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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Tackling Sampling Challenges in Biomolecular Simulations
Molecular dynamics (MD) simulations are a powerful tool to give an atomistic insight into the structure and dynamics of proteins. However, the time scales accessible in standard simulations, which often do not match those in which interesting biological processes occur, limit their predictive capabilities. Many advanced sampling techniques have been proposed over the years to overcome this limitation. This chapter focuses on metadynamics, a method based on the introduction of a time-dependent bias potential to accelerate sampling and recover equilibrium properties of a few descriptors that are able to capture the complexit...
Source: Springer protocols feed by Protein Science - October 24, 2014 Category: Biochemistry Source Type: news
Coarse-Grained Force Fields for Molecular Simulations
Molecular dynamics (MD) simulations at the atomic scale are a powerful tool to study the structure and dynamics of model biological systems. However, because of their high computational cost, the time and length scales of atomistic simulations are limited. Biologically important processes, such as protein folding, ion channel gating, signal transduction, and membrane remodeling, are difficult to investigate using atomistic simulations. Coarse-graining reduces the computational cost of calculations by reducing the number of degrees of freedom in the model, allowing simulations of larger systems for longer times. In the firs...
Source: Springer protocols feed by Protein Science - October 24, 2014 Category: Biochemistry Source Type: news
Membrane-Associated Proteins and Peptides
This chapter discusses the practical aspects of setting up molecular dynamics simulations of membrane-associated proteins and peptides, and the analysis thereof. Topology files for selected lipids are provided and selected analysis tools presented. These include tools for the creation of lipid bilayers of mixed lipid content (DOPE) and easy extraction of lipid coordinates (g_zcoor, g_xycoor), the calculation of helical axes (g_helixaxis) and aromatic order parameters (g_arom), the determination of peptide- or protein-interacting lipids (g_under), and the investigation of lipid-specific interactions through the calculation ...
Source: Springer protocols feed by Protein Science - October 24, 2014 Category: Biochemistry Source Type: news
Molecular Dynamics Simulations of Membrane Proteins
Membrane protein structures are underrepresented in the Protein Data Bank (PDB) due to difficulties associated with expression and crystallization. As such, it is one area where computational studies, particularly Molecular Dynamics (MD) simulations, can provide useful additional information. Recently, there has been substantial progress in the simulation of lipid bilayers and membrane proteins embedded within them. Initial efforts at simulating membrane proteins embedded within a lipid bilayer were relatively slow and interactive processes, but recent advances now mean that the setup and running of membrane protein simula...
Source: Springer protocols feed by Protein Science - October 24, 2014 Category: Biochemistry Source Type: news
Lipid Membranes for Membrane Proteins
The molecular dynamics (MD) simulation of membrane proteins requires the setup of an accurate representation of lipid bilayers. This chapter describes the setup of a lipid bilayer system from scratch using generally available tools, starting with a definition of the lipid molecule POPE, generation of a lipid bilayer, energy minimization, MD simulation, and data analysis. The data analysis includes the calculation of area and volume per lipid, deuterium order parameters, self-diffusion constant, and the electron density profile. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 24, 2014 Category: Biochemistry Source Type: news
Current Status of Protein Force Fields for Molecular Dynamics Simulations
The current status of classical force fields for proteins is reviewed. These include additive force fields as well as the latest developments in the Drude and AMOEBA polarizable force fields. Parametrization strategies developed specifically for the Drude force field are described and compared with the additive CHARMM36 force field. Results from molecular simulations of proteins and small peptides are summarized to illustrate the performance of the Drude and AMOEBA force fields. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 24, 2014 Category: Biochemistry Source Type: news
Transition Path Sampling with Quantum/Classical Mechanics for Reaction Rates
Predicting rates of biochemical reactions through molecular simulations poses a particular challenge for two reasons. First, the process involves bond formation and/or cleavage and thus requires a quantum mechanical (QM) treatment of the reaction center, which can be combined with a more efficient molecular mechanical (MM) description for the remainder of the system, resulting in a QM/MM approach. Second, reaction time scales are typically many orders of magnitude larger than the (sub-)nanosecond scale accessible by QM/MM simulations. Transition path sampling (TPS) allows to efficiently sample the space of dynamic trajecto...
Source: Springer protocols feed by Protein Science - October 24, 2014 Category: Biochemistry Source Type: news
Molecular Dynamics Simulations
Molecular dynamics has evolved from a niche method mainly applicable to model systems into a cornerstone in molecular biology. It provides us with a powerful toolbox that enables us to follow and understand structure and dynamics with extreme detail—literally on scales where individual atoms can be tracked. However, with great power comes great responsibility: Simulations will not magically provide valid results, but it requires a skilled researcher. This chapter introduces you to this, and makes you aware of some potential pitfalls. We focus on the two basic and most used methods; optimizing a structure with energy ...
Source: Springer protocols feed by Protein Science - October 24, 2014 Category: Biochemistry Source Type: news
Receptor Tyrosine Kinases and Drug Resistance: Development and Characterization of In Vitro Models of Resistance to RTK Inhibitors
Aberrant expression of receptor tyrosine kinases (RTKs) has been extensively associated with alterations in the physiological activities of cells. These include cell growth and differentiation, cell death/survival, and the motility of cells which can subsequently lead to emergence of various diseases including cancer. Recent advances in the treatment of cancer have involved using RTKs as therapeutic targets. Unfortunately, the clinical use of receptor tyrosine kinase inhibitors (RTKIs) for the treatment of cancer has been hindered by innate or acquired resistance among some patients, as also experienced with classical chem...
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Receptor Tyrosine Kinase Targeting in Multicellular Spheroids
While growing cells as a monolayer is the traditional method for cell culture, the incorporation of multicellular spheroids into experimental design is becoming increasingly popular. This is due to the understanding that cells grown as spheroids tend to replicate the in vivo situation more reliably than monolayer cells. Thus, the use of multicellular spheroids may be more clinically relevant than monolayer cell cultures. Here, we describe methods for multicellular 3D spheroid generation that may be used to provide samples for receptor tyrosine kinase (and other protein) detection. Methods described include the forced-float...
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Human Tumor Xenografts in Mouse as a Model for Evaluating Therapeutic Efficacy of Monoclonal Antibodies or Antibody-Drug Conjugate Targeting Receptor Tyrosine Kinases
Targeting receptor tyrosine kinases by therapeutic monoclonal antibodies and antibody-drug conjugates has met with tremendous success in clinical oncology. Currently, numerous therapeutic monoclonal antibodies are under preclinical development. The potential for moving candidate antibodies into clinical trials relies heavily on therapeutic efficacy validated by human tumor xenografts in mice. Here we describe methods used to determine therapeutic efficacy of monoclonal antibodies or antibody-drug conjugates specific to human receptor tyrosine kinase using human tumor xenografts in mice as the model. The end point of the st...
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Cell Surface Biotinylation of Receptor Tyrosine Kinases to Investigate Intracellular Trafficking
Cell surface biotinylation is a biochemical approach to covalently bind membrane-impermeable biotin to the extracellular domain of membrane proteins, such as receptor tyrosine kinases (RTKs). Subsequent to ligand incubation periods, activated biotinylated receptors may internalize from the cell surface into early endosomes and then travel through intracellular compartments to either recycle back to the membrane or degrade in lysosomes. The biotin-labeled proteins may be detected through affinity purification with streptavidin agarose resins. This chapter describes methods for cell surface biotinylation to assess RTK traffi...
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Regulation of Receptor Tyrosine Kinases by miRNA: Overexpression of miRNA Using Lentiviral Inducible Expression Vectors
MicroRNAs have the ability to alter and regulate multiple genes, including RTK family members, making them an attractive approach for molecular therapeutic development. We use a pCDNA6.2-EmGFP-microRNA expression vector to overexpress individual mature microRNA and then transfer the expression cassette into a single, inducible lentiviral vector (pINDUCER20). We successfully use this system to create a pINDUCER-EmGFP-miRNA27a expression vector and generate a stable head and neck cancer cell line (UM-SCC-22A) that inducibly expresses miRNA-27a, resulting in targeted epidermal growth factor receptor down regulation. In this c...
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Downregulation of Receptor Tyrosine Kinases Through Ubiquitination: Analysis by Immunodetection
After ligand binding, receptor tyrosine kinases (RTKs) transmit intracellular signals involved in the regulation of various cell events and then attenuate signal transduction. Ubiquitination is a critical step involved in the downregulation of RTK signaling. Here, we describe how to immunodetect the ligand-induced ubiquitination and degradation of TrkA, an RTK, by immunoprecipitation and Western blotting. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
Identification of Receptor Protein Tyrosine Phosphatases (RPTPs) as Regulators of Receptor Tyrosine Kinases (RTKs) Using an RPTP siRNA-RTK Substrate Screen
Receptor tyrosine kinase (RTK) signaling exists in equilibrium between RTK tyrosyl phosphorylation and RTK tyrosyl dephosphorylation. Despite a detailed understanding of RTK tyrosyl phosphorylation, much less is known about RTK tyrosyl dephosphorylation. The receptor PTPs (RPTPs) are outstanding targets for the dephosphorylation of RTKs because of their mutual membrane proximity. In this chapter, we describe how to identify RPTPs that modulate the activity of RTKs using a siRNA screen and commercially available proteomic applications. The validation of putative RTKs as RPTP substrates using substrate-trapping approaches is...
Source: Springer protocols feed by Protein Science - October 17, 2014 Category: Biochemistry Source Type: news
