This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases.

1. Plasmid Construction
<ol>
	<li>After cloning a gene that encodes a protein of interest into an appropriate expression vector, introduce amber mutations at desired locations using a site-directed mutagenesis kit. 
	NOTE: Structural information and surface exposure predictions can serve as useful guides to determine the positions of mutations. Because the efficiency of amber suppression and cross-linking can vary considerably, amber mutations should be introduced at multiple positions. Furthermore, because Bpa is an analog of tyrosine and phenylalanine, try to introduce amber mutations at positions that normally encode large hydrophobic amino acids to minimize perturbations of protein structure and function. Finally, when choosing an expression vector keep in mind that overproducing a protein of interest at a very high level might limit the efficiency of Bpa incorporation.</li>
</ol>
2. Culture Preparation
<ol>
	<li>Use electroporation to transform an appropriate E. coli strain with a plasmid that encodes the amber suppression system (pDULE-pBpa; see refrence4) and a compatible plasmid that encodes the protein of interest with an amber mutation.</li>
	<li>Plate cells on LB agar containing appropriate antibiotics (use 5 &#181;g/ml tetracycline to select for pDULE-pBpa). Incubate the plates O/N at 37&#160;&#176;C.</li>
	<li>On the following day, start O/N cultures from single colonies in 3-5 ml M9 medium14 containing 0.2% glycerol, all of the L-amino acids except methionine and cysteine (40 &#181;g/ml) and antibiotics. Incubate at 37&#160;&#176;C.</li>
	<li>On the next day, centrifuge O/N cultures at 2,500 x g for 5 min at RT to pellet cells. Resuspend the cells in 5 ml fresh M9 medium and repeat the centrifugation. 
	NOTE: This wash step is critical to maintain plasmids that encode ampicillin resistance because small amounts of b lactamase that are present in the medium from O/N cultures degrade freshly added ampicillin before significant cell growth occurs.</li>
	<li>Resuspend the cells in 2 ml fresh medium and measure cell concentration in a spectrophotometer (OD550).</li>
	<li>Add cells to a 250 ml Erlenmeyer flask containing 50 ml M9 medium and antibiotics at OD550=0.03. Incubate the cultures at 37 &#176;C in a shaking water bath.</li>
</ol>
3. Bpa Incorporation and Preparation for Photocrosslinking
NOTE: The protocol described below is for a sample time course that has three time points (0, 1, and 5 min). Adjust the volumes accordingly if a different number of time points is desired. An outline of the procedure is illustrated in Figure&#160;1.
<ol>
	<li>While the cultures are growing, prepare a fresh 1 M (1,000x) solution of Bpa in 1 M NaOH (27 mg Bpa in 100 &#956;l 1 M NaOH). Use a dark walled tube to keep the solution in the dark.</li>
	<li>When the cultures reach early to mid-log phase(OD550=0.2), add 50 &#956;l 1 M Bpa to each culture. Add Bpa one drop at a time while swirling the flask to avoid precipitation. Next, add any inducers that are needed to drive expression of the amber mutant. Continue to incubate the cultures at 37 &#176;C for an additional 30 min.</li>
	<li>During the 30 min induction period label six disposable 15 ml centrifuge tubes with the time point (0 min, 1 min, 5 min) and either &#34;+ UV&#34; or &#34;- UV&#34;. Place the tubes on ice.</li>
	<li>Fill a second ice bucket with ice and place a 6-well tissue culture plate on top of the ice.</li>
	<li>Thaw high specific activity (1,000 mCi/mmol)&#160;35S-methionine and 35S-cysteine (or a premade mixture of the two compounds) for the pulse labeling and prepare (or thaw) a solution of nonradioactive L-cysteine and L-methionine (100 mM each) for the chase.</li>
	<li>Turn on the UV lamp five min&#160;prior to radiolabeling. 
	NOTE: The lamp should be adjustable so that it can be positioned ~3-4 cm from the samples in the multiwell plate.</li>
	<li>Add ice (~2 ml) to each tube labeled &#34;- UV&#34; and to two of the wells in the multiwell plate. 
	NOTE: Ice should be added to a third well immediately before the 5 min&#160;time point. It is essential to place ice directly into the tubes and wells to stop intracellular reactions immediately at each time point and to thereby obtain accurate &#34;snapshots&#34; of a specific biochemical pathway.</li>
	<li>At the end of the 30 min&#160;induction period transfer 25 ml of the culture to a prewarmed 125 ml disposable Erlenmeyer flask. Place the flask in the 37 &#176;C shaking water bath.</li>
</ol>
4. Pulse-chase Labeling and UV Irradiation
NOTE: The pulse-chase labeling and UV irradiation protocol has a lot of steps that must be performed in a timely manner and that require considerable organization and advance preparation. All of the reagents and tubes should be readily accessible. A protocol for an experiment that has 0, 1,&#160;and 5 min&#160;time points is presented below.
<ol>
	<li>At time 0:00 (min:sec) transfer 25 ml of culture into 125 ml flask.</li>
	<li>At time 0:30 add 30 &#181;Ci/ml radioactive amino acids; swirl quickly to mix. Place the flask into the 37&#160;&#176;C shaking water bath.</li>
	<li>At time 1:00 add 250 &#181;l of the 100 mM methionine-cysteine solution; swirl quickly to mix.
	<ol>
		<li>Immediately pipette 4 ml of the culture into one of the wells of the chilled multiwell plate that contains ice. Pipette a second 4 ml aliquot into the 15 ml tube labeled &#34;0 min - UV&#34;. Return the flask to the water bath.</li>
	</ol>
	</li>
	<li>At time 2:00 pipette 4 ml of the culture into the second well of the chilled multiwell plate that contains ice. Pipette another 4 ml aliquot into the 15 ml tube labeled &#34;1 min - UV&#34;.&#160;Return the flask to the water bath.</li>
	<li>At time 6:00 pipette 4 ml of the culture into the third well of the chilled multiwell plate that contains ice. Pipette another 4 ml aliquot into the 15 ml tube labeled &#34;5 min - UV&#34;.</li>
	<li>Place the ice bucket with the multiwell plate under the preheated UV lamp and irradiate each sample individually for 4 min. 
	NOTE: In experiments in which time points are spaced at least 4 min&#160;apart each sample should be irradiated as soon as it is pipetted into the multiwell plate.</li>
	<li>Transfer the cells into the chilled 15 ml tubes labeled &#34;0 min +UV&#34;, 1 min +UV&#34;, etc.</li>
	<li>Centrifuge cells at 2,500 x g for 10 min&#160;at 4&#160;&#176;C.</li>
	<li>Resuspend each sample in 300 &#181;l M9 medium or PBS and add 33 &#181;l 100% cold trichloroacetic acid (TCA) to precipitate proteins. Centrifuge the TCA precipitates in a microfuge at top speed for 10 min&#160;and remove the supernatant. At this point samples can be stored at - 20&#160;&#176;C.</li>
</ol>
5. Immunoprecipitation and Crosslinking Product Detection
<ol>
	<li>Add 50 &#181;l solubilization buffer (15% glycerol, 200 mM Trisbase, 15 mM EDTA, 4% SDS, 2 mM PMSF) to each sample and heat with agitation at 95&#160;&#176;C for 5 min&#160;to solubilize precipitated protein.</li>
	<li>Add 1 ml radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1.0% Igepal CA-630, 0.5% deoxycholate, 0.1% SDS) and perform standard immunoprecipitations15 using one-fifth to one-half of each sample. 
	NOTE: Igepal CA-630 (octylphenyl-polyethylene glycol) is used in place of NP-40, which is no longer commercially available. The two compounds are chemically indistinguishable.</li>
	<li>Resolve immunoprecipitated proteins by SDS-PAGE. Stain, destain,&#160;and dry gels.</li>
	<li>Expose gels to a phosphor screen O/N and detect radioactive proteins (including crosslinking products) with a phosphorimager.</li>
</ol>

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	<li>Wang, L., Xie, J., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+code.">Expanding the genetic code.</a> Annu. Rev. Biophys. Biomol. Struct. 35, 225-249 (2006).</li><li>von Mering, C., 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=Comparative+assessment+of+large-scale+data+sets+of+protein-protein+interactions.">Comparative assessment of large-scale data sets of protein-protein interactions.</a> Nature. 417, 399-403 (2002).</li><li>Chin, J. W., Martin, A. B., King, D. S., Wang, L., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+a+photocrosslinking+amino+acid+to+the+genetic+code+of+Escherichia+coli.">Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli.</a> PNAS. 99, 11020-11024 (2002).</li><li>Farrell, I. S., Toroney, R., Hazen, J. L., Mehr, R. A., Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-cross-linking+interacting+proteins+with+a+genetically+encoded+benzophenone.">Photo-cross-linking interacting proteins with a genetically encoded benzophenone.</a> Nat. Methods. 2 (5), 377-384 (2005).</li><li>Wittelsberger, A., Mierke, D. F., Rosenblatt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+ligand-receptor+interfaces:+approaching+the+resolution+limit+of+benzophenone-based+photaffinity+scanning.">Mapping ligand-receptor interfaces: approaching the resolution limit of benzophenone-based photaffinity scanning.</a> Chem. Biol. Drug Des. 71, 380-383 (2008).</li><li>Ieva, R., Tian, P., Peterson, J. H., Bernstein, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+and+spatially+restricted+interactions+of+assembly+factors+with+an+autotransporter+beta+domain.">Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain.</a> PNAS. 108, 383-391 (2011).</li><li>Wang, F., Robbins, S., Guo, J., Shen, W., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+incorporation+of+unnatural+amino+acids+into+proteins+in+Mycobacterium+tuberculosis.">Genetic incorporation of unnatural amino acids into proteins in Mycobacterium tuberculosis.</a> PLoS One. 5, (2010).</li><li>Deiters, 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=Adding+amino+acids+with+novel+reactivity+to+the+genetic+code+of+Saccharyomycescerevisiae.">Adding amino acids with novel reactivity to the genetic code of Saccharyomycescerevisiae.</a> J. Am. Chem. Soc. 125, 11782-11783 (2003).</li><li>Young, T. S., Ahmad, I., Brock, A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+repertoire+of+the+methylotrophic+yeast+Pichiapastoris.">Expanding the genetic repertoire of the methylotrophic yeast Pichiapastoris.</a> Biochemistry. 48 (12), 2643-2653 (2009).</li><li>Hino, N., 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=Protein+photo-cross-linking+in+mammalian+cells+by+site-specific+incorporation+of+a+photoreactive+amino+acid.">Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid.</a> Nat. Methods. 2 (3), 201-206 (2005).</li><li>Liu, W., Brock, A., Chen, S., Chen, S., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+incorporation+of+unnatural+amino+acids+into+proteins+in+mammalian+cells.">Genetic incorporation of unnatural amino acids into proteins in mammalian cells.</a> Nat. Methods. 4 (3), 239-244 (2007).</li><li>Leyton, D. L., Rossiter, A. E., Henderson, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+self+sufficiency+to+dependence:+mechanisms+and+factors+important+for+autotransporter+biogenesis.">From self sufficiency to dependence: mechanisms and factors important for autotransporter biogenesis.</a> Nat. Rev. Microbiol. 10 (3), 213-225 (2012).</li><li>Ieva, R., Bernstein, H. 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The authors declare that they have no competing financial interests.

This paper describes a method that combines site-specific photocrosslinking with pulse-chase labeling to examine the dynamics of interactions between a protein of interest and other components of a living cell. The method is especially useful to study multistep processes in which the protein interacts sequentially with other factors. Unlike traditional chemical cross-linking approaches, site-specific photocrosslinking provides detailed information about the status of individual segments or even individual residues of a p...

It is often essential to identify and characterize interactions between a protein of interest and other cellular components to define its role in a biological pathway, to understand its assembly, or to elucidate its mechanism of action. A wide variety of approaches are commonly used to study protein-protein interactions, but all have their limitations. The simplest unbiased method to identify proteins that bind to a protein of interest is copurification (or coimmunoprecipitation) but this approach requires that a protein complex remain intact during the purification procedure. Weak or transient protein interactions can be stabilized through chemical cross-linking, but this method typically requires the use of compounds that link proteins through primary amines that are widely scattered in the protein sequence. Complicated cross-linking patterns that are difficult to interpret can be generated, especially when proteins of interest are components of multiprotein complexes. Furthermore, because the spacer arms of chemical cross-linkers generally exceed 10 &#197; in length, covalent bonds can be formed with proteins that are located in proximity to but do not interact directly with the protein of interest. Methods such as affinity chromatography and two-hybrid screens are also often useful to identify protein-protein interactions. The former approach, however, requires reproducing conditions that promote physiologically significant interactions and cannot be used to detect weak interactions. The latter approach requires that binding interactions can be replicated in the host organism and are maintained when a protein of interest and its binding partners are placed in the context of a fusion protein. Two-hybrid methods also tend to generate false-positive results2. Once a protein-protein interaction has been established, considerable work is often required to map the site of interaction. Perhaps the most significant drawback of traditional approaches is that they do not provide any information about the temporal sequence of intermolecular interactions or the kinetics of binding.
This paper describes a simple method that overcomes some of the limitations of other approaches that are used to study protein-protein interactions. Initially a single amber mutation is introduced into the gene that encodes a protein of interest. The amber mutant is then coexpressed with an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii that have been engineered to incorporate the photoactivatable amino acid analog p-benzoylphenylalanine (Bpa) only at amber codons3. Irradiation of cells with long wavelength ultraviolet (UV) light (~365 nm) then facilitates formation of a covalent bond between Bpa and proteins that are within ~3-8 &#197;4,5. In some experimental contexts, cross-links can also be formed between activated Bpa and nonprotein components of cells such as lipids and nucleic acids6. Molecules that are terminated at the amber codon should generally be easily distinguishable from the full-length protein on SDS-PAGE and cannot be cross-linked to other proteins. By subjecting cells to pulse-chase radiolabeling prior to cross-linking and then immunoprecipitating or affinity purifying cross-linking products, the sequence and duration of protein-protein interactions can be established. The ability to generate temporal information about intermolecular interactions is especially valuable to study the progression of a protein of interest through any ordered multistep assembly, trafficking or signaling pathway and to identify pathway intermediates. Like chemical cross-linking, site-specific cross-linking detects protein-protein interactions that occur under physiological conditions, but the introduction of a cross-linker at a single position facilitates the analysis of interactions between individual segments of a protein and other factors. This unique feature of site-specific cross-linking is especially advantageous when the protein of interest has multiple segments or domains that have the potential to interact with distinct binding partners. Furthermore, site-specific cross-linking can be used in combination with methods such as mass spectrometry to map the residues that mediate protein-protein interactions with precision. Although this method is optimized for use in E. coli, the incorporation of photactivatable amino acid analogs into proteins by amber suppression has been reported in Mycobacterium, yeast and mammalian cells7-11. Thus, in principle our method can be used in other systems.
We have used this method extensively to identify intermolecular interactions that facilitate the biogenesis of EspP, an E. coli O157:H7 virulence factor. EspP is a member of a superfamily of proteins known as &#34;autotransporters&#34; that contain a large N-terminal extracellular domain (&#34;passenger domain&#34;) and aC-terminal domain (&#34;b domain&#34;) that folds into a cylindrical b barrel structure and anchors the protein to the outer membrane (OM)12. The mechanism by which the passenger domain is translocated across the OM has been a long-standing mystery. Like all bacterial cell surface proteins, EspP must be transported across the inner membrane, shuttled across the periplasm (the space between the inner and outer membranes), and targeted to the OM in a series of ordered events. Following its translocation across the OM, the EspP passenger domain is also separated from the b domain by a proteolytic cleavage and released from the cell surface. By combining site-specific cross-linking with pulse-chase radiolabeling, we have shown that both domains of the protein initially interact with the molecular chaperone Skp in the periplasm6. Subsequently, discrete segments of the b domain interact in a stereospecific fashion with components of a heterooligomer called the Bam complex, which catalyzes the integration of b barrel proteins into the bacterial OM by an unknown mechanism. Perhaps most interestingly, we have found that the passenger domain is in contact with one of the Bam complex subunits (BamA) as it traverses the OM13. Our results have enabled us to construct a detailed model for autotransporter assembly14 and have implicated the Bam complex in the transport of the passenger domain across the OM.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QuikChange II Site-Directed Mutagenesis Kit</td>
			<td style="width:71px;">Agilent</td>
			<td align="right" style="width:119px;">200521</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BPA (H-p-Bz-Phe-OH)</td>
			<td style="width:71px;">Bachem</td>
			<td style="width:119px;">F-2800</td>
			<td></td>
		</tr>
		<tr height="93">
			<td height="93" style="height:93px;width:216px;">TRAN35S-LABEL, Metabolic Labeling Reagent (35S-L-methionine and 35S-L-cysteine, &#62;1,000 Ci/mmol)</td>
			<td style="width:71px;">MP Biomedicals</td>
			<td align="right" style="width:119px;">51006</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Spectroline SB-100P Super-High-Intensity UV Lamp, 365 nm</td>
			<td style="width:71px;">Spectronics Corporation</td>
			<td style="width:119px;">SB-110P</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Spectroline Replacement Bulb 100S</td>
			<td style="width:71px;">Spectronics Corporation</td>
			<td style="width:119px;">11-992-15</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Falcon Tissue Culture Plate, 6-well</td>
			<td style="width:71px;">Becton Dickinson Labware</td>
			<td align="right" style="width:119px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable 125 ml Erlenmeyer flask</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">430421</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Innova 3100 shaking water bath</td>
			<td style="width:71px;">New Brunswick Scientific</td>
			<td style="width:119px;">n.a.</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We used site-specific photocrosslinking to identify proteins that interact with EspP during its journey to the OM. In the experiments that are shown here, phenylalanine codons at residues 1113 and 1214 were replaced with amber codons. Both positions are located in the bdomain, which integrates into the OM and serves as a membrane anchor. The crystal structure of the EspP&#946; domain16 shows that the two residues are on the periplasmic side of the b barrel but are separated by ~120&#176; (see ...

Watch this Scientific Journal Video about Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking at JoVE.com

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

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 ...

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6.1K Views.  National Institutes of Health. 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 “snapshots” of an ordered assembly pathway in a living cell.