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1.
Bioconjug Chem ; 30(4): 1162-1168, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30869874

RESUMO

PEGylation is the most widely used half-life extension strategy for protein therapeutics. While it imparts a range of attractive attributes PEGylation can impede protein binding and reduce efficacy. A model system to probe the effects of PEGylation on protein binding has practical applications. Here, we present a system based on complex formation between a hexavalent lectin (RSL) and the globular polysaccharide Ficoll PM70 (a type of glycocluster). Mutants of the lectin were used to generate conjugates with 3, 6, or 12 PEG (1 kDa) chains. Using NMR spectroscopy we monitored how the degree of PEGylation impacted the lectin-Ficoll interaction. The binding propensity was observed to decrease with increasing polymer density. Apparently, the extended PEG chains sterically impede the lectin-Ficoll binding. This deduction was supported by molecular dynamics simulations of the protein-polymer conjugates. The implications for protein-surface interactions are discussed.


Assuntos
Lectinas/química , Polietilenoglicóis/química , Polissacarídeos/química , Proteínas de Bactérias/química , Simulação de Dinâmica Molecular , Ligação Proteica , Ralstonia solanacearum/química
2.
Angew Chem Int Ed Engl ; 57(24): 7126-7130, 2018 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-29673020

RESUMO

Here, we provide the first structural characterization of host-guest complexation between cucurbit[7]uril (Q7) and dimethyllysine (KMe2 ) in a model protein. Binding was dominated by complete encapsulation of the dimethylammonium functional group. While selectivity for the most sterically accessible dimethyllysine was observed both in solution and in the solid state, three different modes of Q7-KMe2 complexation were revealed by X-ray crystallography. The crystal structures revealed also entrapped water molecules that solvated the ammonium group within the Q7 cavity. Remarkable Q7-protein assemblies, including inter-locked octahedral cages that comprise 24 protein trimers, occurred in the solid state. Cucurbituril clusters appear to be responsible for these assemblies, suggesting a strategy to generate controlled protein architectures.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Imidazóis/química , Lisina/análogos & derivados , Proteínas/química , Cristalografia por Raios X , Metilação , Modelos Moleculares , Água/química
3.
Biochemistry ; 55(8): 1195-203, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26845253

RESUMO

Sugar binding by a cell surface ∼29 kDa lectin (RSL) from the bacterium Ralstonia solanacearum was characterized by NMR spectroscopy. The complexes formed with four monosaccharides and four fucosides were studied. Complete resonance assignments and backbone dynamics were determined for RSL in the sugar-free form and when bound to l-fucose or d-mannose. RSL was found to interact with both the α- and the ß-anomer of l-fucose and the "fucose like" sugars d-arabinose and l-galactose. Peak splitting was observed for some resonances of the binding site residues. The assignment of the split signals to the α- or ß-anomer was confirmed by comparison with the spectra of RSL bound to methyl-α-l-fucoside or methyl-ß-l-fucoside. The backbone dynamics of RSL were sensitive to the presence of ligand, with the protein adopting a more compact structure upon binding to l-fucose. Taking advantage of tryptophan residues in the binding sites, we show that the indole resonance is an excellent reporter on ligand binding. Each sugar resulted in a distinct signature of chemical shift perturbations, suggesting that tryptophan signals are a sufficient probe of sugar binding.


Assuntos
Proteínas de Bactérias/metabolismo , Fucose/metabolismo , Lectinas/metabolismo , Ralstonia solanacearum/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Fucose/análogos & derivados , Lectinas/química , Manose/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Ralstonia solanacearum/química , Alinhamento de Sequência
4.
Biomacromolecules ; 17(8): 2719-25, 2016 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-27403588

RESUMO

PEGylation, the covalent modification of proteins with polyethylene glycol, is an abundantly used technique to improve the pharmacokinetics of therapeutic proteins. The drawback with this methodology is that the covalently attached PEG can impede the biological activity (e.g., reduced receptor-binding capacity). Protein therapeutics with "disposable" PEG modifiers have potential advantages over the current technology. Here, we show that a protein-polymer "Medusa complex" is formed by the combination of a hexavalent lectin with a glycopolymer. Using NMR spectroscopy, small-angle X-ray scattering (SAXS), size exclusion chromatography, and native gel electrophoresis it was demonstrated that the fucose-binding lectin RSL and a fucose-capped polyethylene glycol (Fuc-PEG) form a multimeric assembly. All of the experimental methods provided evidence of noncovalent PEGylation with a concomitant increase in molecular mass and hydrodynamic radius. The affinity of the protein-polymer complex was determined by ITC and competition experiments to be in the micromolar range, suggesting that such systems have potential biomedical applications.


Assuntos
Lectinas/química , Polietilenoglicóis/química , Cromatografia em Gel , Espectroscopia de Ressonância Magnética , Espalhamento a Baixo Ângulo , Difração de Raios X
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