Springer protocols feed by Protein Science 
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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.In Vivo Optogenetics for Light-Induced Oxidative Stress in Transgenic Zebrafish Expressing the KillerRed Photosensitizer Protein
Optogenetic methods are gaining broad recognition. The zebrafish is particularly useful for these applications as a model vertebrate due to a unique combination of translucent embryos/larvae and efficient transgenesis. Here, we describe a zebrafish model of light-induced cardiac deficiency. Upon illumination with intense green light, the membrane-tethered photosensitizer protein KillerRed acts as a photoinducer of reactive oxygen species which in turn cause changes in heart rate and contractility in hearts that express this transgene. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
In Vivo Cell Tracking Using PhOTO Zebrafish
By combining the strength of previously described in vivo cell tracking methodologies, we have recently generated a set of transgenic zebrafish lines, called “PhOTO (photoconvertible optical tracking of…)” zebrafish. PhOTO zebrafish lines are suitable for cell tracking during highly dynamic events, including gastrulation, tissue regeneration, tumorigenesis, and cancer/disease progression. Global monitoring of cell shape, cell interactions, e.g., cell intercalations, coordinated division, and cell dynamics are accomplished by using fluorescence imaging of nuclear and plasma membrane fluorescent protein la...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Using Photoactivatable GFP to Track Axonal Transport Kinetics
The advent of photoactivatable tools has revolutionized imaging of dynamic cellular processes. One such application is to visualize axonal transport—an intricate and dynamic process by which proteins and other macromolecules are conveyed from their sites of synthesis in the cell bodies to their destinations within axons and synapses. High-quality dynamic imaging of axonal transport using photoactivatable vectors can now be routinely performed using epifluorescence microscopes and CCD cameras that are standard in most laboratories, yet this is largely underutilized. Here we describe detailed protocols for imaging carg...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Structural Basis of Photoswitching in Fluorescent Proteins
Fluorescent proteins have revolutionized life sciences because they allow noninvasive and highly specific labeling of biological samples. The subset of “phototransformable” fluorescent proteins recently attracted a widespread interest, as their fluorescence state can be modified upon excitation at defined wavelengths. The fluorescence emission of Reversibly Switchable Fluorescent Proteins (RSFPs), in particular, can be repeatedly switched on and off. RSFPs enable many new exciting modalities in fluorescence microscopy and biotechnology, including protein tracking, photochromic Förster Resonance Energy Tran...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Photoactivated Adenylyl Cyclases as Optogenetic Modulators of Neuronal Activity
In recent years, optogenetic methods became invaluable tools, particularly in neurobiological research. Most prominently, optogenetic methods utilize microbial rhodopsins to elicit neuronal de- or hyperpolarization. However, other optogenetic tools have emerged that allow influencing neuronal function by different approaches. In this chapter we describe the use of photoactivated adenylyl cyclases (PACs) as modulators of neuronal activity. Using Caenorhabditis elegans as a model organism, this chapter shows how to measure the effect of PAC photoactivation by behavioral and electrophysiological assays, as well as their signi...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
Salvaging Ruins: Reverting Blind Retinas into Functional Visual Sensors
Blindness is one of the most devastating conditions affecting the quality of life. Hereditary degenerative diseases, such as retinitis pigmentosa, are characterized by the progressive loss of photoreceptors, leading to complete blindness. No treatment is known, the current state-of-the-art of restoring vision are implanted electrode arrays. As a recently discovered alternative, optical neuromodulators, such as channelrhodopsin, allow new strategies for treating these diseases by imparting light-sensitivity onto the remaining retinal neurons after photoreceptor cell death. Retinal degeneration is a heterogeneous set of dise...
Source: Springer protocols feed by Protein Science - April 11, 2014 Category: Biochemistry Source Type: news
A Practical Guide for Nuclear Resonance Vibrational Spectroscopy (NRVS) of Biochemical Samples and Model Compounds
Nuclear resonance vibrational spectroscopy (NRVS) has been used by physicists for many years. However, it is still a relatively new technique for bioinorganic users. This technique yields a vibrational spectrum for a specific element, which can be easily interpreted. Furthermore, isotopic labeling allows for site-specific experiments. In this chapter, we discuss how to access specific beamlines, what kind of equipment is used in NRVS, and how the sample should be prepared and the data collected and analyzed. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - March 27, 2014 Category: Biochemistry Source Type: news
Characterizing Millisecond Intermediates in Hemoproteins Using Rapid-Freeze-Quench Resonance Raman Spectroscopy
The combination of rapid freeze quenching (RFQ) with resonance Raman (RR) spectroscopy represents a unique tool with which to investigate the nature of short-lived intermediates formed during the enzymatic reactions of metalloproteins. Commercially available equipment allows trapping of intermediates within a millisecond to second time scale for low-temperature RR analysis resulting in the direct detection of metal–ligand vibrations and porphyrin skeletal vibrations in hemoproteins. This chapter briefly discusses RFQ-RR studies carried out previously in our laboratory and presents, as a practical example, protocols f...
Source: Springer protocols feed by Protein Science - March 27, 2014 Category: Biochemistry Source Type: news
FTIR Spectroscopy of Metalloproteins
Absorption of infrared radiation by proteins gives important information about their structure and function. The most intense infrared bands correspond to the overlap of all the peptide bond absorption. Additionally, in many metalloproteins their prosthetic groups have intrinsic ligands or bind substrates/inhibitors that absorb intensively in the infrared. Here, we describe thoroughly several Fourier transform infrared methods for studying structure–function relationships in metalloproteins, using hydrogenases as an example. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - March 27, 2014 Category: Biochemistry Source Type: news
Electrochemistry of Metalloproteins: Protein Film Electrochemistry for the Study of E. coli [NiFe]-Hydrogenase-1
Protein film electrochemistry is a technique which allows the direct control of redox-active enzymes, providing particularly detailed information on their catalytic properties. The enzyme is deposited onto a working electrode tip, and through control of the applied potential the enzyme activity is monitored as electrical current, allowing for direct study of inherent activity as electrons are transferred to and from the enzyme redox center(s). No mediators are used. Because the only enzyme present in the experiment is bound at the electrode surface, gaseous and liquid phase inhibitors can be introduced and removed whilst t...
Source: Springer protocols feed by Protein Science - March 27, 2014 Category: Biochemistry Source Type: news
Cell-Free Synthesis of the H-Cluster: A Model for the In Vitro Assembly of Metalloprotein Metal Centers
Many organometallic cofactors are highly complex and require multiple accessory proteins for both their assembly and transfer to a target protein. A cell-free system in which the biosynthetic pathway for a prosthetic group has been fully or even partially reconstructed enables investigations of the reaction sequence as well as the cofactor itself. As a model for the in vitro assembly of protein-bound metal centers, we describe a procedure for the cell-free synthesis of the H-cluster in the context of producing purified and active [FeFe] hydrogenase samples for spectroscopic studies. In general terms, this in vitro system i...
Source: Springer protocols feed by Protein Science - March 27, 2014 Category: Biochemistry Source Type: news
Techniques for the Production, Isolation, and Analysis of Iron–Sulfur Proteins
Iron–sulfur clusters constitute a group of cofactors found in many proteins that play key roles in an exceptionally wide range of metabolic processes. The chemical reactivity of iron–sulfur clusters means that they can be particularly prone to damage when removed from the protective environment of the cell. In general, the key to obtaining an intact, biologically active iron–sulfur cluster-containing protein is to maintain a strictly anaerobic environment throughout the entire process of protein purification and analysis. For many proteins, particularly those with more labile clusters, it is essential. (S...
Source: Springer protocols feed by Protein Science - March 27, 2014 Category: Biochemistry Source Type: news
Expression and Purification of NifB Proteins from Aerobic and Anaerobic Sources
We describe here the methods for NifB purification from cells of the strict aerobic nitrogen-fixing bacterium Azotobacter vinelandii, the facultative anaerobic nitrogen-fixing bacterium Klebsiella pneumoniae, and the facultative anaerobic non-nitrogen fixing bacterium Escherichia coli recombinantly expressing a nifB gene of thermophilic origin. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - March 27, 2014 Category: Biochemistry Source Type: news
Purification of O2-Sensitive Metalloproteins
The most dependable factor to perform successful biochemical experiments in an O2-free environment is the experience required to set up an efficient laboratory, to properly manipulate samples, to anticipate potential O2-related problems, and to maintain the complex laboratory setup operative. There is a long list of O2-related issues that may ruin your experiments. We provide here a guide to minimize these risks. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - March 27, 2014 Category: Biochemistry Source Type: news
Advances in Research on Metalloproteins
Metal ions play essential roles in biological processes. Ions such as K+ and Na+ are important in ion transport, and Mg2+, Ca2+, and Zn2+ are important chelators in many processes, including phosphotransfer and harvesting of light for energy metabolism. The transition metals readily undergo one-electron chemistry, and in this capacity they function uniquely in biological processes such as long-range and inner sphere electron transfer. They also facilitate many one-electron chemical reactions involving free radical intermediates. Iron, being the dominant element in the earth and a transition metal, most frequently participa...
Source: Springer protocols feed by Protein Science - March 27, 2014 Category: Biochemistry Source Type: news
