RESUMEN
A novel statistical analysis of paramagnetic relaxation enhancement (PRE) and paramagnetic relaxation interference (PRI) based nuclear magnetic resonance (NMR) data is proposed based on the computation of correlation matrices. The technique is demonstrated with an example of the intrinsically disordered proteins (IDPs) osteopontin (OPN) and brain acid soluble protein 1 (BASP1). The correlation analysis visualizes in detail the subtleties of conformational averaging in IDPs and highlights the presence of correlated structural fluctuations of individual sub-domains in IDPs.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Osteopontina/química , Osteopontina/metabolismo , Estructura Terciaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/metabolismoRESUMEN
Hyperpolarized water can selectively enhance NMR signals of rapidly exchanging protons in osteopontin (OPN), a metastasis-associated intrinsically disordered protein (IDP), at near-physiological pH and temperature. The transfer of magnetization from hyperpolarized water is limited to solvent-exposed residues and therefore selectively enhances signals in 1 H-15 N correlation spectra. Binding to the polysaccharide heparin was found to induce the unfolding of preformed structural elements in OPN.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Osteopontina/química , Polisacáridos/química , Agua/química , Concentración de Iones de Hidrógeno , Protones , Solventes/química , TemperaturaRESUMEN
Incorporation of myristic acid onto the Nâ terminus of a protein is a crucial modification that promotes membrane binding and correct localization of important components of signaling pathways. Recombinant expression of N-myristoylated proteins in Escherichia coli can be achieved by co-expressing yeast N-myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12-carbon fatty acid lauric acid can also occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the brain acid soluble proteinâ 1 and the 1-185 N-terminal region of c-Src, we show the significant, and protein-specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl-free samples of myristoylated proteins in both rich and minimal media.
Asunto(s)
Ácido Mirístico/química , Proteínas/química , Ácido Mirístico/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , SolucionesRESUMEN
Hyperpolarization is generated by dissolution dynamic nuclear polarization (d-DNP) using a polymer-based polarizing agent dubbed FLAP (filterable labeled agents for polarization). It consists of a thermo-responsive poly(N-isopropylacrylamide), also known as pNiPAM-COOH, labeled with nitroxide radicals. The polymer powder is impregnated with an arbitrary solution of interest and frozen as is. Dissolution is followed by a simple filtration, leading to hyperpolarized solutions free from any contaminants. We demonstrated the use of FLAP to hyperpolarize partially deuterated water up to P((1) H)=6 % with a long relaxation T1 >36â s characteristic of high purity. Water hyperpolarization can be transferred to drugs, metabolites, or proteins that are waiting in an NMR spectrometer, either by exchange of labile protons or through intermolecular Overhauser effects. We also show that FLAPs are suitable polarizing agents for (13) C-labeled metabolites such as pyruvate, acetate, and alanine.
Asunto(s)
Resinas Acrílicas/química , Proteínas/química , Agua/química , Acetatos/química , Alanina/química , Isótopos de Carbono , Deuterio , Radicales Libres/química , Congelación , Espectroscopía de Resonancia Magnética , Metabolómica/métodos , Óxidos de Nitrógeno/química , Preparaciones Farmacéuticas/química , Protones , Piruvatos/química , SolubilidadRESUMEN
A graph theoretical analysis of nuclear magnetic resonance (NMR) data of six different protein interactions has been presented. The representation of the protein interaction data as a graph or network reveals that all of the studied interactions are based on a common functional concept. They all involve a single densely packed hub of functionally correlated residues that mediate the ligand binding events. This is found independent of the kind of protein (folded or unfolded) or ligand (protein, polymer or small molecule). Furthermore, the power of the graph analysis is demonstrated at the examples of the Calmodulin (CaM)/Calcium and the Cold Shock Protein A (CspA)/RNA interaction. The presented approach enables the precise determination of multiple binding sites for the respective ligand molecules.
Asunto(s)
Calcio/química , Calmodulina/química , Proteínas y Péptidos de Choque por Frío/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , HumanosRESUMEN
GAP-43 is a 25 kDa neuronal intrinsically disordered protein, highly abundant in the neuronal growth cone during development and regeneration. The exact molecular function(s) of GAP-43 remains unclear but it appears to be involved in growth cone guidance and actin cytoskeleton organization. Therefore, GAP-43 seems to play an important role in neurotransmitter vesicle fusion and recycling, long-term potentiation, spatial memory formation and learning. Here we report the nearly complete assignment of recombinant human GAP-43.