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2.
Mol Immunol ; 166: 16-28, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38181455

RESUMO

Over 500 million people worldwide are affected by diabetes mellitus, a chronic disease that leads to high blood glucose levels and causes severe side effects. The predominant biological marker for diagnosis of diabetes is glycated haemoglobin (GHb). In human blood the predominant reducing sugar, glucose, irreversibly conjugates onto accessible amine groups within Hb. Most methods for diagnosis and monitoring of diabetes selectively detect N-terminal glycation at Val-1 on the ß-globin chain, but not glycation at other sites. Detection of other glycated epitopes of GHb has the potential to provide new information on the extent, duration and timing of elevated glucose, facilitating personalised diagnosis and intelligent diabetic control. In this work, a new anti-GHb Fab antibody (Fab-1) specific for haemoglobin A1c (HbA1c) with nanomolar affinity was discovered via epitope-directed immunisation and phage display. A single chain variable fragment (scFv) antibody derived from Fab-1 retained affinity and specificity for HbA1c, and affinity was enhanced tenfold upon addition of an enhanced green fluorescent protein tag. Both the scFv and Fab-1 recognised an epitope within HbA1c that was distinct from ß-Val-1, and our data suggest that this epitope may include glycation at Lys-66 in the ß-globin chain. To our knowledge, this is the first report of an scFv/Fab anti-glycated epitope antibody that recognises a non-A1c epitope in GHb, and confirms that fructosamine attached to different, discrete glycation sites within the same protein can be resolved from one another by immunoassay.


Assuntos
Diabetes Mellitus , Anticorpos de Cadeia Única , Oxibato de Sódio , Humanos , Hemoglobinas Glicadas , Epitopos , Glucose , Globinas beta
3.
PLoS One ; 17(9): e0274415, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36178936

RESUMO

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1-4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12-80%) and specificities (14-100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.


Assuntos
Infecções por HIV , Mycobacterium tuberculosis , Tuberculose dos Linfonodos , Adulto , Testes Diagnósticos de Rotina/métodos , Epitopos , Infecções por HIV/diagnóstico , Humanos , Lipopolissacarídeos , Filogenia , Estudos Retrospectivos , Sensibilidade e Especificidade
4.
Emerg Infect Dis ; 27(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33256890

RESUMO

We investigated the dynamics of seroconversion in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. During March 29-May 22, 2020, we collected serum samples and associated clinical data from 177 persons in London, UK, who had SARS-CoV-2 infection. We measured IgG against SARS-CoV-2 and compared antibody levels with patient outcomes, demographic information, and laboratory characteristics. We found that 2.0%-8.5% of persons did not seroconvert 3-6 weeks after infection. Persons who seroconverted were older, were more likely to have concurrent conditions, and had higher levels of inflammatory markers. Non-White persons had higher antibody concentrations than those who identified as White; these concentrations did not decline during follow-up. Serologic assay results correlated with disease outcome, race, and other risk factors for severe SARS-CoV-2 infection. Serologic assays can be used in surveillance to clarify the duration and protective nature of humoral responses to SARS-CoV-2 infection.


Assuntos
COVID-19/sangue , COVID-19/imunologia , Imunoglobulina G/sangue , SARS-CoV-2 , Soroconversão , Adulto , Idoso , Anticorpos Antivirais/sangue , COVID-19/fisiopatologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Free Radic Biol Med ; 144: 234-244, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31075498

RESUMO

Lipids are susceptible to damage by reactive oxygen species, and from lipid oxidation reactions many short chain lipid peroxidation products can be formed. 4-Hydroxy-2-nonenal (HNE) is one of the most abundant and cytotoxic lipid oxidation products and is known to form covalent adducts with nucleophilic amino acids of proteins. HNE-modified proteins have value as biomarkers and can be detected by antibody-based techniques, but most commercially available antibodies were raised against HNE-keyhole limpet hemocyanin. We used HNE-treated human serum albumin (HSA) to raise sheep antiserum and report for the first time the use of covalently modified peptide arrays to assess epitope binding of antibodies (Abs). Peptide arrays covering the sequence of HSA and treated post peptide synthesis with HNE were used to compare the different binding patterns of a commercial polyclonal antibody (pAb) raised against HNE-treated KLH and an in-house anti-HNE enriched pAb. The results were correlated with analysis of HNE-modified HSA by high-resolution tandem mass spectrometry. Both anti-HNE pAbs were found to bind strongly to eight common peptides on the HNE-treated HSA membranes, suggesting that HNE adducts per se induced an immune response in both cases even though different immunogens were used. Both antibodies bound with the highest affinity to the peptide 365DPHECYAKVFDEFKPLV381, which contains K378 and was also shown to be modified by the mass spectrometry analysis. Overall, the commercial anti-HNE pAb showed better specificity, recognizing nine out of the eleven adducts found by MS/MS, while the in-house enriched pAb only recognizes six. Nevertheless, the in-house pAb recognized specific peptides that were not recognized by the commercial pAb, which suggests the presence of clones uniquely specific to HNE adducts on HSA.


Assuntos
Aldeídos/química , Anticorpos/metabolismo , Mapeamento de Epitopos/métodos , Epitopos/química , Hemocianinas/química , Albumina Sérica Humana/química , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Sítios de Ligação , Epitopos/metabolismo , Hemocianinas/metabolismo , Humanos , Soros Imunes/química , Modelos Moleculares , Oxirredução , Análise Serial de Proteínas , Ligação Proteica , Estrutura Secundária de Proteína , Proteólise , Albumina Sérica Humana/metabolismo , Ovinos , Espectrometria de Massas em Tandem , Tripsina/química
6.
Chembiochem ; 19(18): 1959-1968, 2018 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-29968955

RESUMO

The enzyme carboxypeptidase G2 (CPG2) is used in antibody-directed enzyme prodrug therapy (ADEPT) to catalyse the formation of an active drug from an inert prodrug. Free CPG2 in the bloodstream must be inhibited before administration of the prodrug in order to avoid a systemic reaction in the patient. Although a few small-molecule CPG2 inhibitors have been reported, none has been taken forward thus far. This lack of progress is due in part to a lack of structural understanding of the CPG2 active site as well as the absence of small molecules that can block the active site whilst targeting the complex for clearance. The work described here aimed to address both areas. We report the structural/functional impact of extensive point mutation across the putative CPG2 catalytic site and adjacent regions for the first time, revealing that residues outside the catalytic region (K208A, S210A and T357A) are crucial to enzyme activity. We also describe novel molecules that inhibit CPG2 whilst maintaining the accessibility of galactosylated moieties aimed at targeting the enzyme for clearance. This work acts as a platform for the future development of high-affinity CPG2 inhibitors that occupy new chemical space and will advance the safe application of ADEPT in cancer treatment.


Assuntos
Domínio Catalítico/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , gama-Glutamil Hidrolase/antagonistas & inibidores , gama-Glutamil Hidrolase/metabolismo , Descoberta de Drogas , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , gama-Glutamil Hidrolase/química
7.
Protein Expr Purif ; 127: 44-52, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27374188

RESUMO

Due to its applications in the treatment of cancer and autoimmune diseases, the 42 kDa zinc-dependent metalloenzyme carboxypeptidase G2 (CPG2) is of great therapeutic interest. An X-ray crystal structure of unliganded CPG2 reported in 1997 revealed the domain architecture and informed early rational drug design efforts, however further efforts at co-crystallization of CPG2 with ligands, substrates or inhibitors have not been reported. Thus key features of CPG2 such as the location of the active site, the presence of additional ligand-binding sites, stability, oligomeric state, and the molecular basis of activity remain largely unknown, with the current working understanding of CPG2 activity based primarily on computational modelling. To facilitate renewed efforts in CPG2 structural biology, we report the first high-yield (250 mg L(-1)) recombinant expression (and purification) of soluble and active CPG2 using the Escherichia coli expression system. We used this protocol to produce full-length enzyme, as well as protein fragments corresponding to the individual catalytic and dimerization domains, and the activity and stability of each construct was characterised. We adapted our protocol to allow for uniform incorporation of NMR labels ((13)C, (15)N and (2)H) and present preliminary solution-state NMR spectra of high quality. Taken together, our results offer a route for production and solution-state characterization that supports renewed effort in CPG2 structural biology as well as design of significantly truncated CPG2 proteins, which retain activity while yielding (potentially) improved immunogenicity.


Assuntos
Proteínas de Bactérias , Escherichia coli/metabolismo , Expressão Gênica , Pseudomonas/genética , gama-Glutamil Hidrolase , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/genética , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Pseudomonas/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , gama-Glutamil Hidrolase/biossíntese , gama-Glutamil Hidrolase/química , gama-Glutamil Hidrolase/genética , gama-Glutamil Hidrolase/isolamento & purificação
8.
Free Radic Biol Med ; 75 Suppl 1: S50, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26461400

RESUMO

There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. It is known that patients with inflammatory disease have higher levels of plasma protein nitro-tyrosine than healthy controls. Fibrinogen is an abundant plasma protein, highly susceptible to such oxidative modifications, and is therefore a potential marker for oxidative protein damage. The aim of this study was to map tyrosine nitration in fibrinogen under oxidative conditions and identify susceptible residues. Fibrinogen was oxidised with 0.25mM and 1mM SIN-1, a peroxynitrite generator, and methionine was used to quench excess oxidant in the samples. The carbonyl assay was used to confirm oxidation in the samples. The carbonyl levels were 2.3, 8.72 and 11.5nmol/mg protein in 0, 0.25mM and 1mM SIN-1 samples respectively. The samples were run on a SDS-PAGE gel and tryptically digested before analysis by HPLC MS-MS. All 3 chains of fibrinogen were observed for all treatment conditions. The overall sequence coverage for fibrinogen determined by Mascot was between 60-75%. The oxidised samples showed increases in oxidative modifications in both alpha and beta chains, commonly methionine sulfoxide and tyrosine nitration, correlating with increasing SIN-1 treatment. Tyrosines that were most susceptible were Tyr135 (tryptic peptide YLQEIYNSNNQK) and Tyr277 (tryptic peptide GGSTSYGTGSETESPR), but several other nitrated tyrosines were also identified with high confidence. Identification of these susceptible peptides will allow design of sequences-specific biomarkers of oxidative and nitrative damage to plasma protein in inflammatory conditions.

9.
Analyst ; 137(5): 1130-6, 2012 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-22249441

RESUMO

The peptidoglycan layer of Staphylococcus aureus contains a (Gly)(5) cross-link which is not found in other bacteria, and which could be used to develop a specific immunoassay for detection of S. aureus in MRSA infections. A semi-synthetic route was used to prepare the S. aureus peptidoglycan precursor UDPMurNAc-L-Ala-γ-D-Glu-L-Lys(Gly)(5)-D-Ala-D-Ala, which was covalently attached to carrier protein bovine serum albumin via the UDP nucleotide. Serum raised using this antigen showed specificity for chemically immobilised peptidoglycan monomer containing (Gly)(5), using an ELISA immunoassay. ELISA assays using 0.1 or 1.0 µg samples of cell walls prepared from two MRSA strains and one penicillin-sensitive S. aureus strain, and from three other bacteria, showed the highest response against cell walls containing (Gly)(5), with a particularly high response against cell walls from one MRSA strain. Competition assays to investigate antibody selectivity demonstrated that the antibody response could be most effectively antagonised using ligands containing (Gly)(5). These data demonstrate that it is possible to generate antibodies with high affinity and selectivity for the (Gly)(5) containing monomer in S. aureus peptidoglycan, that could be used to develop an immunoassay for S. aureus.


Assuntos
Parede Celular/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Soros Imunes/imunologia , Oligopeptídeos/síntese química , Oligopeptídeos/imunologia , Peptidoglicano/química , Staphylococcus aureus/citologia , Sequência de Aminoácidos , Animais , Bovinos , Oligopeptídeos/química
10.
J Biol Chem ; 286(28): 25016-26, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21592960

RESUMO

Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where it is routinely detected and quantified by specific immunoassays. Intelligent epitope selection is essential to achieving the required assay performance. We present binding affinity measurements demonstrating that a typical ß3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCGß(66-80) and the closely related luteinizing hormone (LH) fragment LHß(86-100), which differ only by a single amino acid residue. A combination of optical spectroscopic measurements and atomistic computer simulations on these free peptides reveals differences in turn type stabilized by specific hydrogen bonding motifs. We propose that these structural differences are the basis for the observed selectivity in the full protein.


Assuntos
Anticorpos Monoclonais Murinos/química , Gonadotropina Coriônica Humana Subunidade beta/química , Simulação por Computador , Epitopos/química , Peptídeos/química , Animais , Anticorpos Monoclonais Murinos/genética , Gonadotropina Coriônica Humana Subunidade beta/genética , Epitopos/genética , Feminino , Humanos , Imunoensaio , Camundongos , Peptídeos/genética , Gravidez , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
11.
Org Biomol Chem ; 7(1): 76-84, 2009 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-19081949

RESUMO

We have explored a series of trisubstituted acridine-peptide conjugates for their ability to recognize and discriminate between DNA quadruplexes derived from the human telomere, and the c-kit and N-ras proto-oncogenes. Quadruplex affinity was measured as the peptide sequences were varied, together with their substitution position on the acridine, and the identity of the C-terminus (acid or amide). Surface plasmon resonance measurements revealed that all compounds bound to the human telomeric quadruplex with sub-micromolar affinity. Docking calculations from molecular modelling studies were used to model the effects of substituent orientation and peptide sequence. Modelling and experiment were in agreement that placement of the peptide over the face of the acridine is detrimental to binding affinity. The highest degrees of selectivity were observed towards the N-ras quadruplex by compounds capable of forming simultaneous contacts with their acridine and peptide moieties. The ligands that bound best displayed quadruplex affinities in the 1-5 nM range and at least 10-fold discrimination between the quadruplexes studied.


Assuntos
Acridinas/química , Peptídeos/química , Biotinilação , DNA/química , Transferência Ressonante de Energia de Fluorescência , Quadruplex G , Humanos , Cinética , Ligantes , Modelos Químicos , Modelos Moleculares , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Telômero/ultraestrutura , Proteínas ras/metabolismo
12.
J Biol Chem ; 283(50): 34571-9, 2008 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-18842590

RESUMO

MurM and MurN are tRNA-dependent ligases that catalyze the addition of the first (L-Ala/L-Ser) and second (L-Ala) amino acid onto lipid II substrates in the biosynthesis of the peptidoglycan layer of Streptococcus pneumoniae. We have previously characterized the first ligase, MurM (Lloyd, A. J., Gilbey, A. M., Blewett, A. M., De Pascale, G., El Zoeiby, A., Levesque, R. C., Catherwood, A. C., Tomasz, A., Bugg, T. D., Roper, D. I., and Dowson, C. G. (2008) J. Biol. Chem. 283, 6402-6417). In order to characterize the second ligase MurN, we have developed a chemoenzymatic route to prepare the lipid II-Ala and lipid II-Ser substrates. Recombinant MurN enzymes from penicillin-resistant (159) and -sensitive (Pn16) S. pneumoniae were expressed and purified as MBP fusion proteins and reconstituted using a radiochemical assay. MurN ligases from strains 159 and Pn16 both showed a 20-fold higher catalytic efficiency for lipid II-L-Ala over lipid II-l-Ser, with no activity against unmodified lipid II, and similar kinetic parameters were measured for MurN from penicillin-resistant and penicillin-sensitive strains. These results concur with the peptidoglycan analysis of S. pneumoniae, in which the major cross-link observed is L-Ala-L-Ala. The combined action of ligases MurM and MurN is therefore required in order to rationalize the high level of dipeptide cross-links in penicillin-resistant S. pneumoniae, with ligase MurM showing the major difference between penicillin-resistant and penicillin-sensitive strains.


Assuntos
Proteínas de Bactérias/química , Lipídeos/química , Peptídeo Sintases/química , Streptococcus pneumoniae/enzimologia , Alanina/química , Proteínas de Bactérias/metabolismo , Bioquímica/métodos , Catálise , Reagentes de Ligações Cruzadas/química , Resistência Microbiana a Medicamentos , Cinética , Espectrometria de Massas/métodos , Modelos Químicos , Penicilinas/química , Peptídeo Sintases/metabolismo , Proteínas Recombinantes/química , Serina/química
13.
Microbiology (Reading) ; 152(Pt 10): 2959-2967, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17005977

RESUMO

The molecular target for the bacteriolytic E protein from bacteriophage X174, responsible for host cell lysis, is known to be the enzyme phospho-MurNAc-pentapeptide translocase (MraY), an integral membrane protein involved in bacterial cell wall peptidoglycan biosynthesis, with an essential role being played by peptidyl-prolyl isomerase SlyD. A synthetic 37 aa peptide E(pep), containing the N-terminal transmembrane alpha-helix of E, was found to be bacteriolytic against Bacillus licheniformis, and inhibited membrane-bound MraY. The solution conformation of E(pep) was found by circular dichroism (CD) spectroscopy to be 100 % alpha-helical. No change in the CD spectrum was observed upon addition of purified Escherichia coli SlyD, implying that SlyD does not catalyse prolyl isomerization upon E. However, E(pep) was found to be a potent inhibitor of SlyD-catalysed peptidyl-prolyl isomerization (IC(50) 0.15 microM), implying a strong interaction between E and SlyD. E(pep) was found to inhibit E. coli MraY activity when assayed in membranes (IC(50) 0.8 microM); however, no inhibition of solubilized MraY was observed, unlike nucleoside natural product inhibitor tunicamycin. These results imply that the interaction of E with MraY is not at the MraY active site, and suggest that a protein-protein interaction is formed between E and MraY at a site within the transmembrane region.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptidilprolil Isomerase/metabolismo , Mapeamento de Interação de Proteínas , Transferases/metabolismo , Proteínas Virais/metabolismo , Antibacterianos/farmacologia , Bacillus/efeitos dos fármacos , Proteínas de Bactérias/química , Bacteriólise , Dicroísmo Circular , Proteínas de Escherichia coli/antagonistas & inibidores , Modelos Biológicos , Peptídeos/farmacologia , Peptidilprolil Isomerase/antagonistas & inibidores , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transferases/química , Transferases (Outros Grupos de Fosfato Substituídos) , Proteínas Virais/química
14.
Biochemistry ; 45(5): 1393-9, 2006 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-16445281

RESUMO

We have recently described an engineered zinc finger protein (Gq1) that binds with high specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3', and that inhibits the activity of the enzyme telomerase in vitro. Here we report site-directed mutagenesis, biophysical, and molecular modeling studies that provide new insights into quadruplex recognition by the zinc finger scaffold. We show that any one finger of Gq1 can be replaced with the corresponding finger of Zif268, without significant loss of quadruplex affinity or quadruplex versus duplex discrimination. Replacement of two fingers, with one being finger 2, of Gq1 by Zif268 results in significant impairment of quadruplex recognition and loss of discrimination. Molecular modeling suggests that the zinc fingers of Gq1 can bind to the human parallel-stranded quadruplex structure in a stable arrangement, whereas Zif268-quadruplex models show significantly weaker binding energy. Modeling also suggests that an important role of the key protein finger residues in the Gq1-quadruplex complex is to maintain Gq1 in an optimum conformation for quadruplex recognition.


Assuntos
DNA/química , Proteína 1 de Resposta de Crescimento Precoce/química , Conformação de Ácido Nucleico , Proteínas Recombinantes/química , Dedos de Zinco/fisiologia , Cisteína/química , DNA/genética , DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Ensaio de Imunoadsorção Enzimática , Quadruplex G , Histidina/química , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Dedos de Zinco/genética
15.
Mol Biosyst ; 2(10): 484-91, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17216029

RESUMO

UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.


Assuntos
Indicadores e Reagentes/análise , Lipídeos/química , Peptídeos/química , Peptidoglicano/biossíntese , Difosfato de Uridina/análogos & derivados , Proteínas de Bactérias/química , Cisteína/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Ligases/química , Estrutura Molecular , Peptídeo Sintases/química , Transferases/química , Transferases (Outros Grupos de Fosfato Substituídos) , Difosfato de Uridina/genética
16.
Org Biomol Chem ; 2(20): 2925-31, 2004 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-15480456

RESUMO

3,6-Bis-peptide acridine and acridone conjugates have been designed and synthesised to selectively interact with G-quadruplex DNA. The ligand properties are peptide sequence dependent, the highest discrimination being obtained with the FRHR tetrapeptide (up to >50-fold specificity). Molecular modeling studies have helped us rationalise the data and suggest that human telomeric quadruplex DNA can readily accommodate tetrapeptides, and furthermore that FRHR contributes to stabilization of the complex by non-bonded interactions within the TTA loop pockets of the quadruplex. These studies indicate that targeting distinct features of a G-quadruplex with hybrid molecules is a promising strategy for discriminating between quadruplex and duplex DNA.


Assuntos
DNA/química , Peptídeos/química , Acridinas/química , Quadruplex G , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Pirrolidinas/química
17.
J Am Chem Soc ; 125(19): 5594-5, 2003 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-12733873

RESUMO

G-quadruplex DNA-specific ligands were generated using a combinatorial selection of tetrapeptides which were then conjugated to a hemicyanine scaffold. The heterocycle enhanced peptide binding affinity by approximately 1000-fold to give ligands with near micromolar affinity and >40-fold discrimination for quadruplex DNA over duplex.


Assuntos
Carbocianinas/química , DNA/química , Oligopeptídeos/química , Corantes/química , Técnicas de Química Combinatória/métodos , DNA/metabolismo , Quadruplex G , Cinética , Ligantes , Oligopeptídeos/metabolismo , Especificidade por Substrato , Ressonância de Plasmônio de Superfície
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