The presented work was supported in parts by funding from the Canadian Institutes of Health Research / Fragile X Research Foundation of Canada partnership program to RD, the Jerome Lejeune Foundation to RD, the Interdisziplin&#228;res Zentrum f&#252;r klinische Forschung of the University Erlangen-Nuremberg to RD and from the Deutsche Forschungsgemeinschaft to RD and RE. The authors wish to thank Ingrid Zenger for technical assistance with cell culture maintenance as well as Prof. M. Wegner for making a pCMV5-FLAG vector available. The authors further wish to particularly thank Nadja Schroeder for the helpful support at the video shooting.

1. Stock Solutions
<ol>
	<li>Resuspend the lyophilized active motif powder in sterile H2O to a final concentration of 2 &#181;g/&#181;l. Tap carefully for mixing.</li>
	<li>Prepare small aliquots (e.g., 12 &#181;l each, 2 &#181;l are required per reaction) and store them at -20 &#176;C.</li>
</ol>
2. Preparation of Cells
<ol>
	<li>Seed mammalian cells such as HEK293 cells or primary neurons on a 24 well plate in 500 &#181;l growth medium including antibiotics. 
	NOTE: When using neurons, seed the cells at low density and use only the two middle rows of the 24 well plate. Each well will thus have an empty counterpart to harbor the medium during incubation (step 4.2). When handling neurons, always make sure that the cells are not kept outside the incubator for more than a few minutes.</li>
	<li>Culture the cells at appropriate conditions (humidified, 5% CO2 and 37 &#176;C) until the cells are app. 50% confluent. 
	NOTE: When using neurons, culture the cells until the desired developmental stage is reached and change app. 25-50% of the medium twice a week. Medium: Neurobasal medium (containing 1x B27, 5 mM L-glutamine and 1x Penicillin and Streptomycin; compare with ref 16).</li>
</ol>
3. Chariot Complex Formation
<ol>
	<li>Per reaction, dilute 0.1-2 &#181;g of the POI or the corresponding antibody as well as the control protein, or control antibody, respectively, in 50 &#181;l of PBS. 
	NOTE: The technique also works with macromolecular complexes consisting of pre-bound primary and secondary antibodies (cp. &#39;Representative Results&#39;, Figure 1). Mix and spin.</li>
	<li>For each sample, dilute 2 &#181;l Chariot stock solution in 50 &#181;l of PBS using separate tubes in order to avoid self-association of Chariot. Mix and spin.</li>
	<li>Transfer the diluted POI or antibody (step 3.1) to the Chariot dilution by pipetting. Mix and spin.</li>
	<li>Incubate the mixture for 30 min at room temperature (Chariot-POI complexes will form).</li>
</ol>
4. Transfection of Cells
<ol>
	<li>Dilute the&#160;Chariot complexes (step 3.4) in 100 &#181;l pre-warmed growth medium (37 &#176;C, no additives). Remove the medium and wash the cells once using pre-warmed 1x PBS. 
	NOTE: When using neurons, do not discard the medium, it will be reused. Keep it at 37 &#176;C. For washing, use PBS containing 0.5 mM MgCl2 and CaCl2. 
	Suggestion: keep the medium in empty wells while incubating the plate (step 4.5).</li>
	<li>Apply the Chariot complex solution (step 4.1) to the cells and rock the cells gently to ensure an even distribution of the solution. Grow the cells under standard conditions (step 2.2) for 1 hr. Add serum to a final concentration of 10%. When using neurons, add the medium from step 4.1. Grow the cells for 1 to 2 more hours. 
	NOTE: The optimal incubation time depends on the size and properties of the cargo and may need to be adjusted empirically. The following suggestions may serve as a guideline: For peptides, 1 hr is usually sufficient, for proteins 1-2 hr are recommended, for antibodies 2 hr, and for the transfection of neurons with antibodies 4 hr.</li>
	<li>Process cells for analysis as usual. Please note: the technique is compatible with experiments using fixation protocols as well as live imaging.</li>
</ol>
5. Imaging
<ol>
	<li>Using a laser scanning microscope, take z-stacks of cells and/or cell compartments of interest using approximately 0.25-0.5 &#181;m distance. The precise layer distance depends on the size of the structure to be reconstructed and needs to be determined individually.</li>
</ol>
6. Reconstruction
<ol>
	<li>In the following steps, use the Esc bottom to switch between Select and Navigate, Cntr&#160;to select multiple objects by clicking on them and shift&#160;key to cut objects (cp. step 6.10).</li>
	<li>Open the lsm-file in Imaris.</li>
	<li>Using the Display Adjustment for each channel, contrast the picture until the brightest structures reach saturation. Please note that this adjustment will not influence the surface construction, it only serves to assist the experimenter in thresholding the image.</li>
	<li>Click on the Add new Surfaces icon. A wizard will open.</li>
	<li>Select: Segment only a Region of Interest. Proceed to the next step.</li>
	<li>Adjust the margins of the selection box to fit your object of interest. Please bear in mind that the image has 3 dimensions and that the selection needs to be adjusted at the z plane as well. If the rectangular shape of the selection box should not match the object of interest properly, turn the object and/or enlarge the box until all required structures are embedded. Undesired objects may be removed later on (cp. step 6.10). Proceed.</li>
	<li>Select the appropriate channel. The Surface Area Detail Level or sphere diameter should be set to match the structures being reconstructed.&#160; Depending on the signal characteristics, either Absolute Intensity or Local Contrast can be used.&#160; Generally, Local Contrast works better for diffuse signals. In any case, the settings need to be adjusted separately for each signal or structure of interest, respectively.</li>
	<li>Using the Thresholding tool, reconstruct the structure of interest.</li>
	<li>Complete the reconstruction by clicking on the green arrow bottom.</li>
	<li>Adjust the object further by using the pencil tool to cut and remove surfaces as required. It is only possible to cut in north-south direction, therefore, tilt the image in order to cut the desired object.</li>
	<li>Obtain measurements of volume, area and intensities for each surface by selecting Statistics, Detailed Statistics, and Average Values.</li>
	<li>(Optional step) To observe color-coded statistics (cp. Figure 2E and F), select the color tab of the corresponding surface and mark &#39;Statistic Coded&#39; instead of &#39;Base&#39;. Several options will be displayed.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+APP+transgenic+mice:+a+model+of+Alzheimer's+disease+or+simply+overexpression+of+APP?.">The pathology of APP transgenic mice: a model of Alzheimer's disease or simply overexpression of APP?.</a> Histol Histopathol. 24, 83-100 (2009).</li><li>Carlson, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Modification+of+the+Phenotypes+Produced+by+Amyloid+Precursor+Protein+Overexpression+in+Transgenic+Mice.">Genetic Modification of the Phenotypes Produced by Amyloid Precursor Protein Overexpression in Transgenic Mice.</a> Human molecular genetics. 6, 1951-1959 (1997).</li><li>Jiao, X. F., Wang, Z., Kiledjian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+mRNA-secapping+regulator+implicated+in+X-linked+mental+retardation.">Identification of an mRNA-secapping regulator implicated in X-linked mental retardation.</a> Mol. Cell. 24, 713-722 (2006).</li><li>Fukami, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+member+of+a+gene+family+on+Xp22.3,+VCX-A,+is+deleted+in+patients+with+X-linked+nonspecific+mental+retardation.">A member of a gene family on Xp22.3, VCX-A, is deleted in patients with X-linked nonspecific mental retardation.</a> Am. J. Med. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Rho+GTPase+activity+alleviates+chondroitin+sulfate+proteoglycan-dependent+inhibition+of+neurite+extension.">Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycan-dependent inhibition of neurite extension.</a> J Neurosci Res. 77, 299-307 (2004).</li><li>Watanabe, S., Wakasugi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+function+of+human+neuroglobin+is+correlated+with+its+guanine+nucleotide+dissociation+inhibitor+activity.">Neuroprotective function of human neuroglobin is correlated with its guanine nucleotide dissociation inhibitor activity.</a> Biochem Biophys Res Commun. 369, 695-700 (2008).</li><li>Eguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+nuclear+localization+signal+for+nuclear+transport+of+DNA-encapsulating+particles.">Optimization of nuclear localization signal for nuclear transport of DNA-encapsulating particles.</a> J Control Release. 104, 507-519 (2005).</li></ol>

The authors have nothing to disclose.

Here, we present a protocol to study the significance of protein expression levels in controlling cellular functions in a dose driven manner. The protocol described allows for a fine-tuned manipulation of protein expression in various mammalian cell types, including hippocampal neurons, hence facilitating detailed studies of protein function on the cellular level.
Although RNA interference represents a well known strategy to down-regulate POIs, it has its disadvantages (cp. 14). Not...

A tight control of protein expression is essential for every living organism to command its own development as well as to react to environmental signals. Hence, a multitude of mechanisms has been invented during evolution to precisely regulate the expression level of each protein encoded by the app. 20,000 genes existing in any eukaryotic cell at any given time of its life. Taking place at different stages of protein production, regulatory mechanisms range from the management of chromatin structure, transcription and RNA handling to the direction of posttranslational protein modifications, transport and degradation.
It is therefore not surprising that malfunctions in the underlying molecular machineries and altered protein expression levels have been associated with diverse diseases such as cancer or intellectual disabilities. Indeed, looking at the outstanding complexity of neuronal development and mammalian brain function, the sensitivity of these sophisticated systems to alterations in protein expression manifests itself in several well-known intellectual deficits including Alzheimer and Parkinson disease (AD and PD) as well as autism spectrum disorders (ASD) like the Fragile X Syndrome (FXS). The latter disease is characterized by an extensive misexpression of a variety of proteins, which is due to the loss of a single translation regulating protein, FMRP (Fragile X Mental Retardation Protein)1-4. Furthermore, chromosomal rearrangements affecting the Variable charge x linked protein A (VCXA), a protein, that manages mRNA stability and translation by modifying mRNA capping5, have recently been associated with intellectual deficits, while point mutations were not identified in patients with cognitive disabilities as of now6,7, suggesting that the observed mental impairments originate from altered VCXA expression and dysregulated expression of its target proteins. In line with these findings, a study investigating whether de novo copy number variations of genes are associated with ASD established that novel gene duplications and deletions are a significant risk factor for ASD8, thus supporting the idea that elevated or diminished protein expression levels may cause intellectual deficits.
Remarkably, recent research further provided evidence that the expression level of a given protein is precisely adjusted to prevent its aggregation as a consequence of high protein amounts with almost no safety margins9. It has therefore been proposed that even small increments are sufficient to induce diseases such as AD and PD9. Although the variety of molecular machineries contributing to protein expression control suggests a complex regulation scheme in the light of these findings, a study investigating the expression level of over 5,000 mammalian genes10 demonstrated that nature preferred a more parsimonious scheme: The cellular abundance of proteins was shown to be predominantly regulated at the level of translation10, thus illustrating that the management of RNA availability mainly serves to fine-tune protein expression.
Studying the dose of proteins of interest (POIs) is therefore not only important to the understanding of the endogenous functions of a protein, but also to the investigation of many diseases and the development of therapies. Thus, last decades have seen the advance of several strategies using RNA interference to manipulate POI dosage. Though RNA interference is widely used to study protein function and is even being applied in clinical trials to treat cancer or ocular diseases as well as to pursue antiviral therapies in patients11-13, some difficulties may arise which might render the strategy impossible. For example, the seed sequence, which drives the knockdown by homology is comparable short, hence promoting off-target effects. Since highly efficient sequences are rare and need to be found among thousands of options (reviewed in 14), identifying the right sequence can be time-consuming and costly, but results may still be disappointing.
An alternative strategy is to directly target the POI by antibodies. Here, we illustrate the use of the protein carrier Chariot&#160;(manufactured by Active Motif) to reduce cellular protein availability, and the employment of three-dimensional reconstructions to study protein function following knock-down.
The active motif of Chariot, itself a 2.8 kDa peptide, is used to shuttle peptides, proteins and antibodies across membranes of mammalian cells15. The peptide associates with POIs by forming non-covalently coupled macromolecular complexes utilizing hydrophobic interactions, whereupon Chariot-POI complexes are internalized into cells in an endosome-independent manner. Importantly, Chariot was indicated to neither affect the intracellular localization of shuttled proteins, nor to exert cytotoxic effects or to affect the biological activity of its cargo15.

<table><tbody><tr><td>Chariot</td><td>Active Motif</td><td>30025</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Neurobasal medium</td><td>Life technologies</td><td>21103-049</td><td>warm up to 37 &amp;deg;C before using</td></tr><tr><td>1x B27</td><td>Life technologies</td><td>17504044</td><td>store at -20 &amp;deg;C</td></tr><tr><td>L-glutamine</td><td>Life technologies</td><td>25030-149</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Penicillin and Streptomycin</td><td>Life technologies</td><td>15140-122</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Imaris software</td><td>Bitplane</td><td>n.a.</td><td>expensive, but unmatched</td></tr><tr><td>Laser Scanning Microscope</td><td>Zeiss</td><td>n.a.</td><td></td></tr></tbody></table>

studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

In the following paragraphs, exemplary results illustrating a functional knockdown of a POI (Simiate, for further details please see 16,17) using Chariot reagent and antibody interference are presented. The findings demonstrate that diminished expression of Simiate impairs transcriptional activity, and, in a dosage dependent manner, induces apoptosis, culminating in mortality rates of over 99% if higher antibody amounts (&#62; 1 &#181;g antibody) are applied (these discoveries were publis...

Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

studying-protein-function-role-altered-protein-expression-antibody

Research

JoVE Journal

Bioengineering

6.6K Views.  University of Erlangen-Nuremberg. Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

Video: Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Environment

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Biochemistry

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Immunology and Infection

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The linker was created using molecular modeling software as a loop structure. Here, we introduce a protocol forin silico analysis of a complete scFv antibody that interacts with the epidermal growth factor receptor (EGFR). The homology modeling, with Pyrx of protein-protein docking and molecular dynamic simulation of the interacting scFv antibody and EGFR First, the authors used a protein structure modeling program and Python for homology modeling, and the antibody scFv structure was modeled for homology. The investigators downloaded Pyrx software as a platform in the docking study. The Molecular dynamic simulation was run using modeling software. Results show that when the MD simulation was subjected to energy minimization, the protein model had the lowest binding energy (-5.4 kcal/M). In addition, the MD simulation in this study showed that the docked EGFR-scFv antibody was stable for 20-75 ns when the movement of the structure increased sharply to 7.2 &#197;. In conclusion, in silicoanalysiswas performed, and the molecular docking and molecular dynamics simulations of the scFv antibody proved the effectiveness of the designed immune-therapeutic drug scFv as a specific drug therapy for EGFR.

Structural Analyses of An Epidermal Growth Factor Receptor-Specific Single-Chain Fragment Variable via An In Silico Approach

This antibody homology modeling prediction protocol is followed by antibody-receptor Pyrx docking and molecular dynamic simulation. These three primary methods are used to visualize the accurate antibody-receptor binding areas and the binding stability of the final structure.

Single-chain fragment variable (scFv) antibodies were previously constructed of variable light and heavy chains joined by a (Gly4-Ser) 3 linker. The ...

Genetics

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Biology

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Author Spotlight: Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Developmental Biology

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

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