CAF gratefully acknowledges support from NSF, NIH (R01 GM52032; R24 GM069 736), and the US Environmental Protection Agency, EPA (R 832721-010). A portion of this research was made possible with Government support by DoD, Air Force Office of Scientific Research. JS gratefully acknowledges support from NIH (P50GM071508-06). MLBP gratefully acknowledges support from a National Defense Science and Engineering Graduate (NDSEG) Fellowship, 32 CFR 168a. GAK gratefully acknowledges support from a National Science Foundation Graduate Research Fellowship under grant number DGE-1148900.

The authors declare that they have no competing financial interests.

The de novo protein design framework consists of two stages, a sequence selection stage and a validation stage. The framework is robust enough to handle rigid and flexible design templates, and can be applied to single protein design or complex protein design. The framework has been successfully applied to numerous protein systems with applications to dozens of diseases. A number of the designs have been experimentally validated, providing the most potent inhibitors or agonists of some proteins discovered to dat...

De novo protein design is the identification of protein sequences that will yield a desired tertiary structure with improved properties or function. Since the native fold of a protein is the conformation which lies at the free energy minimum, de novo protein design seeks sequences that will have a free energy minimum in the target fold. This problem was first described by Drexler1 and Pabo2 and was referred to as the "inverse folding problem." However, unlike the protein folding problem, where a sequence can yield only one folded structure solution, the de novo protein design problem exhibits degeneracy. Many different amino acid sequences can yield the same tertiary structure and function.
While protein design has traditionally been performed experimentally through rational design and directed evolution, computational methods have more recently been employed to overcome the limited search space inherent in experimental methods. A variety of computational methods have been used, including deterministic methods, stochastic methods, and probabilistic methods.3,4 Early computational methods used fixed-backbone templates to make the problem easier to solve.5-7 With the advent of faster processors, high performance computing, and more efficient algorithms, backbone flexibility has been incorporated by using an ensemble of fixed-backbone templates8-14 or by incorporating true backbone flexibility by expressing the template in terms of ranges of atom-to-atom distances and dihedral angles.15,16
This paper describes in detail Protein WISDOM, an online tool that has been made available to the academic community to utilize our computational de novo protein design framework. This framework has been applied to the design of numerous proteins, for therapeutic use targeting diseases such as HIV, cancer, complement diseases, and other autoimmune disorders. Many of the predicted peptides were experimentally validated, demonstrating the power of the method. Table 1 provides a summary of the different proteins that have been designed including the size of the protein or peptide, the number of predictions, and experimental validation.
<table border="1">
	<tbody>
		<tr>
			<td>Protein Design</td>
			<td>Protein Length</td>
			<td># of Computational Predictions</td>
			<td># of Experimental Validations</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Full sequence design of human beta-defensin-2</td>
			<td>41</td>
			<td>340</td>
			<td>&nbsp;</td>
			<td>(17)</td>
		</tr>
		<tr>
			<td>Compstatin inhibitors of human C3</td>
			<td>13</td>
			<td>28</td>
			<td>3/3</td>
			<td>(18, 19)</td>
		</tr>
		<tr>
			<td>Compstatin analogues that bind to rat C3c</td>
			<td>13</td>
			<td>5</td>
			<td>&nbsp;</td>
			<td>(20)</td>
		</tr>
		<tr>
			<td>Compstatin analogues with di-serine extension</td>
			<td>15</td>
			<td>8</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stabilizing structure of compstatin analog W4A9</td>
			<td>13</td>
			<td>18</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C3a receptor agonists and antagonists</td>
			<td>77</td>
			<td>20</td>
			<td>4/7</td>
			<td>(21)</td>
		</tr>
		<tr>
			<td>C5a receptor agonists and antagonists</td>
			<td>74</td>
			<td>61</td>
			<td>2/61</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HIV-1 gp14 inhibitors</td>
			<td>12</td>
			<td>6</td>
			<td>4/5</td>
			<td>(22)</td>
		</tr>
		<tr>
			<td>HIV-1 gp120 inhibitors</td>
			<td>9</td>
			<td>14</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bak inhibitors of Bcl-x L and Bcl-2</td>
			<td>16-18</td>
			<td>10</td>
			<td>5/5</td>
			<td>(23)</td>
		</tr>
		<tr>
			<td>Inhibitors of ERK2</td>
			<td>11</td>
			<td>25</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Inhibitors of EZH2</td>
			<td>21</td>
			<td>17</td>
			<td>10/10</td>
			<td>(24)</td>
		</tr>
		<tr>
			<td>Inhibitors of LSD1 and LSD2</td>
			<td>16</td>
			<td>41</td>
			<td>17/20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Inhibitors of HLA-DR1</td>
			<td>13</td>
			<td>6</td>
			<td>&nbsp;</td>
			<td>(25)</td>
		</tr>
		<tr>
			<td>Inhibitors of PNP</td>
			<td>5</td>
			<td>13</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 1. Summary of designed proteins and peptides using the de novo protein design framework. The # of computational predictions is presented as the number of favorable predictions (i.e. fold specificities above a certain cutoff or approximate binding affinities greater than the native sequence). The # of experimental validations gives two numbers: the first is the number of predictions that were experimentally validated while the second is the total number of predictions that were tested experimentally.
Design of human-beta-defensin-2 (h&beta;D-2) was performed to enhance the peptide's antimicrobial property.17 For this design, we considered two cases: 1) up to 10 mutations along h&beta;D-2 and 2) full sequence design of all h&beta;D-2 residue positions except the Cysteines (8, 15, 20, 30, 37, and 38). Three different design templates and three different sequence selection models were utilized in the design. High levels of similarity in mutations were observed between the weighted average and distance bin models for both the 10 mutation design and the full sequence design. Additionally, a large number of sequences were found to have more favorable calculated Fold Specificity values than the native sequence.
Complement system inhibitors (of C3, C3a, and C5a) were designed to combat a number of immune diseases such as stroke, heart attack, Alzheimer's disease, asthma, rheumatoid arthritis, rejection of xenotransplantation, adult respiratory disease, psoriasis, and Crohn's disease. Three compstatin inhibitors of C3c predicted by the protein design framework plus three rationally designed sequences were experimentally validated to be better binders than the native compstatin.18,19
Further studies examined the loss of activity of compstatin against non-primate C3c and designed a number of candidate rat and mouse C3c inhibitors. Five sequences were shown to have more favorable association free energies with rat C3c than the W4A9 compstatin mutant known to inhibit C3c. This is due to a new salt bridge formation by Arg1.20 Eight sequences with an N-terminal extension were predicted to be better binders than W4A9 with a di-Serine extension. Finally, 18 compstatin sequences were predicted to stabilize the bound conformation of W4A9, providing strong candidates for primate and non-primate C3c inhibitors.
In addition to C3c inhibitors, C3a and C5a receptor agonists and antagonists were designed based upon the structures of C3a and C5a. Seven C3a sequences predicted by the model were experimentally tested. Two of the sequences were potent agonists while two others were partial agonists.21 The two potent agonists showed a 58-fold improvement over a previously discovered "superagonist". The design of C5a receptor agonists and antagonists provided a set of 61 sequences. All the sequences were synthesized and two were found to be novel C5a agonists.
Fusion inhibitors of HIV-1, the virus that causes AIDS, were designed to prevent HIV-1 from infecting cells. The first design targeted gp41, an envelope glycoprotein of HIV-1. The protein design framework predicted six sequences that were better binders than the native sequence. Four of these predicted sequences were experimentally validated to inhibit HIV-1 with the best sequence having an IC50 as low as 29 &mu;M. This sequence showed a 3-15 fold improvement over the native sequence and had no loss of activity against an Enfuvirtide-resistant virus strain.22 The second design targeted gp120, another envelope glycoprotein of HIV-1. Fourteen sequences were predicted to be binders of gp120 and provide additional potential fusion inhibitors of HIV-1.
Numerous proteins linked to cancer provided promising targets for cancer therapeutics. Bcl-2 and Bcl-xL are anti-apoptotic proteins that prevent cell death. Inhibitors of these two proteins were designed to induce cell death in cancer cells. Ten sequences were predicted to be better binders than the native, and these results captured previous experimental and mutagenesis results.23 Another target protein, ERK2, is involved in signal-transduction cascades that make it a promising target for antiproliferative cancer therapies. Twenty-five sequences were predicted to be inhibitors of ERK2.
Histone methyltransferases and demethylases dynamically control histone methylation, which has been linked to many cancer types including prostate, breast, lymphoma, myeloma, bladder, colon, skin, liver, endometrial, lung, and gastric. The de novo protein design framework identified 17 inhibitors of EZH2 (a Lysine methyltransferase) and of the ten experimentally tested, all were found to inhibit EZH2.24 The most potent peptide had an IC50 of about 13 &mu;M, was equally effective with elevated enzyme concentrations, and did not compete with the cofactor. These peptides were the first set of inhibitors of EZH2. 53 inhibitors of LSD1 (a demethylase) were predicted by the framework and of the 20 experimentally tested, 17 were inhibitors of LSD1 and 18 were inhibitors of LSD2. The best inhibitors had IC50 values below 1 &mu;M, making them the most potent peptidic inhibitors discovered to date.
The final two protein systems provided targets for treating various autoimmune diseases such as Coeliac disease, diabetes mellitus type 1, systemic lupus erythematosus, Sj&ouml;gren's syndrome, Churg-Strauss Syndrome, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, rheumatoid arthritis, and allergies. None of these potential inhibitors have been experimentally validated, however the framework predicted six sequences that bind to HLA-DR1 and 13 sequences that bind to PNP.
Table 2 summarizes experimentally validated inhibitors and agonists predicted using the de novo protein design framework. The approximate binding affinity metric was used to predict nine of the sequences (inhibitors of human C3c, HIV-1 gp41, EZH2, LSD1, and LSD2), while the fold specificity metric was used to identify four of the sequences (agonists/antagonists of C3aR). These peptides highlight the success of the de novo protein design framework, particularly the added approximate binding affinity metric. The framework is extremely versatile in its applicability. Six different proteins linked to twenty-five different diseases have been successfully designed and experimentally validated.
<table border="1" cellpadding="1" cellspacing="1" height="147" width="856">
	<tbody>
		<tr>
			<td>Name</td>
			<td>IC50</td>
			<td>EC50</td>
			<td>Protein Target</td>
			<td>Applicable Diseases</td>
		</tr>
		<tr>
			<td>SQ027</td>
			<td>0.94 &mu;M</td>
			<td>&nbsp;</td>
			<td>human C3c</td>
			<td colspan="1" rowspan="7">stroke, heart attack, Alzheimer's disease, asthma, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis, diabetes type I, Crohn's disease, pancreatitis, and cystic fibrosis</td>
		</tr>
		<tr>
			<td>SQ086</td>
			<td>1.98 &mu;M</td>
			<td>&nbsp;</td>
			<td>human C3c</td>
		</tr>
		<tr>
			<td>SQ059</td>
			<td>4.73 &mu;M</td>
			<td>&nbsp;</td>
			<td>human C3c</td>
		</tr>
		<tr>
			<td>SQ110-4</td>
			<td>&nbsp;</td>
			<td>15.2 nM</td>
			<td>C3aR</td>
		</tr>
		<tr>
			<td>SQ060-4</td>
			<td>&nbsp;</td>
			<td>36.4 nM</td>
			<td>C3aR</td>
		</tr>
		<tr>
			<td>SQ007-5</td>
			<td>15.4 nM</td>
			<td>&nbsp;</td>
			<td>C3aR</td>
		</tr>
		<tr>
			<td>SQ002-5</td>
			<td>26.1 nM</td>
			<td>&nbsp;</td>
			<td>C3aR</td>
		</tr>
		<tr>
			<td>SQ435</td>
			<td>29 - 253 &mu;M</td>
			<td>&nbsp;</td>
			<td>HIV-1 gp41</td>
			<td>AIDS</td>
		</tr>
		<tr>
			<td>SQ037</td>
			<td>13.57 &mu;M</td>
			<td>&nbsp;</td>
			<td>EZH2</td>
			<td colspan="1" rowspan="5">prostate, breast, lymphoma, myeloma, bladder, colon, skin, liver, endometrial, lung, and gastric cancers</td>
		</tr>
		<tr>
			<td>SQ011-1</td>
			<td>0.521 &mu;M</td>
			<td>&nbsp;</td>
			<td>LSD1</td>
		</tr>
		<tr>
			<td>SQ016-1</td>
			<td>0.249 &mu;M</td>
			<td>&nbsp;</td>
			<td>LSD1</td>
		</tr>
		<tr>
			<td>SQ026-1</td>
			<td>2.51 &mu;M</td>
			<td>&nbsp;</td>
			<td>LSD2</td>
		</tr>
		<tr>
			<td>SQ015-1</td>
			<td>1.332 &mu;M</td>
			<td>&nbsp;</td>
			<td>LSD2</td>
		</tr>
	</tbody>
</table>
Table 2. Computationally predicted and experimentally validated peptides targeting various diseases.

protein wisdom: a workbench for in silico de novo design of biomolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

De Novo Design of Entry Inhibitors for HIV-1
The de novo design framework implemented in Protein WISDOM has been used for the design of inhibitor peptides for several important therapeutic systems (Tables 1 and 2). One system of note is the design of peptides to inhibit HIV-1 entry to the host cell receptor CD4, which is here used as a representative system to demonstrate the practical use of the Protein WISDOM interface. The peptides were designed to target the...

Watch this Scientific Journal Video about Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules at JoVE.com

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

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 (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

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16.9K Views.  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.

Video: Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single assembly step and prevents biofouling on silicon and gold surfaces. The SAM is used to coat microelectrodes patterned with standard, positive-tone lithography. Using the microtubule as an example, we apply a DC voltage to induce electrophoretic migration to the SAM-coated electrode in a reversible manner. A flow chamber is used for imaging the electrophoretic migration and microtubule patterning in situ using epifluorescence microscopy. This method is generally applicable to biomolecule patterning, as it employs electrophoresis to immobilize target molecules and thus does not require specific molecular interactions. Further, it avoids problems encountered when attempting to pattern the SAM molecules directly using lithographic techniques. The compatibility with electron beam lithography allows this method to be used to pattern biomolecules at the nanoscale.

We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the nanoscale.

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient expression of the transcription factor YAP. With this method, luminal differentiated (LD) cells of the mouse mammary gland are converted into cells that exhibit molecular and functional properties of mammary SCs. YAP also turns fully differentiated pancreatic exocrine cells into pancreatic duct-like progenitors. Similarly, to endogenous, natural SCs, YAP-induced stem-like cells ("ySCs") can be eventually expanded as organoid cultures long term in vitro, without further need of ectopic YAP/TAZ, as ySCs are endowed with a heritable self-renewing SC-like state. 
The reprogramming procedure presented here offers the possibility to generate and expand in vitro progenitor cells of various tissue sources starting from differentiated cells. The straightforward expansion of somatic cells ex vivo has implications for regenerative medicine, for understanding mechanisms of tumor initiation and, more in general, for cell and developmental biology studies.

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Availability of somatic SCs is crucial for regenerative medicine, disease modeling and to gain insight into SC properties. Here we present experimental strategies to reprogram, in vitro, differentiated adult cells into their corresponding expandable tissue-specific stem/progenitor cells by the transient expression of the single transcriptional co-activator YAP.

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...