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1.
Cell ; 163(3): 620-8, 2015 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-26456111

RESUMEN

Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy and enable structural studies in biologically complex environments. Here, we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an intrinsically disordered region, the yeast prion protein Sup35. We added an exogenously prepared isotopically labeled protein to deuterated lysates, rendering the biological environment "invisible" and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible, and such contexts can influence protein structure.


Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Factores de Terminación de Péptidos/química , Priones/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Factores de Terminación de Péptidos/metabolismo , Priones/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Biochemistry ; 56(36): 4850-4859, 2017 09 12.
Artículo en Inglés | MEDLINE | ID: mdl-28792214

RESUMEN

A mechanistic understanding of Aß aggregation and high-resolution structures of Aß fibrils and oligomers are vital to elucidating relevant details of neurodegeneration in Alzheimer's disease, which will facilitate the rational design of diagnostic and therapeutic protocols. The most detailed and reproducible insights into structure and kinetics have been achieved using Aß peptides produced by recombinant expression, which results in an additional methionine at the N-terminus. While the length of the C-terminus is well established to have a profound impact on the peptide's aggregation propensity, structure, and neurotoxicity, the impact of the N-terminal methionine on the aggregation pathways and structure is unclear. For this reason, we have developed a protocol to produce recombinant Aß1-42, sans the N-terminal methionine, using an N-terminal small ubiquitin-like modifier-Aß1-42 fusion protein in reasonable yield, with which we compared aggregation kinetics with AßM01-42 containing the additional methionine residue. The data revealed that Aß1-42 and AßM01-42 aggregate with similar rates and by the same mechanism, in which the generation of new aggregates is dominated by secondary nucleation of monomers on the surface of fibrils. We also recorded magic angle spinning nuclear magnetic resonance spectra that demonstrated that excellent spectral resolution is maintained with both AßM01-42 and Aß1-42 and that the chemical shifts are virtually identical in dipolar recoupling experiments that provide information about rigid residues. Collectively, these results indicate that the structure of the fibril core is unaffected by N-terminal methionine. This is consistent with the recent structures of AßM01-42 in which M0 is located at the terminus of a disordered 14-amino acid N-terminal tail.


Asunto(s)
Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Análisis Espectral/métodos
3.
J Am Chem Soc ; 137(46): 14673-85, 2015 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-26535489

RESUMEN

Amyloid ß-protein (Aß) sequence length variants with varying aggregation propensity coexist in vivo, where coaggregation and cross-catalysis phenomena may affect the aggregation process. Until recently, naturally occurring amyloid ß-protein (Aß) variants were believed to begin at or after the canonical ß-secretase cleavage site within the amyloid ß-protein precursor. However, N-terminally extended forms of Aß (NTE-Aß) were recently discovered and may contribute to Alzheimer's disease. Here, we have used thioflavin T fluorescence to study the aggregation kinetics of Aß42 variants with N-terminal extensions of 5-40 residues, and transmission electron microscopy to analyze the end states. We find that all variants form amyloid fibrils of similar morphology as Aß42, but the half-time of aggregation (t1/2) increases exponentially with extension length. Monte Carlo simulations of model peptides suggest that the retardation is due to an underlying general physicochemical effect involving reduced frequency of productive molecular encounters. Indeed, global kinetic analyses reveal that NTE-Aß42s form fibrils via the same mechanism as Aß42, but all microscopic rate constants (primary and secondary nucleation, elongation) are reduced for the N-terminally extended variants. Still, Aß42 and NTE-Aß42 coaggregate to form mixed fibrils and fibrils of either Aß42 or NTE-Aß42 catalyze aggregation of all monomers. NTE-Aß42 monomers display reduced aggregation rate with all kinds of seeds implying that extended termini interfere with the ability of monomers to nucleate or elongate. Cross-seeding or coaggregation may therefore represent an important contribution in the in vivo formation of assemblies believed to be important in disease.


Asunto(s)
Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Dicroismo Circular , Cinética , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Método de Montecarlo
4.
Chem Biol ; 21(2): 295-305, 2014 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-24485763

RESUMEN

Yeast prions are self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. Using magic-angle spinning nuclear magnetic resonance spectroscopy, we provide a detailed look at the dynamic properties of these forms over a broad range of timescales. We establish that different prion strains have distinct amyloid structures, with many side chains in different chemical environments. Surprisingly, the prion strain with a larger fraction of rigid residues also has a larger fraction of highly mobile residues. Differences in mobility correlate with differences in interaction with the prion-partitioning factor Hsp104 in vivo, perhaps explaining strain-specific differences in inheritance.


Asunto(s)
Amiloide/metabolismo , Chaperonas Moleculares/metabolismo , Priones/metabolismo , Secuencia de Aminoácidos , Amiloide/química , Sitios de Unión , Chaperonas Moleculares/química , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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