共查询到20条相似文献,搜索用时 15 毫秒
1.
Sabrina Deroo Philippe Fournier Dietmar Theisen Nicolas H.C. Brons Hans Deckmyn Claude P. Muller 《Letters in Peptide Science》1998,5(2-3):159-162
Phage displayed random 6-mer libraries were screened with a monoclonal antibody specific for a minimized linear 7-mer epitope of the measles virus hemagglutinin protein. No clone with the wild-type sequence was selected and most clones contained a sequence motif not found in the wild-type sequence. Two mimotopes (LYMPQLS, SEMPQLP) were synthesized which inhibited binding to the measles virus 95–135 times better than a wild-type peptide. Sequence comparison of proteins with known 3D-structure indicates that the epitope corresponds to an -helix, while the best mimotopes have no predicted helix propensity. The proline is thought to be required for inducing a turn necessary for mimicking part of the -helix. The higher intrinsic stability of such a mimotope may explain its improved binding and may be more suitable in immunogenicity experiments. 相似文献
2.
A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response. 总被引:2,自引:1,他引:1 下载免费PDF全文
A solid-phase 8-mer random combinatorial peptide library was used to generate a panel of mimotopes of an epitope recognized by a monoclonal antibody to the F protein of measles virus (MV). An inhibition immunoassay was used to show that these peptides were bound by the monoclonal antibody with different affinities. BALB/c mice were coimmunized with the individual mimotopes and a T-helper epitope peptide (from MV fusion protein), and the reactivity of the induced anti-mimotope antibodies with the corresponding peptides and with MV was determined. The affinities of the antibodies with the homologous peptides ranged from 8.9 x 10(5) to 4.4 x 10(7) liters/mol. However, only one of the anti-mimotope antibodies cross-reacted with MV in an enzyme-linked immunosorbent assay and inhibited MV plaque formation. Coimmunization of mice with this mimotope and the T-helper epitope peptide induced an antibody response which conferred protection against fatal encephalitis induced following challenge with MV and with the structurally related canine distemper virus. These results indicate that peptide libraries can be used to identify mimotopes of conformational epitopes and that appropriate immunization with these mimotopes can induce protective antibody responses. 相似文献
3.
The X-ray crystallographic structure of HIV-1 capsid protein suggests that the dimer interface of the dimerization domain is mainly formed from a putative α-helix structure of 14 amino acids (Gag residues 311–324) and lies directly C-terminal to the capsid major homology region. We found that a deletion mutation in the α-helix drastically reduces virus particle production. Alanine-scanning mutagenetic analysis indicated that substitution mutations at residues Q311, V313, K314, W316, and M317 all impair virus particle production markedly. Membrane flotation assays suggested that some mutations in the dimer interface have slight effects on the efficient binding of Gag to membranes. Indirect immunofluorescence studies revealed that mutants defective in virus production exhibit a subcellular distribution pattern similar to that of wild-type. However, velocity sedimentation analysis showed that mutations significantly impairing virus particle production were also detrimental to Gag multimerization, suggesting that the impaired virus production may be due to a defect in Gag multimerization. These results support the proposal that residues in the capsid dimer interface play a crucial role in promoting Gag multimerization, possibly by facilitating stable Gag–Gag interactions. 相似文献
4.
Multidrug resistance to anti-cancer drugs is a major medical problem. Resistance is manifested largely by the product of
the human MDR1 gene, P-glycoprotein, an ABC transporter that is an integral membrane protein of 1280 amino acids arranged into two homologous
halves, each comprising 6 putative transmembrane α-helices and an ATP binding domain. Despite the plethora of data from site-directed,
scanning and domain replacement mutagenesis, epitope mapping and photoaffinity labeling, a clear structural model for P-glycoprotein
remains largely elusive. In this report, we propose a new model for P-glycoprotein that is supported by the vast body of previous
data. The model comprises 2 membrane-embedded 16-strand β-barrels, attached by short loops to two 6-helix bundles beneath
each barrel. Each ATP binding domain contributes 2 β-strands and 1 α-helix to the structure. This model, together with an
analysis of the amino acid sequence alignment of P-glycoprotein isoforms, is used to delineate drug binding and translocation
sites. We show that the locations of these sites are consistent with mutational, kinetic and labeling data.
Received: 18 February 1998/Revised: 2 September 1998 相似文献
5.
Shimba N Kariya E Tate S Kaji H Kainosho M 《Journal of structural and functional genomics》2000,1(1):26-42
The effect of substituting Pro25, located in the α-helical region of the cystatin A structure, with Ser has been studied. The structures of wild type and
P25S cystatin A were determined by multidimensional NMR spectroscopy under comparable conditions. These two structures were
virtually identical, and the α-helix between Glu15-Lys30 exists with uninterrupted continuity, with a slight bend at residue 25. In order to characterize the possible substitution
effects of Pro25 with Ser on the α-helix, the chemical shifts of the amide nitrogens and protons, the generalized order parameters obtained
by the analyses of the 15N-1H relaxation data, the amide proton exchange rates, and the NOE networks among the α-helical and surrounding residues were
carefully compared. None of these parameters indicated any significant static or dynamic structural differences between the
α-helical regions of the wild-type and P25S cystatin A proteins. We therefore conclude that our previous structure of the
wild-type cystatin A, in which the α-helix exhibited a sharp kink at Pro25, must be revised. The asymmetric distribution of hydrophobic interactions between the side-chain residues of the α-helix
and the rolled β-sheet surface, as revealed by NOEs, may be responsible for the slight bend of the α-helix in both variants
and for the destabilized hydrogen bonding of the α-helical residues that follow Pro25/Ser25, as evidenced by increased amide exchange rates.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
6.
Tonarelli Georgina Lottersberger Javier Salvetti Jorge L. Jacchieri Saul Silva-Lucca Rosemeire A. Beltramini Leila M. 《International journal of peptide research and therapeutics》2000,7(4):217-224
Summary In order to study the correlation between secondary structure and bioaffinity of long and modified sequences of p24 protein
from HIV-1, three peptides containing the minimal size epitope from region 208–217 (EAAEWDRVHP) were prepared. It was found
that peptide p24-1n, an elongated native sequence, has the lowest KD and a predominant α-helix structure in the presence of trifluoroethanol. Three peptides containing another epitope from region
293–302 (FRDYVDRFK) were also synthesized. We have observed that modifications on the native sequence p24-2n (region 287–308)
increased the α-helix content and this was correlated with an improvement of the recognition by antibodies in ELSA.
Abbreviations: CD, circular dichroism; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; ELSA, enzyme linked immunosorbent assay; EDT,
1,2-ethanedithiol; Fmoc, fluorenylmethoxycarbonyl; FAB-MS, fast atom bombardment mass spectrometry; HPLC, high pressure liquid
chromatography; MeCN, acetonitrile; NMM, N-methylmorpholine; PyBOP, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate;
SELCOM, self consistent method; TMB, 3,3′,5,5′-tetramethylbenzidine; TFA, trifluoroacetic acid; TFE, trifluoroethanol; Aminoacid
symbols denote the L-configurations. Abbreviations for amino acids and nomenclature of peptides follow the recommendations
of the IUPAC-IUB Commission on Biochemical Nomenclature (Eur. J. Biochem., 138 (1984) 9). 相似文献
7.
Identification of an immunodominant neutralizing and protective epitope from measles virus fusion protein by using human sera from acute infection. 下载免费PDF全文
S F Atabani O E Obeid D Chargelegue P Aaby H Whittle M W Steward 《Journal of virology》1997,71(10):7240-7245
Polyclonal sera obtained from African children with acute measles were used to screen a panel of 15-mer overlapping peptides representing the sequence of measles virus (MV) fusion (F) protein. An immunodominant antigenic region from the F protein (p32; amino acids 388 to 402) was found to represent an amino acid sequence within the highly conserved cysteine-rich domain of the F protein of paramyxoviruses. Epitope mapping of this peptide indicated that the complete 15-amino-acid sequence was necessary for high-affinity interaction with anti-MV antibodies. Immunization of two strains of mice with the p32 peptide indicated that it was immunogenic and could induce antipeptide antibodies which cross-reacted with and neutralized MV infectivity in vitro. Moreover, passive transfer of antipeptide antibodies conferred significant protection against fatal rodent-adapted MV-induced encephalitis in susceptible mice. These results indicate that this epitope represents a candidate for inclusion in a future peptide vaccine for measles. 相似文献
8.
Aimed at understanding the crucially important structural features for the integrity of α-helical mimicry by βγ-sequences,
an α-amino acid sequence in a native peptide was substituted by differently arranged βγ-sequences. The self- and hetero-assembly
of a series of αβγ-chimeric sequences based on a 33-residue GCN4-derived peptide was investigated by means of molecular dynamics,
circular dichroism, and a disulfide exchange assay. Despite the native-like behavior of βγ alternating sequences such as retention
of α-helix dipole and the formation of 13-membered α-helix turns, the αβγ-chimeras with different βγ substitution patterns
do not equally mimic the structural behavior of the native parent peptide in solution. The preservation of the key residue
contacts such as van der Waals interactions and intrahelical H-bonding, which can be met only by particular substitution patterns,
thermodynamically favor the adoption of coiled coil folding motif. In this study, we show how successfully the destabilizing
structural consequences of α → βγ modification can be harnessed by reducing the solvent-exposed hydrophobic surface area and
placing of suitably long and bulky helix-forming side chains at the hydrophobic core. The pairing of αβγ-chimeric sequences
with the native wild-type are thermodynamically allowed in the case of ideal arrangement of β- and γ-residues. This indicates
a similarity in local side chain packing of β- and γ-amino acids at the helical interface of αβγ-chimeras and the native α-peptide.
Consequently, the backbone extended residues are able to participate in classical “knob-into-hole” packing with native α-peptide. 相似文献
9.
Chagnaud J. L. Moynet D. Londos-Gagliardi D. Bezian J. H. Vincendeau P. Fleury H. Guillemain B. 《International journal of peptide research and therapeutics》2001,8(2):95-106
Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs).
Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed
against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries,
displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and
4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4
mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained
heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino
acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide
library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus
sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized
a continoous topographic epitope. 相似文献
10.
Rudolf MP Zuercher AW Nechansky A Ruf C Vogel M Miescher SM Stadler BM Kricek F 《Journal of immunology (Baltimore, Md. : 1950)》2000,165(2):813-819
IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine. 相似文献
11.
Ollesch J Poschner BC Nikolaus J Hofmann MW Herrmann A Gerwert K Langosch D 《European biophysics journal : EBJ》2008,37(4):435-445
LV-peptides were designed as membrane-spanning low-complexity model structures that mimic fusion protein transmembrane domains.
These peptides harbor a hydrophobic core sequence that consists of helix-promoting and helix-destabilizing residues at different
ratios. Previously, the fusogenicity of these peptides has been shown to increase with the conformational flexibility of their
hydrophobic cores as determined in isotropic solution. Here, we examined the secondary structure, orientation, and distribution
of LV-peptides in membranes. Our results reveal that the peptides are homogeneously distributed within the membranes of giant
unilamellar liposomes and capable of fusing them. Increasing the valine content of the core up to the level of the β-branched
residue content of SNARE TMDs (∼50%) enhances fusogenicity while maintaining a largely α-helical structure in liposomal membranes.
A further increase in valine content or introduction of a glycine/proline pair favors β-sheet formation. In planar bilayers,
the α-helices adopt oblique angles relative to the bilayer normal and the ratio of α-helix to β-sheet responds more sensitively
to valine content. We propose that the fusogenic conformation of LV-peptides is likely to correspond to a membrane-spanning
α-helix. β-Sheet formation in membranes may be considered a side-reaction whose extent reflects conformational flexibility
of the core. 相似文献
12.
Xuejun Ma Rong Hu Hai Lü Kaikun Wei Lilan Zhang Shuixing Xue Yunde Hou 《中国科学C辑(英文版)》1999,42(2):191-201
Human interferon-α1c/86D (IFNα1c/86D) was functionally displayed on the surface of the filamentous bacteriophage using a phagemid vector system (pCANTAB5E). The key amino acid residues involved in the receptor binding were further defined with phage displayed 6-mer peptide library and two neutralizing antibodies against linear epitopeson the IFN-α1b, indicating that residues 30, 33, 34, (AB-loop) and residues 124, 126, 127 (D helix, DE-loop) were more critical than the adjacent residues for recognition of receptor. In addition, a cassette mutagenesis library was generated by fully randomizing the sequence of the four positions 29, 31, 32 and 35 in AB-loop, and used to select phage-IFN variants with WISH-based panning method. Three phage-IFN variants were isolated to possess more antiviral activity in the range of 4–16-fold than parental phage-IFN after IPTG-induced soluble expression. The results suggest that phage displayed phage-IFN α1c/86D variants with increased specific activity might be obtained after purification procedures. 相似文献
13.
Mauricio Urquiza Johanna Melo-Cardenas Tatiana Guevara Ignacia Echeverria Isabel C. Rodriguez Magnolia Vanegas Mario Amzel Manuel E. Patarroyo 《Amino acids》2010,39(5):1507-1519
We tested the hypothesis that stabilizing α-helix of Epstein–Barr virus gH-derived peptide 11438 used for binding human cells
will increase its biological activity. Non-stable α-helix of peptide 11438 was unfolded in an entropy-driven process, despite
the opposing effect of the enthalpy factor. Adding and/or changing amino acids in peptide 11438 allowed the designing of peptides
33207, 33208 and 33210; peptides 33208 and 33210 displayed higher helical content due to a decreased unfolding entropy change
as was determined by AGADIR, molecular dynamics and circular dichroism analysis. Peptides 33207, 33208 and 33210 inhibited
EBV invasion of peripheral blood mononuclear cells and displayed epitopes more similar to native protein than peptide 11438;
these peptides could be useful for detecting antibodies induced by native gH protein since they displayed high reactivity
with anti-EBV antibodies. Anti-peptide 33207 antibodies showed higher reactivity with EBV than anti-peptide 11438 antibodies
being useful for inducing antibodies against EBV. Anti-peptide 33210 antibodies inhibit EBV invasion of epithelial cells better
than anti-peptide 11438 antibodies. Peptide 33210 bound to normal T lymphocytes and Raji cells stronger than peptide 11438
and also induced apoptosis of monocytes and Raji cells but not of normal T cells in a similar way to EBV-gH. Peptide 33210
inhibited the monocytes’ development toward dendritic cells better than EBV and peptide 11438. In conclusion, stabilizing
the α-helix in peptides 33208 and 33210 designed from peptide 11438 increased the antigenicity and the ability of the antibodies
induced by peptides of inhibiting EBV invasion of host cells. 相似文献
14.
Monoclonal antibodies with broad specificity for hepatitis C virus hypervariable region 1 variants can recognize viral particles 总被引:6,自引:0,他引:6
Cerino A Meola A Segagni L Furione M Marciano S Triyatni M Liang TJ Nicosia A Mondelli MU 《Journal of immunology (Baltimore, Md. : 1950)》2001,167(7):3878-3886
The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is a highly heterogeneous sequence that is promiscuously recognized by human sera via binding to amino acid residues with conserved physicochemical properties. We generated a panel of mAbs from mice immunized with HVR1 surrogate peptides (mimotopes) affinity-selected with sera from HCV-infected patients from a phage display library. A high number of specific clones was obtained after immunization with a pool of nine mimotopes, and the resulting mAbs were shown to recognize several 16- and 27-mer peptides derived from natural HVR1 sequences isolated from patients with acute and chronic HCV infection, suggesting that HVR1 mimotopes were efficient antigenic and immunogenic mimics of naturally occurring HCV variants. Moreover, most mAbs were shown to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded HVR1. In addition, a highly promiscuous mAb was able to specifically capture bona fide viral particles (circulating HCV RNA) as well as rHCV-like particles assembled in insect cells expressing structural viral polypeptides derived from an HCV 1a isolate. These findings demonstrate that it is possible to induce a broadly cross-reactive clonal Ab response to multiple HCV variants. In consideration of the potentially important role of HVR1 in virus binding to cellular receptor(s), such a mechanism could be exploited for induction of neutralizing Abs specific for a large repertoire of viral variants. 相似文献
15.
ON Rogacheva BF Shchegolev VE Stefanov GA Zakharov EV Savvateeva-Popova 《Biochemistry. Biokhimii?a》2012,77(5):456-464
Protein-ligand docking and molecular dynamics studies have shown that the key event initiated by 3′:5′-AMP binding to the
A- and B-domains of protein kinase A Iα regulatory subunit is formation of a hydrogen bond between 3′:5′-AMP and A202(A326)
(the residue in parentheses being from the B-domain). The A202(A326) amide group movement associated with the bond formation
leads to reorganization of the phosphate binding cassette (PBC) (the short 310-helix becomes the long α-helix). This process results in L203(L327) displacement and finally causes hinge (B-helix) rotation.
The L203(L327) displacement and packing into the hydrophobic pocket formed by the PBC and β2β3-loop also depends on the β2β3-loop
conformation. The correct conformation is maintained by R, I, E, but not K at position 209(333) of the A- and B-domains. So,
the R209K and R333K mutants have problems with reaching B-conformation. The apo-form of the 3′:5′-AMP-binding domain also
undergoes transition from H- to B-conformation. In this case, the movement of A202(A326) amide group seems to be a result
of reorganization of the PBC into a more stable α-helix. 相似文献
16.
MA XuejunHU Rong LU Hai WEI KaikunZHANG LilanXUE Shuixing HOU Yunde 《中国科学:生命科学英文版》1999,42(2):191-201
Human interferon-α1c/86D (IFNα1c/86D) was functionally displayed on the surface of the filamentous bacteriophage using a phagemid
vector system (pCANTAB5E). The key amino acid residues involved in the receptor binding were further defined with phage displayed
6-mer peptide library and two neutralizing antibodies against linear epitopeson the IFN-α1b, indicating that residues 30,
33, 34, (AB-loop) and residues 124, 126, 127 (D helix, DE-loop) were more critical than the adjacent residues for recognition
of receptor. In addition, a cassette mutagenesis library was generated by fully randomizing the sequence of the four positions
29, 31, 32 and 35 in AB-loop, and used to select phage-IFN variants with WISH-based panning method. Three phage-IFN variants
were isolated to possess more antiviral activity in the range of 4–16-fold than parental phage-IFN after IPTG-induced soluble
expression. The results suggest that phage displayed phage-IFN α1c/86D variants with increased specific activity might be
obtained after purification procedures.
Project supported by the China National Expert Committee for Biotechnology Development. 相似文献
17.
Yvonne van de Wal Yvonne M. C. Kooy Jan Woulter Drijfhout Reinout Amons Frits Koning 《Immunogenetics》1996,44(4):246-253
Genetic susceptibility to coeliac disease (CD) is strongly associated with the expression of theHLA-DQ2 (α1*0501, β1*0201) allele. There is evidence that this DQ2 molecule plays a role in the pathogenesis of CD as a restriction element for
gliadin-specific T cells in the gut. However, it remains largely unclear which fragments of gliadin can actually be presented
by the disease-associated DQ dimer. With a view to identifying possible CD-inducing antigens, we studied the peptide binding
properties of DQ2. For this purpose, peptides bound to HLA-DQ2 were isolated and characterized. Dominant peptides were found
to be derived from two self-proteins: in addition to several sizevariants of the invariant chain (li)-derived CLIP peptide,
a relatively large amount of an major histocompatibility complex (MHC) class I-derived peptide was found. Analogues of this
naturally processed epitope (MHClα46–63) were tested in a cell-free peptide binding competition assay to investigate the requirements
for binding to DQ2. First, a core sequence of 10 amino acids within the MHClα46–63 peptide was identified. By subsequent single
amino acid substitution analysis of this core sequence, five putative anchor residues were identified at relative positions
P1, P4, P6, P7, and P9. Replacement by the large, positively charged Lys at these positions resulted in a dramatic loss of
binding. However, several other non-conservative substitutions had little or no discernable effect on the binding capacity
of the peptides.
Substitutions at P1 and P4 were most critical, suggesting a more prominent role as anchor residues. Structural features of
the DQ2 molecule that may relate to the binding motif and to gluten sensitivity are discussed. 相似文献
18.
Desai P. Coutinho E. Srivastava S. Haq W. Katti S.B. 《International journal of peptide research and therapeutics》2002,9(1):21-34
Summary Allergic bronchopulmonary aspergillosis (ABPA), caused byAspergillus fumigatus, is a complication of allergic asthma. Asp f 1 secreted byA. fumigatus is reported to be a major allergen/antigen involved in pathogenesis of aspergillosis. A 11-mer immunodominant epitope (Leu-Asn-Pro-Lys-Thr5-Asn-Lys-Trp-Glu-Asp10-Lys) of Asp f 1 has shown immunoreactivity with specific IgG and IgE antibodies in the sera of patients with ABPA in ELISA
inhibition assay. Various studies have suggested that the peptide has a potential use in the development of ELISA based diagnostic
kit for early diagnosis of infections caused byA. fumigatus. In view of these interesting properties of the undecapeptide we have embarked on an investigation of its conformation to
understand the relationship between structure and immunoreactivity. NMR and molecular modeling studies of the peptide suggest
a structure with a β-turn spanning residues Asn6-Glu9 in water at pH 4.0, a β-pleated sheet in DMSO and α-helix in 40% HFA. 相似文献
19.
Conformation of 60-residue peptide fragment from N-terminal of porcine kidney fructose 1,6-bisphosphatase 总被引:1,自引:0,他引:1
Limited digestion of fructose 1,6-bisphosphatase with subtilisin produces an S-peptide with an about 60-residue peptide fragment that is non-covalently associated with the enzyme. The 60-residue peptide fragment con-sists of the most part of allosteric site for AMP binding. It could be separated from S-protein by gel filtration with a Sephadex G-75 column equilibrated with 9% formic acid. According to X-ray diffraction results the S-peptide consists of two α-helices without β-strand and the α-helix content is about 60% in the 60-residue-peptide fragment. When the enzyme is subjected to limited proteolysis with subtilisin, the secondary structure of the enzyme does not show a de-tectable change in CD spectrum. The CD spectra of the isolated S-peptide were measured under different concentra-tions. In the absence of GuHCl, S-peptide had 30% a-helix and 38.5% turn-like structure but had no β-strand, sug-gesting that the N-terminal 60-residue fragment, which is synthesized initially by ribosome, would fo 相似文献
20.
To properly characterize protective polyclonal antibody responses, it is necessary to examine epitope specificity. Most antibody epitopes are conformational in nature and, thus, cannot be identified using synthetic linear peptides. Cyclic peptides can function as mimetics of conformational epitopes (termed mimotopes), thereby providing targets, which can be selected by immunoaffinity purification. However, the management of large collections of random cyclic peptides is cumbersome. Filamentous bacteriophage provides a useful scaffold for the expression of random peptides (termed phage display) facilitating both the production and manipulation of complex peptide libraries. Immunoaffinity selection of phage displaying random cyclic peptides is an effective strategy for isolating mimotopes with specificity for a given antiserum. Further epitope prediction based on mimotope sequence is not trivial since mimotopes generally display only small homologies with the target protein. Large numbers of unique mimotopes are required to provide sufficient sequence coverage to elucidate the target epitope. We have developed a method based on pattern recognition theory to deal with the complexity of large collections of conformational mimotopes. The analysis consists of two phases: 1) The learning phase where a large collection of epitope-specific mimotopes is analyzed to identify epitope specific "signs" and 2) The identification phase where immunoaffinity-selected mimotopes are interrogated for the presence of the epitope specific "signs" and assigned to specific epitopes. We are currently using computational methods to define epitope "signs" without the need for prior knowledge of specific mimotopes. This technology provides an important tool for characterizing the breadth of antibody specificities within polyclonal antisera. 相似文献