-8HR column. The results of amino acid analysis and sequence determination revealed that only 2 Lys residues at positions 82 and 115 out of 11 Lys residues in notexin were modified. The incorporation of PLP into the protein was accompanied by the loss of 53% lethal toxicity, but the modified notexin showed an about 1.2-fold increase in enzymatic activity. However, the secondary structure of the toxin molecule did not significantly change after modification with PLP as revealed by the CD spectra, and the antigenicity of PLP derivative remained unchanged. The modified derivative retained its affinity for Ca2+, indicating that the modified Lys residues did not participate in Ca2+ binding. These results indicate that modification of Lys residues causes a differential effect on the enzymatic activity and lethal toxicity of notexin, and suggest that notexin might possess two functional sites, one responsible for the catalytic activity and the other associated with its lethal effect. 相似文献
4.
Z. He N. Petesch K.-P. Voges W. Röben K.D. Philipson 《The Journal of membrane biology》1997,156(2):149-156
The Na+-Ca2+ exchanger plays an important role in cardiac contractility by moving Ca2+ across the plasma membrane during excitation-contraction coupling. A 20 amino acid peptide, XIP, synthesized to mimic a region
of the exchanger, inhibits exchange activity. We identify here amino acid residues important for inhibitory function. Effects
of modified peptides on Na+-Ca2+ exchange activity were determined. Exchange activity was assessed as 45Ca2+ uptake into Na+-loaded cardiac sarcolemmal vesicles. We find that the entire length of XIP is important for maximal potency, though the major
inhibitory components are between residues 5 and 16. Basic and aromatic residues are most important for the inhibitory function
of XIP. Substitutions of arginine 12 and arginine 14 with alanine or glutamine dramatically decrease the potency of XIP, suggesting
that these residues play a key role in possible charge-charge interactions. Substitutions of other basic residues with alanines
or glutamines had less effect on the potency of XIP. All aromatic residues participate in binding with the exchanger, probably
via hydrophobic interactions as indicated by tryptophan fluorescence. A tyrosine is required at position 6 for maximal inhibition
and phenylalanine 5 and tyrosine 8 can only be replaced by other aromatic residues. Tyrosine 10 and tyrosine 13 can be replaced
with other bulky residues. A specific conformation of XIP, with structural constrains provided by all parts of the molecule,
is required for optimal inhibitory function.
Received: 19 September 1996/Revised: 20 November 1996 相似文献
5.
We have determined the number of histidine residues available for chemical modification with the specific reagent diethylpyrocarbonate in both bovine and goat -crystallins. Results indicate that there are two distinctly different classes of histidine residues in the native protein. Out of 300 total histidine residues in the protein (on the basis of 800-kDa protein molecular weight) about 170±2 residues have been found to be modified by the reagent. The remaining 130±2 residues are modified when the protein is partially denatured in 1.5 M guanidine hydrochloride. The H+-titration behavior of the histidine residues in the protein corroborates this result. The observed differential accessibility of histidine residues may be important in maintaining the surface hydrophobicity of the aggregate as well as in stabilizing its quaternary structure. 相似文献
6.
Selective modification of cytidine, uridine, guanosine and pseudouridine residues in Escherichia coli leucine transfer ribonucleic acid 总被引:4,自引:0,他引:4
The specificity of methoxyamine for the cytidine residues in an Escherichia coli leuoine transfer RNA (tRNA1leu is described in detail. Of the six non-hydrogen-bonded cytidine residues in the clover-leaf model of this tRNA, four are very reactive (C-35, 53, 85 and 86) and two are unreactive (C-67 and 79).The specificity of l-cyclohexyl-3-[2-morpholino-(4)-ethyl]carbodiimide methotosylate for the uridine, guanosine and pseudouridine residues in the leucine tRNA was also investigated. The carbodiimide completely modified four uridine residues (U-33, 34, 50 and 51) and partially modified G-37 and Ψ-39. For technical reasons, the sites of partial modification in loop I of the tRNA were difficult to establish. There was no modification of base residues in loop IV nor of U-59 at the base of stem e of the tRNA.The modification patterns described for the leucine tRNA are compared with those observed for the E. coli initiator tRNA1met and su+III tyrosine tRNA. Several general conclusions regarding tRNA conformation are made. In particular, the evidence supporting a diversity of anticodon loop structures amongst tRNAs is discussed. 相似文献
7.
High-resolution 1H-nmr analyses are presented for partially modified retro-inverso derivatives of peptides with emphasis on enkephalins. Studies in DMSO-d6 reveal the unique as well as common characteristics of the non-amino acid residues incorporated into these modified peptides. The complete assignment of 1,1-diaminoalkyl and malonyl, as well as of amino acid, residues provides the basis for the exploration of the conformational features induced in peptides by such topochemical modifications. 相似文献
8.
G. Polidoro D. Di Cola C. Di Ilio L. Politi R. Scandurra 《Molecular and cellular biochemistry》1976,11(3):155-159
Summary Holo and apoenzyme of aspartate aminotransferase from beef kidney are 80% inactivated by photoxidation in the presence of 2 × 10–6
m tetraiodofluroescein with the modification of two histidine residues per enzyme protomer. At a higher concentration (1 × 10–5
m) a tyrosine residue is also modified. The keto substrates, ketoglutarate and oxalacetate, protect the enzyme from photoxidation.Diethylpyrocarbonate modifies three histidine residues per enzyme protomer and reduces the activity only 10%. These results suggest that the two histidine residues photoxidized through the sensitizer, are located in the active site of the enzyme, at least one of these appears to be involved in ketosubstrate binding. The other three histidines modified by diethylpyrocarbonate are likely located on the enzyme surface and are not involved in the catalytic activity of the enzyme.This work is part of a program supported by a grant from the Consiglio Nazionale delle Ricerche. 相似文献
9.
Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue 总被引:3,自引:3,他引:0
Choong Yee Soon Maxwell G. Shepherd Patrick A. Sullivan 《The Biochemical journal》1977,165(2):385-393
1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [14C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the 14C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished. 相似文献
10.
Oligonucleotides containing a guanosine residue on the 5′ or the 3′ side of tri- and tetranucleotides were prepared. The guanosine residue was modified with the chemical carcinogen N-2-acetylaminofluorene and the control and modified oligonucleotides were tested for their ability to stimulate 14C-labeled amino-acyl-tRNA binding to ribosomes. The effects of the modification are twofold. The first is that if the guanosine residue to which the drug is eovalently bound is part of a codon the oligonucleotide is completely inactive in the ribosomal binding assay. The second is that if an adenosine residue is adjacent to either the 5′ or 3′ side of the modified guanosine, as in (Ap)3G or G(pA)3, there is partial inhibition of 14C-labeled lysyl-tRNA binding to ribosomes. This inhibitory effect extends only to the function of the immediately adjacent adenosine since the chemical modification of guanosine residues in (Ap)4G or G(pA)4 did not impair their ability to code for lysine. In contrast to these findings if there is a uridine residue adjacent to the modified guanosine, as in (Up)3G or G(pU)3 there is no effect on 14C-labeled phenylalanyl-tRNA binding to ribosomes. Proton magnetic resonance spectra of UpG, GpU and the corresponding dinners in which the guanosine residue was modified with the drug failed to indicate a stacking interaction between the fluorene moiety and the adjacent uridine residue. This is in contrast to previous studies demonstrating a strong stacking interaction between fluorene and adjacent adenosine residues. Taken together these results indicate that acetylaminofluorene modification of guanosine next to an adenosine residue in oligonucleotide inhibits its ribosomal binding capacity. The stacking interaction with adjacent adenosine, and not with adjacent uridine residues, in oligonucleotides probably accounts for the effects observed in the ribosomal binding assay. These data are consistent with our previously described “base displacement” model. 相似文献
11.
Matthew J. McKay Ashley N. Martfeld Anna A. De Angelis Stanley J. Opella Denise V. Greathouse Roger E. Koeppe 《Biophysical journal》2018,114(11):2617-2629
Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW5,19ALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific 2H- and 15N-labeled residues. GW5,19ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as 2H quadrupolar or 15N-1H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW5,19ALP23, the modified GW4,20ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the 2H and 15N NMR signals confirm that the extent of dynamic averaging, particularly rotational “slippage” about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional 2H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment. 相似文献
12.
??-Crystallin, a member of the small heat shock protein family is the major protein of mammalian eye lens and is a molecular chaperone. As there is no protein turn over in the lens, stability of ??-crystallin is one of the most crucial factors for its survival and function. We previously reported that the molecular chaperone-like activity and stability of ??-crystallin dramatically increased in the presence of Zn2+ (Biochemistry, 2008). We also reported that each subunit of ??-crystallin could bind multiple zinc ions through inter-subunit bridging giving rise to enhanced stability (Biopolymers, 2011). The amino acid residues involved in zinc binding were not known. Since cysteine residues were not responsible for binding to Zn2+, we tried to identify the histidine residues bound to zinc ions. We modified recombinant ??A- and ??B-crystallin with diethylpyrocarbonate (DEPC) a histidine modifying reagent, in presence and absence of Zn2+ followed by tryptic digestion. The residues modified by DEPC were identified through peptide mass matching by MALDI mass spectrometry. We have clearly identified H79, H107 and H115 of ??A-crystallin and H104, H111 and H119 of ??B-crystallin as the Zn2+ binding residues. The significance of the histidine rich sequence region of ??-crystallin for its stability is discussed. 相似文献
13.
Mhamed Sa?d El Kebbaj Norbert Latruffe Yves Gaudemer 《Biochemical and biophysical research communications》1982,108(1):42-50
In order to titrate and understand the role of arginyl residues of D-β-hydroxybutyrate dehydrogenase, arginyl specific reagents: butanedione, 1,2-cyclohexanedione and phenylglyoxal were incubated with three different forms of the enzyme; native enzyme (inner mitochondrial membrane bound), purified apoenzyme (phospholipid -free) and phospholipid-enzyme complex (reconstituted active form).After complete inactivation of the enzyme by [14C]-phenylglyoxal, the number of modified arginyl residues was different: one with the lipid-free apoenzyme and three with the phospholipid-enzyme complex, suggesting a conformational change of the enzyme triggered by the presence of phospholipids.After exhaustive chemical modification either of the apoenzyme or of the phospholipid-enzyme complex with [14C]-phenylglyoxal, four arginyl residues were titrated indicating that these residues are located in the hydrophilic part of the enzyme, not interacting with phospholipids.Reconstituted enzyme inactivated by butanedione could no longer bind a pseudosubstrate (succinate) which indicates that an arginyl residue is involved in the enzyme-substrate complex formation.The values of second order rate constants of D-β-hydroxybutyrate dehydrogenase inactivation by butanedione and 1,2-cyclohexanedione were unchanged with the three enzyme forms, suggesting that phospholipids are not involved in the substrate binding mechanism. 相似文献
14.
15.
Carlos Alemn 《Biopolymers》1994,34(7):841-847
A quantum mechanical study to compare the ability of α-aminoisobutyric acid (Aib), de-hydroalanine (ΔAla), and alanine (Ala) residues to stabilize helical conformations has been performed. To address the study, the oligopeptides Xn (X = Aib, ΔAla and Ala), where n varies from 1 to 6, were computed with the AM1 semiempirical method. The results show that the residues modified at the Cα carbon atom, Aib and ΔAla, are better helical formers than Ala. Thus, a cooperative energy effect was found for both residues, and especially for ΔAla. These terms permit the understanding the different conformational behaviors between Ala and its Cα-modified residues Aib and ΔAla. This trend is important for de novo protein design, where Aib and ΔAla must be considered useful residues in the design of synthetic helical motifs. © 1994 John Wiley & Sons, Inc. 相似文献
16.
《Bioscience, biotechnology, and biochemistry》2013,77(4):716-721
Ricin A-chain, a protein that inactivates ribosomes by a specific RNA N-glycosidase activity, has been shown to be inactivated by chemical modification of a few arginine residues. When two or fewer arginine residues in the A-chain were modified with [14C]phenylglyoxal, arginines at positions of 193, 196, 213, and 234/235 were found to be modified, from amino acid compositions and radioactivities of the modified peptides that were obtained by cyanogen bromide cleavage followed by tryptic and chymotryptic digestion. All these arginines have side chains outside the active site cleft; the side chain of Arg213 is adjacent to the edge of the cleft, while other modified arginines are located on the opposite side of the cleft. Kinetic analysis showed that the modification of two arginine residues caused a 8-fold loss in kcat with a 3-fold increase in Km, suggesting that this modification mainly decrease the rate of depurination with an additional effect on the affinity for ribosomes. Neither the environment of tryptophan 211 at the bottom of the cleft nor an interaction of adenine with the cleft was changed by this modification, as judged by fluorescence spectroscopy, suggesting that a conformational change of the catalytic site does not occur upon the modification. These results, taken together with other works, suggest that some of the above arginine residues outside the active site cleft may additively contribute to the catalysis of depurination and/or the initial formation of the A-chain/ribosome complex. 相似文献
17.
One of the histidine residues in swine pepsinogen can be modified in a rapid reaction with diethyl pyrocarbonate (DEP). At the same time, the potential proteolytic activity of the zymogen is reduced to 40% of its initial value. Reaction at the histine residue is reversed by neutral hydroxylamine, but the loss in activity is not. Fractionation of DEP-pepsinogen activation mixtures gave fully active pepsin in reduced yield, not modified enzyme with reduced specific activity. This was taken to indicate that the reaction had produced a mixture of products. Irreversible incorporation of [14C]DEP indicates that carbethoxy groups are incorporated into the molecule at amino acids other than histidine. The positions of these carbethoxy groups were determined by tryptic digestion and hplc. Modification was found to have occurred, in nonstoichiometric amounts, at lysine residues 3 and 9 and at leucine 1. Control experiments showed that activation was not affected by reaction at leucine 1, indicating that inactivation is caused by reaction at one or more of the lysine residues. 相似文献
18.
The Lys residues in the 75-residue Ca2+-binding protein calbindin D9k were reductively methylated with13C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca2+- and apo-forms of the protein, the13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca2+-calbindin D9k and apo-calbindin D9k were obtained by combiningpH titration experiments and (1H,13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca2+- and apo-forms of calbindin D9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D9k has a very similar structure compared to Ca2+-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of thepKa values of the Lys residues. 相似文献
19.
《Archives of biochemistry and biophysics》1985,242(1):231-239
Modified α-d-(1 → 4)-glucans containing a small proportion of 14C-labeled 2-deoxy-d-glucose or 2-amino-2-deoxy-d-glucose were examined as substrates for porcine pancreatic α-amylase (PPA). Cyclomaltoheptaose containing single 2-deoxy-d-glucose residues, synthesized by incubation of 2-deoxyglucosylglycogen with cyclomaltodextrin glucanotransferase in the presence of Triton X-100, was hydrolyzed by PPA to produce 2-deoxy-d-glucose; two isomers of 2-deoxymaltose, and a mixture of modified maltotrioses. These results indicate that 2-deoxy-d-glucose may be productively bound at all five subsites of the PPA active site. Reaction kinetics and the distribution of products formed suggest, however, that productive binding of the modified residue does not occur readily at the point of catalytic attack (subsite 3) and that the preferred position of hydrolysis of modified substrates may be different from that of unmodified substrates. Results of PPA hydrolysis of glycogen containing [14C]-2-amino-2-deoxy-d-glucose showed that a modified trisaccharide and a modified disaccharide were the smallest substituted products formed. Analysis of these products indicated that they did not contain modified residues at their reducing ends. Formation of the observed 2-amino-2-deoxy-maltooligosaccharides is consistent with a scheme where productive binding of 2-amino-2-deoxy-d-glucose is allowed at subsites 1, 2, 4, and 5, but not at subsite 3, the subsite at which hydrolysis occurs. 相似文献
20.
The Lys residues in the 75-residue Ca2+-binding protein calbindin D9k were reductively methylated with13C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca2+- and apo-forms of the protein, the13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca2+-calbindin D9k and apo-calbindin D9k were obtained by combiningpH titration experiments and (1H,13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca2+- and apo-forms of calbindin D9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D9k has a very similar structure compared to Ca2+-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of thepKa values of the Lys residues. 相似文献