1Department of Physiology, Jules Stein Eye Institute and Howard Hughes Medical Institute, University of California, Los Angeles
Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.
Published January 28, 2013. Keywords: Biochemistry, Molecular Biology, Genetics, Cellular Biology, Molecular Biology, Anatomy, Physiology, Ophthalmology, Proteomics, Proteins, Membrane Transport Proteins, Vitamin A, retinoid, RBP complex, membrane transport, membrane receptor, STRA6, retinol binding protein
1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 3School of Medicine, University of Washington
An in vitro method for preparing functional glucocorticoid receptor (GR)•hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.
Published September 21, 2011. Keywords: Biochemistry, glucocorticoid receptor, hsp90, molecular chaperone protein, in vitro reconstitution, steroid binding, biochemistry, immunoadsorption, immunoprecipitation, Experion, western blot
1Pharmacology & Chemical Biology Department, University of Pittsburgh School of Medicine
Here we demonstrate the use of fluorescent Alexa dye coupled to α-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds α-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.
Published March 28, 2014. Keywords: Neuroscience, α-bungarotoxin, binding site, endocytosis, immunostaining, rodent hippocampal neurons, receptor, trafficking, plasma membrane
1Department of Chemical and Biological Engineering, Princeton University
We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (http://www.proteinwisdom.org). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.
Published July 25, 2013. Keywords: Genetics, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
1Center for Computational Medicine and Bioinformatics, University of Michigan, 2Center for Bioinformatics and Department of Molecular Bioscience, University of Kansas
Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.
Published November 3, 2011. Keywords: Biochemistry, On-line server, I-TASSER, protein structure prediction, function prediction
1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford
Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.
Published March 19, 2014. Keywords: Virology, Heterologous protein expression, insect cells, Spodoptera frugiperda, baculovirus, protein purification, kindlin, cell adhesion
1Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University
The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its ε subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with ε’s inhibitory C-terminal domain.
Published February 18, 2014. Keywords: Chemistry, ATP synthase, Bio-Layer Interferometry, Ligand-induced conformational change, Biomolecular Interaction Analysis, Allosteric regulation, Enzyme inhibition
1School of Life Sciences, University of Nottingham
Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.
Published October 17, 2014. Keywords: Chemistry, Kinesin, ATP turnover, mantATP, mantADP, stopped-flow fluorescence, microtubules, enzyme kinetics, nucleotide
1Department of Physics and Astronomy, VU University Amsterdam
This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.
Published October 10, 2014. Keywords: Biochemistry, Reconstitution, Photosynthesis, Chlorophyll, Carotenoids, Light Harvesting Protein, Chlamydomonas reinhardtii, Arabidopsis thaliana
1Physical Biosciences Division, Lawrence Berkeley National Laboratory
This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.
Published July 21, 2014. Keywords: Genetics, DNA-Affinity-Purified-chip, response regulator, transcription factor binding site, two component system, signal transduction, Desulfovibrio, lactate utilization regulator, ChIP-chip