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

The overall goal of the following experiment is to examine the dynamics of protein protein interactions inside a living cell. This is achieved by first expressing a protein of interest in e coli, incorporating the amino acid analog benzoyl phenyl alanine into the protein at a specific location and performing pulse chase radio labeling to tag a small population of synchronously synthesized protein molecules. As a second step.

Aliquots of cells collected at various time points during the chase are ve irradiated to trigger the formation of covalent bonds between the protein of interest and any factors that it is interacting with. Next proteins are immuno precipitated with specific antibodies and resolved by SDS poly acrylamide gel electrophoresis in order to identify interacting factors, results are obtained that show changes in the interactions between the protein of interest and other intracellular factors over time based on changes in the electrophoretic mobility of cross-linking products. The main advantage of this technique over existing methods, such as co immunoprecipitation or chemical cross-linking, is its ability to generate temporal information about interactions that occur between a specific residue in a protein of interest and other molecules.

Such data is especially valuable when studying a progression of a protein through a multi-step assembly pathway and to identify assembly intermediates. Though this method has been optimized for use in e coli, it can also be used in other systems, including other bacteria, yeast, and mammalian cells. Details for culture preparation can be found in the text protocol.

Briefly prepare five milliliters of M nine medium containing 0.2%glycerol all of the el amino acids except methionine and cystine and antibiotics begin overnight cultures by adding e coli transformed with a plasmid that encodes for the protein of interest with an amber mutation and the plasmid that encodes for the amber suppression system incubate at 37 degrees Celsius. On the day of the experiment, add an appropriate amount of cells from the overnight culture to a 250 milliliter erlenmeyer flask containing 50 milliliters of M nine medium and antibiotics to obtain an optical density at 550 nanometers of 0.03. Incubate the cultures at 37 degrees Celsius in a shaking water bath.

When the cultures reach early to mid log phase, add 50 microliters of one molar benzoyl phenylalanine or 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 degrees Celsius for an additional 30 minutes during the 30 minute induction period. Label six disposable 15 milliliter centrifuge tubes with the time point and either plus UV or minus uv. Place the labeled tubes on ice.

Place a six well tissue culture plate on top of a second ice bucket filled with ice. Turn on the UV lamp five minutes prior to radio labeling. Then add approximately two milliliters of ice to each tube labeled minus UV and to two of the wells in the multi-well plate.

It is essential to air ice directly into the tubes and wells to stop intracellular reactions immediately at each time point and to thereby obtain an accurate snapshot of the biochemical pathway At the end of the 30 minute induction period. Transfer 25 milliliters of the culture to a pre warmed 125 milliliter disposable erlenmeyer flask. Place the flask in the 37 degrees Celsius shaking water bath.

The steps in pulse chase labeling and UV irradiation must be performed in a timely manner and require considerable organization and advanced preparation. Add 30 micro curies per milliliter of S 35 labeled methionine and cysteine to the culture and swirl quickly to mix at time. One minute zero seconds.

Add 250 microliters of a non-radioactive 100 millimolar methionine cysteine solution and swirl quickly. To mix immediately pipette four milliliters of the culture into one of the wells of the chilled multi-well plate that contains ice pipette a second four milliliter aliquot into the 15 milliliter tube labeled zero minutes minus UV at time. Two minutes zero seconds.

Pipette four milliliters of the culture into a second well of the chilled multi-well plate that contains ice. Then pipette another four milliliter aliquot into the 15 milliliter tube labeled one minute minus UV immediately before the five minute time point at approximately two milliliters of ice to a third well in the multi-well plate at times six minutes zero seconds. Pipette four milliliters of the culture into the third well of the chilled multi-well plate that contains ice pipe.

Had another four milliliter aliquot into the 15 milliliter tube labeled five minutes minus uv. Placed the ice bucket with the multi-well plate under the preheated UV lamp and irradiate each sample individually for four minutes. Transfer the cells into the chilled 15 milliliter tubes labeled zero minutes plus uv, one minutes plus UV and so forth.

Then centrifuge cells at 2, 500 times G for 10 minutes at four degrees Celsius. Resuspend each sample in 300 microliters of M nine medium or PBS and 33 microliters of 100%cold trichloroacetic acid or TCA to precipitate the proteins centrifuge the TCA precipitates in a micro fuge at top speed for 10 minutes. Before removing the sate, add 50 microliters of solubilization buffer to each sample and heat with agitation at 95 degrees Celsius for five minutes to solubilize the precipitated protein.

After adding one milliliter of radio immunoprecipitation assay buffer performs standard immuno precipitations using one fifth to one half of each sample. Resolve the immuno precipitated proteins by sodium ESAL sulfate, poly acrylamide gel electrophoresis or SDS page. Finally, expose the stained and dried gels to a phospho screen overnight and to detect radioactive proteins including cross-linking products with a phospho imager site-Specific photo cross linking was used to identify proteins that interact with ESPP during its journey to the outer membrane.

For this experiment, pH alanine codons at residues 1113 and 1214 of the ESPP beta domain were replaced with Amber codons. The crystal structure of the ESPP beta domain shows that the two residues are both on the peri plasmic side of the beta barrel, separated by approximately 120 degrees. Two different polypeptides were immuno precipitated from both irradiated and control cells by the C terminal anti ESPP and serum.

Initially, an approximately 135 kilodalton precursor form of the protein that contains covalently linked passenger and beta domains predominated at later time points. The level of PRO ESPP declined and the free beta domain began to predominate as the passenger domain was trans located across the outer membrane and separated from the beta domain by a proteolytic cleavage. The UV I radiated samples, however clearly had higher molecular weight bands that were not present in the control samples.

These bands result from the cross-linking of PRO ESPP to interacting proteins based on the mobility of these polypeptides, the sizes of the interacting proteins were estimated to test whether they might correspond to known outer membrane protein assembly factors. Additional immuno precipitations using anti cera generated against the putative interacting partners were performed. An approximate 150 kilodalton polypeptide that was observed when BPA was incorporated at both residues 1113 and 1214 could be immuno precipitated with an antiserum against the 17 kilodalton peri plasmic chaperone skip.

Furthermore, we found that larger polypeptides that were observed when BPA was incorporated at only one of the two positions could be immuno precipitated with anti cera against the BAM complex subunits, BAM B and BAM D respectively. After performing this procedure, cross-linking products can be purified and other methods including mass spectrometry can be performed to help identify factors that interact with a protein of interest. After watching with video, you should have a good understanding of how to successfully combine pulse chase labeling with site specific photo cross linking to study the dynamics of interaction of your protein, of interest with other molecules inside the living cell.