RESUMEN
Lipidated proteins are an emerging class of hybrid biomaterials that can integrate the functional capabilities of proteins into precisely engineered nano-biomaterials with potential applications in biotechnology, nanoscience, and biomedical engineering. For instance, fatty-acid-modified elastin-like polypeptides (FAMEs) combine the hierarchical assembly of lipids with the thermoresponsive character of elastin-like polypeptides (ELPs) to form nanocarriers with emergent temperature-dependent structural (shape or size) characteristics. Here, we report the biophysical underpinnings of thermoresponsive behavior of FAMEs using computational nanoscopy, spectroscopy, scattering, and microscopy. This integrated approach revealed that temperature and molecular syntax alter the structure, contact, and hydration of lipid, lipidation site, and protein, aligning with the changes in the nanomorphology of FAMEs. These findings enable a better understanding of the biophysical consequence of lipidation in biology and the rational design of the biomaterials and therapeutics that rival the exquisite hierarchy and capabilities of biological systems.
Asunto(s)
Elastina , Proteínas Intrínsecamente Desordenadas , Elastina/química , Proteínas Intrínsecamente Desordenadas/química , Péptidos/química , Temperatura , Biotecnología , Materiales Biocompatibles/químicaRESUMEN
Membrane proteins are difficult to isolate and purify due to their dependence on the surrounding lipid membrane for structural stability. Detergents are often used to solubilize these proteins, with this approach requiring a careful balance between protein solubilization and denaturation. Determining which detergent is most appropriate for a given protein has largely been done empirically through screening, which requires large amounts of membrane protein and associated resources. Here, we describe an alternative to conventional detergent screening using a computational modeling approach to identify the most likely candidate detergents for solubilizing a protein of interest. We demonstrate our approach using ghrelin O-acyltransferase (GOAT), a member of the membrane-bound O-acyltransferase family of integral membrane enzymes that has not been solubilized or purified in active form. A computationally derived GOAT structural model provides the only structural information required for this approach. Using computational analysis of detergent ability to penetrate phospholipid bilayers and stabilize the GOAT structure, a panel of common detergents were rank-ordered for their proposed ability to solubilize GOAT. The simulations were performed at all-atom resolution for a combined simulation time of 24 µs. Independently, we biologically screened these detergents for their solubilization of fluorescently tagged GOAT constructs. We found computational prediction of protein structural stabilization was the better predictor of detergent solubilization ability, but neither approach was effective for predicting detergents that would support GOAT enzymatic function. The current rapid expansion of membrane protein computational models lacking experimental structural information and our computational detergent screening approach can greatly improve the efficiency of membrane protein detergent solubilization, supporting downstream functional and structural studies.
Asunto(s)
Detergentes , Proteínas de la Membrana , Animales , Detergentes/química , Detergentes/metabolismo , Proteínas de la Membrana/química , Fosfolípidos , Aciltransferasas , Cabras/metabolismo , SolubilidadRESUMEN
Flock House virus (FHV) is a well-characterized model system to study infection mechanisms in non-enveloped viruses. A key stage of the infection cycle is the disruption of the endosomal membrane by a component of the FHV capsid, the membrane active γ peptide. In this study, we perform all-atom molecular dynamics simulations of the 21 N-terminal residues of the γ peptide interacting with membranes of differing compositions. We carry out umbrella sampling calculations to study the folding of the peptide to a helical state in homogenous and heterogeneous membranes consisting of neutral and anionic lipids. From the trajectory data, we evaluate folding energetics and dissect the mechanism of folding in the different membrane environments. We conclude the study by analyzing the extent of configurational sampling by performing time-lagged independent component analysis.
Asunto(s)
Membrana Celular/metabolismo , Nodaviridae/metabolismo , Péptidos/química , Pliegue de Proteína , Proteínas Virales/química , Secuencia de Aminoácidos , Modelos Moleculares , Nodaviridae/fisiología , Péptidos/metabolismo , Estructura Secundaria de Proteína , Proteínas Virales/metabolismoRESUMEN
Using a combination of coarse-grained and atomistic molecular dynamics simulations we have investigated the membrane binding and folding properties of the membrane lytic peptide of Flock House virus (FHV). FHV is an animal virus and an excellent model system for studying cell entry mechanisms in non-enveloped viruses. FHV undergoes a maturation event where the 44 C-terminal amino acids are cleaved from the major capsid protein, forming the membrane lytic (γ) peptides. Under acidic conditions, γ is released from the capsid interior allowing the peptides to bind and disrupt membranes. The first 21 N-terminal residues of γ, termed γ1, have been resolved in the FHV capsid structure and γ1 has been the subject of in vitro studies. γ1 is structurally dynamic as it adopts helical secondary structure inside the capsid and on membranes, but it is disordered in solution. In vitro studies have shown the binding free energies to POPC or POPG membranes are nearly equivalent, but binding to POPC is enthalpically driven, while POPG binding is entropically driven. Through coarse-grained and multiple microsecond all-atom simulations the membrane binding and folding properties of γ1 are investigated against homogeneous and heterogeneous bilayers to elucidate the dependence of the microenvironment on the structural properties of γ1. Our studies provide a rationale for the thermodynamic data and suggest binding of γ1 to POPG bilayers occurs in a disordered state, but γ1 must adopt a helical conformation when binding POPC bilayers.
Asunto(s)
Nodaviridae/metabolismo , Péptidos/metabolismo , Proteínas Virales/metabolismo , Membrana Celular/química , Membrana Dobles de Lípidos/química , Péptidos/química , Unión Proteica , Pliegue de Proteína , Termodinámica , Proteínas Virales/químicaRESUMEN
The PACE force field presents an attractive model for conducting molecular dynamics simulations of membrane-protein systems. PACE is a hybrid model, in which lipids and solvents are coarse-grained consistent with the MARTINI mapping, while proteins are described by a united atom model. However, given PACE is linked to MARTINI, which is widely used to study membranes, the behavior of proteins interacting with membranes has only been limitedly examined in PACE. In this study, PACE is used to examine the behavior of several peptides in membrane environments, namely WALP peptides, melittin and influenza hemagglutinin fusion peptide (HAfp). Overall, we find PACE provides an improvement over MARTINI for modeling helical peptides, based on the membrane insertion energetics for WALP16 and more realistic melittin pore dynamics. Our studies on HAfp, which forms a helical hairpin structure, do not show the hairpin structure to be stable, which may point toward a deficiency in the model. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Péptidos/química , Lípidos de la Membrana/química , Porosidad , Conformación Proteica , Pliegue de Proteína , Solventes/química , TermodinámicaRESUMEN
Flock House virus (FHV) serves as a model system for understanding infection mechanisms utilized by non-enveloped viruses to transport across cellular membranes. During the infection cycle of FHV, a fundamental stage involves disruption of the endosomal membrane by membrane active peptides, following externalization of the peptides from the capsid interior. The FHV lytic agents are the 44 C-terminal amino acids residues of the capsid protein, which are auto-catalytically cleaved during the capsid maturation process. The cleaved peptides are termed γ peptides. In this study, we perform multi-scale molecular dynamics simulations including 40⯵s all-atom molecular dynamics simulations to study the behavior of pre-inserted transmembrane lytic peptides at a high concentration in a neutral membrane. We study the dynamical organization among peptides to form oligomeric bundles in four systems including the wild-type γ peptide and three mutant forms; namely, a truncation mutant in which the 23 C-terminal residues are deleted (γ1), a construct where the 8 C-terminal residues of γ are fused to γ1 (Δ385-399 γ) and a single-point mutant (F402A γ), all of which have been experimentally shown to drastically affect infectivity and lytic activity compared to the wild-type γ. Our results shed light on the actions of varied forms of the FHV lytic peptide including membrane insertion, trans-membrane stability, peptide oligomerization, water permeation activity and dynamic pore formation. Findings from this study provide detailed structural information and rationale for the differences in lytic activity among variants of FHV γ.
Asunto(s)
Membrana Celular/efectos de los fármacos , Simulación de Dinámica Molecular , Nodaviridae/química , Fragmentos de Péptidos/química , Proteínas Virales/química , Membrana Celular/química , Mutación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Unión Proteica , Multimerización de ProteínaRESUMEN
The phospholipid cardiolipin mediates the functional interactions of proteins that reside within energy-conserving biological membranes. However, the molecular basis by which this lipid performs this essential cellular role is not well understood. We address this role of cardiolipin using the multisubunit mitochondrial TIM23 protein transport complex as a model system. The early stages of protein import by this complex require specific interactions between the polypeptide substrate receptor, Tim50, and the membrane-bound channel-forming subunit, Tim23. Using analyses performed in vivo, in isolated mitochondria, and in reductionist nanoscale model membrane systems, we show that the soluble receptor domain of Tim50 interacts with membranes and with specific sites on the Tim23 channel in a manner that is directly modulated by cardiolipin. To obtain structural insights into the nature of these interactions, we obtained the first small-angle x-ray scattering-based structure of the soluble Tim50 receptor in its entirety. Using these structural insights, molecular dynamics simulations combined with a range of biophysical measurements confirmed the role of cardiolipin in driving the association of the Tim50 receptor with lipid bilayers with concomitant structural changes, highlighting the role of key structural elements in mediating this interaction. Together, these results show that cardiolipin is required to mediate specific receptor-channel associations in the TIM23 complex. Our results support a new working model for the dynamic structural changes that occur within the complex during transport. More broadly, this work strongly advances our understanding of how cardiolipin mediates interactions among membrane-associated proteins.