RESUMEN
Membrane proteins are generally divided into two classes. Integral proteins span the lipid bilayer, and peripheral proteins are located at the membrane surface. Here, we provide evidence for membrane proteins of a third class that stabilize lipid pores, most probably as toroidal structures. We examined mutants of the staphylococcal α-hemolysin pore so severely truncated that the protein cannot span a bilayer. Nonetheless, the doughnut-like structures elicited well-defined transmembrane ionic currents by inducing pore formation in the underlying lipids. The formation of lipid pores, produced here by a structurally defined protein, is supported by the lipid and voltage dependences of pore formation, and by molecular dynamics simulations. We discuss the role of stabilized lipid pores in amyloid disease, the action of antimicrobial peptides, and the assembly of the membrane-attack complexes of the immune system.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Modelos Moleculares , Conformación Proteica , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/clasificación , Proteínas de la Membrana/metabolismo , Simulación de Dinámica Molecular , Mutagénesis , Reacción en Cadena de la PolimerasaRESUMEN
Aggregation of transmembrane proteins is important for many biological processes, such as protein sorting and cell signaling, and also for in vitro processes such as two-dimensional crystallization. We have used large-scale simulations to study the lateral organization and dynamics of lipid bilayers containing multiple inserted proteins. Using coarse-grained molecular dynamics simulations, we have studied model membranes comprising â¼7000 lipids and 16 identical copies of model cylindrical proteins of either α-helical or ß-barrel types. Through variation of the lipid tail length and hence the degree of hydrophobic mismatch, our simulations display levels of protein aggregation ranging from negligible to extensive. The nature and extent of aggregation are shown to be influenced by membrane curvature and the shape or orientation of the protein. Interestingly, a model ß-barrel protein aggregates to form one-dimensional strings within the bilayer plane, whereas a model α-helical bundle forms two-dimensional clusters. Overall, it is clear that the nature and extent of membrane protein aggregation is dependent on several aspects of the proteins and lipids, including hydrophobic mismatch, protein class and shape, and membrane curvature.
Asunto(s)
Membrana Celular/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Simulación de Dinámica Molecular , Multimerización de Proteína , Secuencia de Aminoácidos , Membrana Celular/química , Difusión , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Datos de Secuencia Molecular , Porosidad , Estructura Secundaria de Proteína , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismoRESUMEN
Nanopores are under investigation for single-molecule DNA sequencing. The alpha-hemolysin (alphaHL) protein nanopore contains three recognition points capable of nucleobase discrimination in individual immobilized ssDNA molecules. We have modified the recognition point R(1) by extensive mutagenesis of residue 113. Amino acids that provide an energy barrier to ion flow (e.g., bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier weaken it. Amino acids with related side chains produce similar patterns of nucleobase recognition providing a rationale for the redesign of recognition points.
Asunto(s)
ADN de Cadena Simple/química , Proteínas Hemolisinas/química , Sustitución de Aminoácidos , Secuencia de Bases , Modelos Moleculares , MutagénesisRESUMEN
A set of 49 protein nanopore-lipid bilayer systems was explored by means of coarse-grained molecular-dynamics simulations to study the interactions between nanopores and the lipid bilayers in which they are embedded. The seven nanopore species investigated represent the two main structural classes of membrane proteins (alpha-helical and beta-barrel), and the seven different bilayer systems range in thickness from approximately 28 to approximately 43 A. The study focuses on the local effects of hydrophobic mismatch between the nanopore and the lipid bilayer. The effects of nanopore insertion on lipid bilayer thickness, the dependence between hydrophobic thickness and the observed nanopore tilt angle, and the local distribution of lipid types around a nanopore in mixed-lipid bilayers are all analyzed. Different behavior for nanopores of similar hydrophobic length but different geometry is observed. The local lipid bilayer perturbation caused by the inserted nanopores suggests possible mechanisms for both lipid bilayer-induced protein sorting and protein-induced lipid sorting. A correlation between smaller lipid bilayer thickness (larger hydrophobic mismatch) and larger nanopore tilt angle is observed and, in the case of larger hydrophobic mismatches, the simulated tilt angle distribution seems to broaden. Furthermore, both nanopore size and key residue types (e.g., tryptophan) seem to influence the level of protein tilt, emphasizing the reciprocal nature of nanopore-lipid bilayer interactions.
Asunto(s)
Simulación por Computador , Membrana Dobles de Lípidos/química , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Péptidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Fosfatidilcolinas/química , Estructura Secundaria de ProteínaRESUMEN
MOTIVATION: Short interfering RNA (siRNA)-induced RNA interference is an endogenous pathway in sequence-specific gene silencing. The potency of different siRNAs to inhibit a common target varies greatly and features affecting inhibition are of high current interest. The limited success in predicting siRNA potency being reported so far could originate in the small number and the heterogeneity of available datasets in addition to the knowledge-driven, empirical basis on which features thought to be affecting siRNA potency are often chosen. We attempt to overcome these problems by first constructing a meta-dataset of 6483 publicly available siRNAs (targeting mammalian mRNA), the largest to date, and then applying a Bayesian analysis which accommodates feature set uncertainty. A stochastic logistic regression-based algorithm is designed to explore a vast model space of 497 compositional, structural and thermodynamic features, identifying associations with siRNA potency. RESULTS: Our algorithm reveals a number of features associated with siRNA potency that are, to the best of our knowledge, either under reported in literature, such as anti-sense 5' -3' motif 'UCU', or not reported at all, such as the anti-sense 5' -3' motif 'ACGA'. These findings should aid in improving future siRNA potency predictions and might offer further insights into the working of the RNA-induced silencing complex (RISC).