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1.
Anhydrotrypsin in an immobilized form shows strong specific affinity for the peptides containing Arg, Lys, or S-aminoethyl-Cys at their C-termini. By taking advantage of this unique property of the adsorbent, we established an efficient chromatographic procedure useful for selective isolation of C-terminal peptides from protease digests of proteins. The utility was demonstrated in cases of a tryptic digest of Streptomyces subtilisin inhibitor in a reduced and S-carboxymethylated form (RCM-SSI) (with C-terminal Phe) and of a chymotryptic digest of α1-antitrypsin (with C-terminal Lys): the C-terminal peptides were recovered as the breakthrough fraction of the chromatography in the former case and as the adsorptive fraction in the latter. Immobilized anhydrochymotrypsin was also useful for selective adsorption of the C-terminal peptide from the tryptic digest of RCM-SSI. It was further found that immobilized anhydrotrypsin exerts strong affinity even for human oxyhemoglobin and its α-chain (with C-terminal Arg), but not for the β-chain (with C-terminal His) and hemoglobin-haptoglobin complex. Thus the adsorbent may be applicable also to the isolation of macromolecular ligands and to the analysis of macromolecular interactions.  相似文献   

2.
We present here an approach to C-terminal sequencing of proteins by the procedure consisting of the following: (1) derivatization of the C-terminal α-carboxyl group with 3-aminopropyltris(2,4,6-trimethoxyphenyl)-phosphonium bromide (TMPP-propylamine) through oxazolone chemistry, (2) enzymatic proteolysis of the TMPP-derivatized protein, and (3) MALDI-MS/MS analysis of the peptide mixture, in which the C-terminal peptide incorporating the TMPP group is preferably detected. In this protocol, it is possible to choose any endoproteinase such as trypsin, GluC, and AspN for digestion so that a C-terminal peptide with length appropriate for mass spectrometric sequencing could be generated. The peptide labeled with TMPP-propylamine at the C terminus tends to exhibit y-type ions in MS/MS spectra, allowing manual sequence interpretation with the simplified fragmentation pattern. The efficacy of the method was verified with five proteins, which demonstrated that the C-terminal peptides were readily distinguishable by their peak intensity and characteristic mass signature peak in MALDI-PSD analysis.  相似文献   

3.
1-Aminocyclopropane-1-carboxylic oxidase (ACCO) is a non-heme iron(II)-containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C-terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C-terminal part is reported. Site-directed spin-labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy was used. Mutagenesis experiments were performed to generate active enzymes bearing two paramagnetic labels (nitroxide radicals) anchored on cysteine residues, one in the main core and one in the C-terminal part. Inter-spin distance distributions were measured by pulsed EPR spectroscopy and compared with the results of molecular dynamics simulations. The results reveal the existence of a flexibility of the C-terminal part. This flexibility generates several conformations of the C-terminal part of ACCO that correspond neither to the existing crystal structures nor to the modelled structures. This highly dynamic region of ACCO raises questions on its exact function during enzymatic activity.  相似文献   

4.
Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H]- and [M - 2H]2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode.  相似文献   

5.
Lin  Min  Li  Xinxin  Zhang  Hongfeng  Ji  Wenquan  Shi  Yuhan  Li  Qinran  Zhang  Chenxi  Deng  Qiliang 《Chromatographia》2022,85(8):743-754
Chromatographia - The C-terminal epitope imprinted polymers on the silica were prepared by reversible addition-fragmentation chain transfer (RAFT) strategy with C-terminal nonapeptide of cytochrome...  相似文献   

6.
In order to investigate the effect of his-tag on glutamine synthetase (GS, EC 6.3.1.2) from Corynebacterium glutamicum, recombinant Escherichia coli strains overexpressing GSIM, HGSIM (GS fused with N-terminal his-tag), GSIMH (GS fused with C-terminal his-tag), and HGSIMH (GS fused with N-terminal & C-terminal his-tags) were constructed, respectively. Under similar expression conditions, GSIM and HGSIM were partially solubly expressed; no soluble GSIMH and HGSIMH were observed, based on the result of SDS-PAGE. Gel filtration of purified soluble HGSIM showed that hexamers and dedocamers coexisted in the quaternary structure of GS from C. glutamicum. Combined this result with the analysis of two GS crystal structure models, we hypothesized that C-terminal residues participated in GS folding after translation on ribosome. After the folding process, C-terminal residues were released again and exposed to solvent. Fused C-terminal his-tag interrupted the GS to fold into its correct conformation so that inclusion bodies formed.  相似文献   

7.
A simple and effective strategy for preparing fluorophore-labelled PNA is described. A C-terminal S-t-butylmercaptocysteine-derivatized PNA was prepared on solid-phase using Fmoc chemistry. Selective deprotection of the S-t-butylmercapto group on-bead, allowed the free thiol to be reacted with a fluorophore derivatized via an iodoacetamido or maleimido linker. Subsequent cleavage and sidechain deprotection yielded C-terminal labelled PNA in good yield and purity. Dual labelled PNA was also prepared by using both C-terminal (-SH) and N-terminal (-NH(2)) labelling chemistries.  相似文献   

8.
The dissociation of deprotonated peptides containing hydroxyl side chains was studied by electrospray ionization coupled with Fourier transform ion cyclotron resonance (ESI-FTICR) via sustained off-resonance irradiation collision induced dissociation (SORI-CID). Dissociation under post-source decay (PSD) conditions was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). This work included hexapeptides with one residue of serine, threonine, or tyrosine and five inert alanine residues. During SORI-CID and PSD, dissociation of [M-H](-) yielded c- and y-ions. Side-chain losses of formaldehyde (HCHO) from serine-containing peptides, acetaldehyde (CH(3)CHO) from threonine-containing peptides, and 4-methylene-2,5-cycohexadienone (C(7)H(6)O) from tyrosine-containing peptides were generally observed in the negative ion PSD and SORI-CID spectra. Side-chain loss occurs much less from tyrosine-containing peptides than from serine- and threonine-containing peptides. This is probably due to the bulky side chain of tyrosine, resulting in steric hindrance and poor geometry for dissociation reactions. Additionally, a selective cleavage leading to the elimination of the C-terminal residue from [M-H](-) was observed from the peptides with serine and threonine at the C-terminus. This cleavage does not occur in the dissociation of peptides with an amide group at the C-terminus or peptides with neutral or basic residues at the C-terminus. It also does not occur with tyrosine at the C-terminus. Both the C-terminal carboxylic acid group and the hydroxyl side chain of the C-terminal residue must play important roles in the mechanism of C-terminal residue loss. A mechanism involving both the C-terminal carboxylic acid group and a hydroxyl side chain of serine and threonine is proposed.  相似文献   

9.
In this study, we investigated the functional role of arginines in the C-terminal (65?C67) of BmK AGP-SYPU1, an analgesic peptide from the Chinese scorpion Buthus martensii Karsch. Using site-directed mutagenesis, arginines at the C-terminal (65?C66) were deleted or added to the C-terminal (67). The genes for three mutants of BmK AGP-SYPU1 were obtained by PCR. An analgesic activity assay was used to evaluate the role of arginine residues in the analgesic activity. The three-dimensional structure of BmK AGP-SYPU1 was established by homology modeling. As a result, we showed that the arginines in the C-terminal are crucial for the analgesic activity and may be located at analgesic functional sites. Our work has implications for further modification of scorpion toxins to obtain new analgesic peptides with enhanced activity.  相似文献   

10.
The computationally engineered peptide amidase exhibits great promising potential in the C-terminal modification of peptides using prop-2-yn-1-amine (PYA) or prop-2-en-1-amine (PEA) as the nucleophile. Subsequently, modified peptides could be further functionalized via click reaction without elaborate isolation of the intermediate.  相似文献   

11.
The product ion spectra of approximately 200 dipeptides were acquired under low-energy conditions using a triple quadrupole mass spectrometer. The spectra of dipeptides containing an N-terminal arginine (R), histidine (H), or lysine (K) were observed to yield a b(1) + H(2)O ion corresponding to the protonated basic amino acid. This was equivalent to the y(1)-ion in the corresponding C-terminal isomer. The formation of a b(1) + H(2)O ion was not a significant fragmentation channel in any dipeptides analyzed including those containing a C-terminal basic amino acid unless they also contained an N-terminal basic amino acid. Occurring simultaneously and under equal energy conditions an apparent b(1)-ion was formed, which has its corresponding C-terminal equivalent in the y(1)-H(2)O ion. Energy resolved mass spectrometry (ERMS), deuterium labeling, and accurate mass experiments as well as data reported were used to show the relationships between the b(1)+H(2)O and b(1)-ions in the dipeptides containing an N-terminal basic amino acid and the y(1) and y(1)-H(2)O ions in the corresponding C-terminal isomers.  相似文献   

12.
The dipeptidyl urea 1 composed of two dipeptide chains bearing the C-terminal pyridyl moiety (-L-Ala-L-Pro-NHPy) was prepared. Two molecules of 1 are revealed to be held together by six intermolecular hydrogen bonds to form a hydrogen-bonded duplex by the single-crystal X-ray structure determination. Proton magnetic resonance nuclear Overhauser effect (NOE) study indicates the hydrogen-bonded duplex even in solution. Furthermore, a shuttle-like molecular dynamics based on recombination of the hydrogen bonds was observed. The dipeptidyl urea composed of two dipeptide chains bearing the C-terminal pyrenyl moiety (-L-Ala-L-Pro-NHCH(2)Pyr) exhibited both monomer and eximer emissions in the fluorescence spectra, supporting the formation of a duplex. A combination of the C-terminal amide NH function in each side and the designed sequence of hydrogen-bonding sites are considered to be a crucial factor for the duplex formation.  相似文献   

13.
The growth of amyloid fibrils is studied by replica exchange molecular dynamics in an implicit solvent. Our data indicate that extremely long simulation times (at least a few hundred ns) are necessary to study the thermodynamics of fibril elongation in detail. However some aspects of the aggregation process are already accessible on the time scales available in the present study. A peak in the specific heat indicates a docking temperature of T(dock) ≈ 320 K. Irreversible locking requires lower temperatures with the locking temperature estimated as T(lock) ≈ 280 K. In our simulation the fibril grows from both sides with the C-terminal of the incoming monomer attaching to the C-terminal of the peptides in the fibril forming a β-sheet on the fibril edge. Our simulation indicates that the C-terminal is crucial for aggregation.  相似文献   

14.
The detailed primary sequences of the N-terminal moiety (72 amino acid residues) and of the C-terminal end (23 amino acid residues) of human milk lysozyme (129 residues) are reported. A tentative complete structure of the enzyme is compared to hen and duck egg-white lysozymes which are very near related proteins despite many amino acid replacements (around 50), the insertion of an additional residue in the N-terminal and a deletion in the C-terminal moiety.  相似文献   

15.
To study the role of the C-terminal domains in the photocycle of a light sensor histidine kinase (Ppr) having a photoactive yellow protein (PYP) domain as the photosensor domain, we analyzed the photocycles of the PYP domain of Ppr (Ppr-PYP) and full-length Ppr. The gene fragment for Ppr-PYP was expressed in Escherichia coli, and it was chemically reconstituted with p-coumaric acid; the full-length gene of Ppr was coexpressed with tyrosine ammonia-lyase and p-coumaric acid ligase for biosynthesis in cells. The light/dark difference spectra of Ppr-PYP were pH sensitive. They were represented as a linear combination of two independent difference spectra analogous to the PYP(L)/dark and PYP(M)/dark difference spectra of PYP from Halorhodospira halophila, suggesting that the pH dependence of the difference spectra is explained by the equilibrium shift between the PYP(L)- and PYP(M)-like intermediates. The light/dark difference spectrum of Ppr showed the equilibrium shift toward PYP(L) compared with that of Ppr-PYP. Kinetic measurements of the photocycles of Ppr and Ppr-PYP revealed that the C-terminal domains accelerate the recovery of the dark state. These observations suggest an interaction between the C-terminal domains and the PYP domain during the photocycle, by which light signals captured by the PYP domain are transferred to the C-terminal domains.  相似文献   

16.
A direct oxo-ester peptide ligation method has been developed. Through the use of an activated C-terminal para nitrophenyl ester (1), it is possible to achieve direct cysteine ligations (1 + 2 --> 4). Peptide substrates incorporating bulky C-terminal amino acids (1) can be accommodated with high reaction efficiency.  相似文献   

17.
The development of hydrogels resulting from the self-assembly of low molecular weight (LMW) hydrogelators is a rapidly expanding area of study. Fluorenylmethoxycarbonyl (Fmoc) protected aromatic amino acids derived from phenylalanine (Phe) have been shown to be highly effective LMW hydrogelators. It has been found that side chain functionalization of Fmoc-Phe exerts a significant effect on the self-assembly and hydrogelation behavior of these molecules; fluorinated derivatives, including pentafluorophenylalanine (F(5)-Phe) and 3-F-phenylalanine (3-F-Phe), spontaneously self-assemble into fibrils that form a hydrogel network upon dissolution into water. In this study, Fmoc-F(5)-Phe-OH and Fmoc-3-F-Phe-OH were used to characterize the role of the C-terminal carboxylic acid on the self-assembly and hydrogelation of these derivatives. The C-terminal carboxylic acid moieties of Fmoc-F(5)-Phe-OH and Fmoc-3-F-Phe-OH were converted to C-terminal amide and methyl ester groups in order to perturb the hydrophobicity and hydrogen bond capacity of the C-terminus. Self-assembly and hydrogelation of these derivatives was investigated in comparison to the parent carboxylic acid compounds at neutral and acidic pH. It was found that hydrogelation of the C-terminal acids was highly sensitive to solvent pH, which influences the charge state of the terminal group. Rigid hydrogels form at pH 3.5, but at pH 7 hydrogel rigidity is dramatically weakened. C-terminal esters self-assembled into fibrils only slowly and failed to form hydrogels due to the higher hydrophobicity of these derivatives. C-terminal amide derivatives assembled much more rapidly than the parent carboxylic acids at both acidic and neutral pH, but the resultant hydrogels were unstable to shear stress as a function of the lower water solubility of the amide functionality. Co-assembly of acid and amide functionalized monomers was also explored in order to characterize the properties of hybrid hydrogels; these gels were rigid in unbuffered water but significantly weaker in phosphate buffered saline. These results highlight the complex nature of monomer/solvent interactions and their ultimate influence on self-assembly and hydrogelation, and provide insight that will facilitate the development of optimal amino acid LMW hydrogelators for gelation of complex buffered media.  相似文献   

18.
Abeta(1-40) and Abeta(1-42) are the main forms of amyloid beta (Abeta) peptides in the brain of Alzheimer's patients; however, the latter possesses much stronger aggregation and deposition propensity than the former, which is partially attributed to the more unfolded C-terminus of Abeta(1-42) than that of Abeta(1-40). To explore the physical basis underlying the different dynamic behaviors of both Abeta peptides, parallel molecular dynamics (MD) simulations on Abeta(1-40) and Abeta(1-42) were performed to investigate their thermal unfolding processes. It is revealed that the addition of residues 41 and 42 in Abeta(1-42) disrupts the C-terminal hydrophobic core, which triggers the unraveling of the C-terminal helix of Abeta(1-42). This conclusion is supported by the MD simulation on the I41A mutant of Abeta(1-42), in which the C-terminal helix possesses relatively higher conformational stability than that of wild type Abeta(1-42) owing to the change in hydrophobic interaction patterns.  相似文献   

19.
By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1 + H2O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2O and NH3) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a ym-1 ion, i.e., to the loss of the N-terminal residue following the a1-ym–1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.  相似文献   

20.
Protein isoprenylation, an important post-translational modification with a lipid, involves the selective attachment of two types of isoprenoids, farnesyl (C15) and geranylgeranyl (C20). The isoprenoid is linked via a thioether bond to the C-terminal cysteine residue of a variety of cellular proteins, including the heterotrimeric G protein gamma-subunits. One member of the G protein family, transducin (Talpha/Tbetagamma), plays a central role in visual transduction, and the structure-function relationship has been extensively studied with purified proteins, predominantly with bovine transducin that was shown to be farnesylated at the C-terminal cysteine residue of the gamma-subunit (Tgamma). We report here the structure of the C-terminal modification of mouse Tgamma, which has not yet been elucidated owing to the low amount of protein that can be isolated from the mouse retina. Electrospray ionization mass spectrometry (ESI-MS) of the high-performance liquid chromatography (HPLC)-purified Tgamma was in good agreement with the calculated mass of the farnesylated and methylated form of mouse Tgamma (Pro1-Cys70). A 'top-down' analysis of intact Tgamma using an ESI hybrid quadrupole time-of-flight (TOF) tandem mass spectrometer provided isoprenyl-specific ions that were observed to produce ions separated by 204 Da from the conventional (unmodified) precursor ion or the C-terminal sequence ions. Such characteristic fragmentation on an isoprenoid observed in top-down analysis could be useful in general for determining the type of isoprenylation as well as probing the site of modification in the protein sequence.  相似文献   

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