共查询到20条相似文献,搜索用时 15 毫秒
1.
D Mohanty R Elber D Thirumalai D Beglov B Roux 《Canadian Metallurgical Quarterly》1997,272(3):423-442
The folding of Ser-Tyr-Pro-Phe-Asp-Val (SYPFDV), and sequence variants of this peptide (SYPYD and SYPFD) are studied computationally in an explicit water environment. An atomically detailed model of the peptide is embedded in a sphere of TIP3P water molecules and its optimal structure is computed by simulated annealing. At distances from the peptide that are beyond a few solvation shells, a continuum solvent model is employed. The simulations are performed using a mean field approach that enhances the efficiency of sampling peptide conformations. The computations predict a small number of conformations as plausible folded structures. All have a type VI turn conformation for the peptide backbone, similar to that found using NMR. However, some of the structures differ from the experimentally proposed ones in the packing of the proline ring with the aromatic residues. The second most populated structure has, in addition to a correctly folded backbone, the same hydrophobic packing as the conformation measured by NMR. Our simulations suggest a kinetic mechanism that consists of three separate stages. The time-scales associated with these stages are distinct and depend differently on temperature. Electrostatic interactions play an initial role in guiding the peptide chain to a roughly correct structure as measured by the end-to-end distance. At the same time or later the backbone torsions rearrange due to local tendency of the proline ring to form a turn: this step depends on solvation forces and is helped by loose hydrophobic interactions. In the final step, hydrophobic residues pack against each other. We also show the existence of an off the pathway intermediate, suggesting that even in the folding of a small peptide "misfolded" structures can form. The simulations clearly show that parallel folding paths are involved. Our findings suggest that the process of peptide folding shares many of the features expected for the significantly larger protein molecules. 相似文献
2.
The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a 15N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an alpha-helix content of approximately 42%. The alpha-helix comprises an uninterrupted hydrophobic stretch of < or = 12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15-75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled 15N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of 15N solid-state NMR spectroscopy (10(-4) seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed. 相似文献
3.
Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable. 相似文献
4.
AA Bergwerff W Stark G Fendrich R Knecht MJ Blommers W Maerki EA Kragten J van Oostrum 《Canadian Metallurgical Quarterly》1998,253(3):560-575
The production of recombinant leech-derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non-glycosylated and glycosylated rLTDI. Monosaccharide analysis and a-mannosidase treatment demonstrated that glycosylated rLDTI was exclusively alpha-mannosylated. A trypsin digest of reduced and S-carboxymethylated glycosylated rLDTI was separated on a reverse-phase HPLC column. Glycopeptides identified by a combination of matrix-assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno-oligosaccharides with a degree of polymerization ranging over 1-3 and 1-13 depending on the yeast strain. In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high-pH anion-exchange chromatography on a CarboPac MA1 column, and analyzed by one- and two-dimensional 1H-NMR spectroscopy. Besides the major oligosaccharide Man alpha1-2Man-ol, the (for yeast protein O-glycosylation) unusual Man alpha1-3Man alpha1-2Man-ol was determined. The solution conformation of glycosylated rLDTI was investigated by two-dimensional NMR spectroscopy. Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements. Analysis of the chemical shifts showed that amino acids Val32-Ser33, Ser36-Ser39 and Thr42 were affected by the O-mannosylation. In addition, changes in chemical shift were observed within the beta-hairpin peptide regions Val13-Ser16 and Gly18-Tyr21 attributed to direct interactions of the mannose residue at Ser36. Furthermore, the protein-linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop. 相似文献
5.
L Kirnarsky M Nomoto Y Ikematsu H Hassan EP Bennett RL Cerny H Clausen MA Hollingsworth S Sherman 《Canadian Metallurgical Quarterly》1998,37(37):12811-12817
The structures of three nine-residue peptide substrates that show differential kinetics of O-linked glycosylation catalyzed by distinct recombinant uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) were investigated by NMR spectroscopy. A combined use of NMR data, molecular modeling techniques, and kinetic data may explain some structural features required for O-glycosylation of these substrates by two GalNAc transferases, GalNAc-T1 and GalNAc-T3. In the proposed model, the formation of an extended backbone structure at the threonine residue to be glycosylated is likely to enhance the O-glycosylation process. The segment of extended structure includes the reactive residue in a beta-like or an inverse gamma-turn conformation and flanking residues in a beta-strand conformation. The hydroxyl group of the threonine to be glycosylated is exposed to solvent, and both the amide proton and carbonyl oxygen of the peptide backbone are exposed to solvent. The exchange rate of the amide proton for the reactive threonine correlated well with substrate efficiency, leading us to hypothesize that this proton may serve as a donor for hydrogen bonding with the active site of the enzyme. The oxygens of the residue to be glycosylated and several flanking residues may also be involved in a set of hydrogen bonds with the GalNAc-T1 and -T3 transferases. 相似文献
6.
E Barbar M Hare V Daragan G Barany C Woodward 《Canadian Metallurgical Quarterly》1998,37(21):7822-7833
A single-disulfide variant of bovine pancreatic trypsin inhibitor (BPTI), [14-38]Abu, is a partially folded ensemble which includes two, and in one case three, conformations that interconvert slowly enough to exhibit separate cross-peaks in the amide region of homonuclear and heteronuclear NMR spectra. Each conformation is itself composed of many subconformations in rapid equilibrium. Partially folded BPTI undergoes local motions that are slow, noncooperative, independent fluctuations of short segments within the chain. Cooperative global unfolding of the ensemble is also observed. Heteronuclear NMR has been used to measure interconversion rate constants of partially folded conformational substates; the rate constants differ for each residue and vary over an order of magnitude. For local fluctuation, the forward rate constants for amide protons of the antiparallel beta-sheet are significantly smaller than the rest of the molecule, consistent with other indications that this is the most stable part of the partially folded protein. The reverse rate constants also vary; they are the highest for Ala 27 in the turn between the strands in the sheet and for Phe 33 in the antiparallel beta-sheet. Global unfolding interconversion rate constants vary over a 3-fold range, consistent with previously observed deviations from two-state behavior. Fast backbone dynamics, from T1, T2, and NOE relaxation parameters, are obtained for the slowly interchanging conformations in the partially folded ensemble. Clear differences are observed between the two conformations; one is more flexible and less compact than the other. In the more flexible and disordered partially folded conformation, intermediate exchange is detected for some backbone amides, namely, those in the central beta-sheet and the turn. These same sheet and turn residues are more ordered in the globally denatured ensemble as well. Our results suggest that the turn initiates formation of a partially folded ensemble in which the slow-exchange core is the most stable region and in which segmental fluctuations reflect multiple nuclei for folding of the rest of the molecule. 相似文献
7.
FY Dupradeau G Le Flem JM Wieruszeski M Calin V Larreta-Garde JP Monti 《Canadian Metallurgical Quarterly》1999,1429(2):446-458
Glycerol, employed to mimic biological media with restricted water activity, has been shown to modify the activity of subtilisin BPN', an endopeptidase, towards the oxidized insulin B-chain, a well-studied substrate (FEBS Lett., 279 (1991) 123-131). Without minimizing the role of the microenvironment on the enzyme, we have studied the effect of glycerol addition on the structure of the enzyme substrate by homonuclear NMR spectroscopy and simulated annealing. Our results show that, in water, the oxidized insulin B-chain tertiary structure loses its central helix (residues B9-B19) and presents a folded structure with a flexible turn (residues B18-B24) in the beta-turn region of the insulin B-chain; whereas, in glycerol, the peptide is more rigid and is not folded. Moreover, in our experimental conditions, glycerol favors beta-strand secondary structure formation. Following these results, hypotheses about the differences observed in enzymatic activity on this substrate in glycerol have been postulated. 相似文献
8.
The behaviour of amino acid side-chains in proteins in solution has been characterised by analysing NMR 3JHalphaH beta coupling constants and crystallographic chi1 torsion angles. Side-chains both in the core of native folded proteins and in situations where there is an absence of close packing including the random coil state have been considered. An analysis of experimental 3JHalphaH beta coupling constant data for ten proteins shows that in the core of native proteins a very close similarity is observed between the chi1 conformations adopted in solution and in crystals. There is clear evidence, however, for significant motional averaging about the chi1 torsion angles in solution. Using a model of a Gaussian distribution about the average torsion angles the extent of these fluctuations has been quantified; the standard deviation for the motion is 26 degrees, the fluctuations about chi1 in the protein core being similar in size to those found for main-chain phi torsion angles in solution. From the distribution of chi1 torsion angles in a data base of protein crystal structures, torsion angle populations and coupling constants have been predicted for a random coil polypeptide. Significant variations in the chi1 distributions for different amino acids give differences in the predicted coupling constants; for 3JHalphaH beta, for example, values of 5.1 and 5.7 Hz are predicted for serine compared with 4.9 and 9.9 Hz for leucine. Experimental data for short unstructured peptides show an excellent agreement with the predictions, indicating that the overall chi1 distributions in protein crystals reflect the local preferences of the amino acids. Predictions from the protein data base therefore provide an important framework for interpreting experimental data for non-native protein conformations and for residues on the surface of folded proteins. 相似文献
9.
The helix-forming tendency of seven peptide fragments corresponding with the entire sequence of the FK506 binding protein (FKBP) has been investigated in aqueous buffer and in 2,2,2-trifluoroethanol (TFE) using CD and NMR spectroscopy. All fragments exhibited random coil conformations in aqueous buffer, whereas the amount of helix induced in the peptide fragments by TFE varied. The fragment with the highest degree of helicity in TFE corresponded with the single (alpha-helix in native FKBP. Fragments corresponding with beta-strands 2 and 3 also exhibited strong propensity towards helix formation. In contrast, the fragment corresponding with beta-strand 1 did not form helix in TFE. The inherent helix-forming tendencies are interpreted in light of the native structure to suggest possible folding nucleation sites. Conformational sampling in each peptide fragment was also compared with that observed in urea-denatured FKBP. With the exception of the fragment corresponding with beta-strand 2, the formation of helical structures in the peptide fragments in TFE was correlated with the observation of turn and/or helix conformers in urea-unfolded FKBP. Surprisingly, peptide fragments in aqueous solution were less structured than the corresponding regions in urea-denatured FKBP. The conformational differences between the peptide fragments and unfolded FKBP were not due to the urea buffer or to differences in their rotational correlation times. We conclude that local amino acid interactions are not generally sufficient to account for the formation of non-random conformations in unfolded FKBP. Formation of non-random structures in unfolded FKBP may require stabilization of incipient turn or helical conformations through transient contact with non-local non-polar residues. 相似文献
10.
SF Conrad IJ Byeon AM DiGeorge MD Lairmore MD Tsai PT Kaumaya 《Canadian Metallurgical Quarterly》1995,1(2):83-92
The identification and characterization of epitopes of human T-lymphotropic virus type 1 (HTLV-I), which elicit an effective humoral or cell-mediated immune response, remains a central obstacle to the development of a peptide-based vaccine against the virus infection. The objective of the studies presented here was to examine the influence of N-linked glycosylation on peptide structure and immunogenicity. We engineered the 233-253 sequence of gp46 of HTLV-I to contain an N-acetylglucosamine (GlcNAc) residue at Asn244. Secondary structure prediction using computer algorithms indicated that this peptide may contain a beta-turn at residues 242-246. Recent work with model glycopeptides suggests that beta-turn conformation in peptides may be induced, and probably is stabilized, by the presence of even a single sugar residue. In the present study, the structures of the 233-253 peptide, SC1, and the 233-253(Asn244-GlcNAc) glycopeptide, SC2, were determined. Similar conformation was exhibited by both the glycosylated and nonglycosylated peptide displaying a beta-turn at residues 243-246 and extended-chain structure at the peptide/glycopeptide termini. Both peptides were engineered into chimeric constructs with a promiscuous T-cell epitope from measles virus and were used as immunogens in rabbits. Both chimeric peptides were highly immunogenic in rabbits, producing high-titered antibodies as early as primary + three weeks. The antibodies generated against either construct were able to bind to whole virus (ELISA) and to gp46 (radioimmunoprecipitation assay). Additionally, human sera of individuals known to be positive for HTLV-I recognized both the glycosylated and nonglycosylated constructs. It appears that the 233-253 peptide is able to adopt a conformation that mimics the structure in native gp46, and addition of a GlcNAc residue at Asn244 does not affect the conformational preference or stability of this construct; nor does glycosylation alter immunogenicity but instead appears to enhance immune recognition. 相似文献
11.
Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding. 相似文献
12.
Tryptic digests of ovine submaxillary apomucin were fractionated by gel filtration and ion exchange chromatography to give 14 peptide fractions. Three purified tryptic peptides, representing 106 of the 650 residues in apomucin, were submitted to automated sequence analysis. The NH2-terminal 50 of the 74 residues in one peptide and the entire sequence of the other two hexadecapeptides were established. These studies suggest that purified ovine submaxillary, mucin is chemically homogeneous, containing a unique primary structure without substantial repeating sequences in its polypeptide chain. The sequences adjacent to 28 known O-glycosidically substituted seryl and threonyl residues were compared. No homologies were apparent around the glycosylated seryl and threonyl residues which might define the specificity of the UDP-N-acetylgalactosaminyl:mucin polypeptide transferase that incorporates N-acetylgalactosamine into O-glycosidic linkage in glycoproteins. However, there appears to be a minimum size requirement for glycosylation, because the transferase catalyzes glycosylation of tryptic peptides efficiently, while chymotryptic and thermolytic peptides were much poorer substrates for the transferase. 相似文献
13.
The conformational properties adopted by angiotensin II in a phospholipid micelle solution were studied by NMR spectroscopy and molecular modelling. The octapeptide was found to assume a well-defined hairpin structure with its C- and N-termini approaching to within 0.76 nm of each other. Three of the residues had fixed side chain configurations; Tyr4 (g+), His6 (g-) and Val3 (g-). Consequently, the His6 and Tyr4 aromatic rings were consistently close together. Conformers containing a cis His6-Pro7 peptide bond were observed for the peptide in a purely aqueous sample but completely disappeared when lipid vesicles were added to the sample. This result is explained by the existence of a very stable hydrogen bond between the Phe8 NH and the His6 carbonyl group of the lipid-solvated trans isomer, resulting in the formation of an inverse gamma turn centered on Pro7. 1H-NMR selective line broadening was apparent for several of the angiotensin II protons upon titration of an aqueous sample with less than stoichiometric amounts of 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer vesicles. The data obtained were consistent with the structure derived for micelle-bound angiotensin II, indicating that conformations adopted by the peptide hormone in the presence of micelles and lipid-bilayer vesicles are similar. 相似文献
14.
TE Creighton CJ Bagley L Cooper NJ Darby RB Freedman J Kemmink A Sheikh 《Canadian Metallurgical Quarterly》1993,232(4):1176-1196
15.
Euglena chloroplast protein precursors are transported as integral membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus prior to chloroplast localization. All Euglena chloroplast protein precursors have functionally similar bipartite presequences composed of an N-terminal signal peptide domain and a stromal targeting domain containing a hydrophobic region approximately 60 amino acids from the predicted signal peptidase cleavage site. Asparagine-linked glycosylation reporters and presequence deletion constructs of the precursor to the Euglena light-harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) were used to identify presequence regions translocated into the ER lumen and stop transfer membrane anchor domains. An asparagine-linked glycosylation site present at amino acid 148 of pLHCPII near the N terminus of mature LHCPII was not glycosylated in vitro by canine microsomes while an asparagine-linked glycosylation site inserted at amino acid 40 was. The asparagine at amino acid 148 was glycosylated upon deletion of amino acids 46-146, which contain the stromal targeting domain, indicating that the hydrophobic region within this domain functions as a stop transfer membrane anchor sequence. Protease protection assays indicated that for all constructs, mature LHCPII was not translocated across the microsomal membrane. Taken together with the structural similarity of all Euglena presequences, these results demonstrate that chloroplast precursors are anchored within ER and Golgi transport vesicles by the stromal targeting domain hydrophobic region oriented with the presequence N terminus formed by signal peptidase cleavage in the vesicle lumen and the mature protein in the cytoplasm. 相似文献
16.
R Guerois F Cordier-Ochsenbein F Baleux T Huynh-Dinh JM Neumann A Sanson 《Canadian Metallurgical Quarterly》1998,7(7):1506-1515
The conformational properties of an 18 residues peptide spanning the entire sequence, L1KTPA5QFDAD10ELRAA15MKG, of the first helix (A-helix) of domain 2 of annexin I, were thoroughly investigated. This fragment exhibits several singular features, and in particular, two successive potential capping boxes, T3xxQ6 and D8xxE11. The former corresponds to the native hydrogen bond network stabilizing the alpha helix N-terminus in the protein; the latter is a non-native capping box able to break the helix at residue D8, and is observed in the domain 2 partially folded state. Using 2D-NMR techniques, we showed that two main populations of conformers coexist in aqueous solution. The first corresponds to a single helix extending from T3 to K17. The second corresponds to a broken helix at residue Ds. Four mutants, T3A, F7A, D8A, and E11A, were designed to further analyze the role of key amino acids in the equilibrium between the two ensembles of conformers. The sensitivity of NMR parameters to account for the variations in the populations of conformers was evaluated for each peptide. Our data show the delta13Calpha chemical shift to be the most relevant parameter. We used it to estimate the population ratio in the various peptides between the two main ensembles of conformers, the full helix and the broken helix. For the WT, E11A, and F7A peptides, these ratios are respectively 35/65, 60/40, 60/40. Our results were compared to the data obtained from helix/coil transition algorithms. 相似文献
17.
Three short protein sequences have been guided by computer to folds resembling their crystal structures. Initially, peptide fragment conformations ranging in size from 9 to 25 residues were selected from a database of known protein structures. A fragment was selected if it was compatible with a segment of the sequence to be folded, as judged by three-dimensional profile scores. By linking the selected fragment conformations together, hundreds of trial structures were generated of the same length and sequence as the protein to be folded. These starting trial structures were then improved by an evolutionary algorithm. Selection pressure for improving the structures was provided by an energy function that was designed to guide the conformational search procedure toward the correct structure. We find that by evolution of only 400 structures for fewer than 1400 generations, the overall fold of some small helical proteins can be computed from the sequence, with deviations from observed structures of 2.5-4.0 A for C alpha atoms. 相似文献
18.
E Mahé P Vossen HW Van den Hooven D Le-Nguyen J Vervoort PJ De Wit 《Canadian Metallurgical Quarterly》1998,52(6):482-494
The race-specific peptide elicitor AVR9 of the fungal pathogen Cladosporium fulvum specifically induces a hypersensitive response in tomato genotypes carrying the complementary resistance gene Cf-9. The total chemical syntheses of this 28-residue AVR9 peptide containing three disulfide bonds, and of three mutant peptides [R8K]AVR9, [F10A]AVR9 and [F21A]AVR9, have been accomplished. The syntheses were carried out using a stepwise solid-phase approach based on tBoc chemistry. The disulfide bridges were formed by air oxidation. The correctness of the chemical structure of all folded synthetic peptides was confirmed by combined NMR and MS analyses. The biological activity and a number of physicochemical properties of folded synthetic AVR9 are identical to those of native fungal 28-residue AVR9. The overall conformations of the folded synthetic mutant peptides were comparable to that of synthetic wild-type AVR9 as demonstrated by NMR spectroscopy. Mutant [R8K]AVR9 showed a threefold higher, and mutant [F10A]AVR9 a threefold lower necrosis-inducing activity when compared to synthetic wild-type AVR9. However, mutant [F21A]AVR9 showed hardly any necrosis-inducing activity. Affinity for polyclonal antibodies raised against native fungal AVR9 is positively correlated with the necrosis-inducing activity of the synthetic AVR9 peptides ([R8K]AVR9 > wild-type AVR9 > [F10A]AVR9 > [F21A]AVR9). 相似文献
19.
BD Scott MK Sharma JM Levett CC Marinelli RA Kieso PG Schmid RE Kerber 《Canadian Metallurgical Quarterly》1993,125(4):1047-1053
Olfactomedin is the major glycoprotein of the extracellular mucous matrix of frog olfactory neuroepithelium. It is responsible for the primary architecture of this extracellular matrix by forming via intermolecular disulfide bonds polymers, which are covered with evenly spaced carbohydrate groups. To study glycosylation of olfactomedin, we raised antibodies against the mature protein and antibodies against a region adjacent to an N-linked glycosylation site near its amino terminus. The latter antibodies cannot bind when this site is glycosylated and reveal precursors of olfactomedin in the perinuclear regions of Bowman's glands. In contrast, antiserum against the mature protein stains acinar regions of glands and the ciliary surface. Enzymatic deglycosylation of olfactomedin shows stepwise removal of carbohydrate and reveals a 51-kDa deglycosylated form. Our results indicate that, prior to secretion, most, if not all, of the six potential N-linked glycosylation sites of olfactomedin are glycosylated with carbohydrate moieties of about 8-10 sugar residues. 相似文献