Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Mol Cell Proteomics ; 20: 100098, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34022432

RESUMO

CysE and CysK, the last two enzymes of the cysteine biosynthetic pathway, engage in a bienzyme complex, cysteine synthase, with yet incompletely characterized three-dimensional structure and regulatory function. Being absent in mammals, the two enzymes and their complex are attractive targets for antibacterial drugs. We have used hydrogen/deuterium exchange MS to unveil how complex formation affects the conformational dynamics of CysK and CysE. Our results support a model where CysE is present in solution as a dimer of trimers, and each trimer can bind one CysK homodimer. When CysK binds to one CysE monomer, intratrimer allosteric communication ensures conformational and dynamic symmetry within the trimer. Furthermore, a long-range allosteric signal propagates through CysE to induce stabilization of the interface between the two CysE trimers, preparing the second trimer for binding the second CysK with a nonrandom orientation. These results provide new molecular insights into the allosteric formation of the cysteine synthase complex and could help guide antibacterial drug design.


Assuntos
Cisteína Sintase/química , Cromatografia Líquida , Deutério , Medição da Troca de Deutério , Hidrogênio , Espectrometria de Massas
2.
Artigo em Inglês | MEDLINE | ID: mdl-32719787

RESUMO

Peptide-N 4-(N-acetyl-ß-glucosaminyl) asparagine amidases (PNGases, N-glycanases, EC 3.5.1.52) are indispensable tools in releasing N-glycans from glycoproteins. So far, only a limited number of PNGase candidates are available for the structural analysis of glycoproteins and their glycan moieties. Herein, a panel of 13 novel PNGase H+ candidates (the suffix H+ refers to the acidic pH optimum of these acidobacterial PNGases) was tested in their recombinant form for their deglycosylation performance. One candidate (originating from the bacterial species Dyella japonica) showed superior properties both in solution-phase and immobilized on amino-, epoxy- and nitrilotriacetate resins when compared to currently acidic available PNGases. The high expression yield compared to a previously described PNGase H+, broad substrate specificity, and good storage stability of this novel N-glycanase makes it a valuable tool for the analysis of protein glycosylation.

3.
Commun Biol ; 3(1): 181, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313072

RESUMO

Albumin has an average plasma half-life of three weeks and is thus an attractive carrier to improve the pharmacokinetics of fused therapeutics. The half-life is regulated by FcRn, a cellular receptor that protects against intracellular degradation. To tailor-design the therapeutic use of albumin, it is crucial to understand how structural alterations in albumin affect FcRn binding and transport properties. In the blood, the last C-terminal residue (L585) of albumin may be enzymatically cleaved. Here we demonstrate that removal of the L585 residue causes structural stabilization in regions of the principal FcRn binding domain and reduces receptor binding. In line with this, a short half-life of only 3.5 days was measured for cleaved albumin lacking L585 in a patient with acute pancreatitis. Thus, we reveal the structural requirement of an intact C-terminal end of albumin for a long plasma half-life, which has implications for design of albumin-based therapeutics.


Assuntos
Albumina Sérica Humana/metabolismo , Amilases/sangue , Animais , Carboxipeptidases A/sangue , Meia-Vida , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Lipase/sangue , Masculino , Camundongos Transgênicos , Pâncreas/enzimologia , Pancreatite/sangue , Pancreatite/enzimologia , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Proteólise , Receptores Fc/genética , Receptores Fc/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/genética , Relação Estrutura-Atividade
4.
FEBS J ; 286(9): 1734-1751, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30675761

RESUMO

Recently, an enzymatic reaction was utilized to covalently link the N and C termini of membrane scaffold proteins to produce circularized nanodiscs that were more homogeneous and stable than standard nanodiscs. We continue this development and aim for obtaining high yields of stable and monodisperse nanodiscs for structural studies of membrane proteins by solution small-angle scattering techniques. Based on the template MSP1E3D1, we designed an optimized membrane scaffold protein (His-lsMSP1E3D1) with a sortase recognition motif and high abundance of solubility-enhancing negative charges. With these modifications, we show that high protein expression is maintained and that the circularization reaction is efficient, such that we obtain a high yield of circularized membrane scaffold protein (csMSP1E3D1) and downstream circularized nanodiscs. We characterize the circularized protein and corresponding nanodiscs biophysically by small-angle X-ray scattering, size-exclusion chromatography, circular dichroism spectroscopy, and light scattering and compare to noncircularized samples. First, we show that circularized and noncircularized (lsMSP1E3D1) nanodiscs are structurally similar and have the expected nanodisc structure. Second, we show that lsMSP1E3D1 nanodiscs are more stable compared to the template MSP1E3D1 nanodiscs as an effect of the extra negative charges and that csMSP1E3D1 nanodiscs have further improved stability as an effect of circularization. Finally, we show that a membrane protein can be efficiently incorporated in csMSP1E3D1 nanodiscs. Large-scale production methods for circularized nanodiscs with improved thermal and temporal stability will facilitate better access to the nanodisc technology and enable applications at physiologically relevant temperatures.


Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanoestruturas/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cromatografia por Troca Iônica , Dicroísmo Circular , Bicamadas Lipídicas/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Fosfatidilcolinas/química , Agregados Proteicos , Desnaturação Proteica , Proteínas Recombinantes/química , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Solubilidade , Soluções , Thermotoga maritima/química , Difração de Raios X
5.
J Pharm Sci ; 107(6): 1690-1700, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29452143

RESUMO

Sexually transmitted Chlamydia trachomatis infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ctrachomatis major outer membrane protein (MOMP) is highly immunogenic but is a challenging vaccine candidate by being an integral membrane protein, and the immunogenicity depends on a correctly folded structure. We investigated the biophysical properties of the recombinant MOMP-based fusion antigen CTH522, which is tested in early human clinical trials. It consists of a truncated and cysteine-free version of MOMP fused to 4 variable domains from serovars D-G. In the native state, CTH522 did not exist as a monomer but showed an unusual self-assembly into nanoparticles with a negative zeta potential. In contrast to the ß-barrel structure of MOMP, native CTH522 contained no well-defined secondary structural elements, and no thermal transitions were measurable. Chemical unfolding resulted in monomers that upon removal of the denaturant self-assembled into higher order structures, comparable to the structure of the native protein. The conformation of CTH522 in nanoparticles is thus not entirely random and contains structural elements stabilized via denaturant-disruptable hydrophobic interactions. In conclusion, CTH522 has an unusual quaternary structure of supramolecular self-assemblies.


Assuntos
Antígenos de Bactérias/química , Vacinas Bacterianas/química , Chlamydia trachomatis/química , Nanopartículas/química , Porinas/química , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Infecções por Chlamydia/prevenção & controle , Chlamydia trachomatis/imunologia , Humanos , Porinas/imunologia , Agregados Proteicos , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia
6.
Mol Cell Proteomics ; 16(3): 451-456, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28062799

RESUMO

The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism of the neonatal Fc receptor (FcRn). For long serum half-life of therapeutic IgGs, the highly pH-dependent interaction with FcRn needs to be balanced to allow efficient FcRn binding and release at slightly acidic pH and physiological pH, respectively. Some IgGs, like the antibody briakinumab has an unusually short half-life of ∼8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two-pronged IgG-FcRn binding mechanism involving direct FcRn interactions with both the Fc region and the Fab regions of briakinumab, and correlate the occurrence of excessive FcRn binding to an unusually strong Fab-FcRn interaction.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Sítios de Ligação , Medição da Troca de Deutério/métodos , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Espectrometria de Massas/métodos , Modelos Moleculares , Ligação Proteica , Estabilidade Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...