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1.
J Am Chem Soc ; 139(42): 14829-14832, 2017 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-28990386

RESUMO

Based on the saposin-A (SapA) scaffold protein, we demonstrate the suitability of a size-adaptable phospholipid membrane-mimetic system for solution NMR studies of membrane proteins (MPs) under close-to-native conditions. The Salipro nanoparticle size can be tuned over a wide pH range by adjusting the saposin-to-lipid stoichiometry, enabling maintenance of sufficiently high amounts of phospholipid in the Salipro nanoparticle to mimic a realistic membrane environment while controlling the overall size to enable solution NMR for a range of MPs. Three representative MPs, including one G-protein-coupled receptor, were successfully incorporated into SapA-dimyristoylphosphatidylcholine nanoparticles and studied by solution NMR spectroscopy.


Assuntos
Biomimética , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Membranas Artificiais , Fosfolipídeos/química , Dimiristoilfosfatidilcolina/química , Concentração de Íons de Hidrogênio , Nanopartículas/química , Nanopartículas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Saposinas/química , Saposinas/metabolismo
2.
Molecules ; 22(8)2017 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-28813004

RESUMO

Galectins are ß-galactoside-binding proteins implicated in a myriad of biological functions. Despite their highly conserved carbohydrate binding motifs with essentially identical structures, their affinities for lactose, a common galectin inhibitor, vary significantly. Here, we aimed to examine the molecular basis of differential lactose affinities amongst galectins using solution-based techniques. Consistent dissociation constants of lactose binding were derived from nuclear magnetic resonance (NMR) spectroscopy, intrinsic tryptophan fluorescence, isothermal titration calorimetry and bio-layer interferometry for human galectin-1 (hGal1), galectin-7 (hGal7), and the N-terminal and C-terminal domains of galectin-8 (hGal8NTD and hGal8CTD, respectively). Furthermore, the dissociation rates of lactose binding were extracted from NMR lineshape analyses. Structural mapping of chemical shift perturbations revealed long-range perturbations upon lactose binding for hGal1 and hGal8NTD. We further demonstrated using the NMR-based hydrogen-deuterium exchange (HDX) that lactose binding increases the exchange rates of residues located on the opposite side of the ligand-binding pocket for hGal1 and hGal8NTD, indicative of allostery. Additionally, lactose binding induces significant stabilisation of hGal8CTD across the entire domain. Our results suggested that lactose binding reduced the internal dynamics of hGal8CTD on a very slow timescale (minutes and slower) at the expense of reduced binding affinity due to the unfavourable loss of conformational entropy.


Assuntos
Galectina 1/química , Galectinas/química , Lactose/química , Sítios de Ligação , Calorimetria , Medição da Troca de Deutério , Entropia , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos
3.
J Am Chem Soc ; 138(14): 4787-95, 2016 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-27010847

RESUMO

In this study, we report the structure and function of a lectin from the sea mollusk Crenomytilus grayanus collected from the sublittoral zone of Peter the Great Bay of the Sea of Japan. The crystal structure of C. grayanus lectin (CGL) was solved to a resolution of 1.08 Å, revealing a ß-trefoil fold that dimerizes into a dumbbell-shaped quaternary structure. Analysis of the crystal CGL structures bound to galactose, galactosamine, and globotriose Gb3 indicated that each CGL can bind three ligands through a carbohydrate-binding motif involving an extensive histidine- and water-mediated hydrogen bond network. CGL binding to Gb3 is further enhanced by additional side-chain-mediated hydrogen bonds in each of the three ligand-binding sites. NMR titrations revealed that the three binding sites have distinct microscopic affinities toward galactose and galactosamine. Cell viability assays showed that CGL recognizes Gb3 on the surface of breast cancer cells, leading to cell death. Our findings suggest the use of this lectin in cancer diagnosis and treatment.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Bivalves/química , Lectinas/química , Lectinas/farmacologia , Trissacarídeos/química , Sequência de Aminoácidos , Animais , Antineoplásicos/metabolismo , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sequência de Carboidratos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Lectinas/metabolismo , Células MCF-7 , Modelos Moleculares , Estrutura Secundária de Proteína , Trissacarídeos/metabolismo
4.
Biomol NMR Assign ; 9(2): 427-30, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26126590

RESUMO

Galectins recognize ß-galectosides to promote a variety of cellular functions. Despite their sequence variations, all galectins share the same carbohydrate recognition domains (CRD) and their modes of ligand recognition at a structural level are essentially identical. Human galectin 8 plays an important role in numerous cancer and immune responses. It consists of two CRDs that are connected via a flexible linker. The substrate affinities and specificities of the N- and C-terminal domains are quite different. In order to investigate the structural basis of their substrate specificities, we complete the NMR (1)H, (13)C, and (15)N chemical shift assignments of C-terminal domain of human galectin-8 (hG8C).


Assuntos
Galectinas/química , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
5.
Biomol NMR Assign ; 8(2): 287-9, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23824732

RESUMO

Many knotted proteins have been discovered recently, but the folding process of which remains elusive. HP0242 is a hypothetical protein from Helicobacter pylori, which is a model system for studying the folding pathway of a knotted protein. In this study, we report the (1)H, (13)C, and (15)N chemical shift assignments of HP0242. The results will enable us to further investigate HP0242 by NMR experiments.


Assuntos
Proteínas de Bactérias/química , Helicobacter pylori , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
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