Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Summary

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

Abstract

A transcription factor (TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two σ factors, namely σ⁶⁶ and σ²⁸, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Introduction

Transcription, which produces RNA molecules using DNA as template, is the very first step of gene expression. Bacterial RNA synthesis begins following the binding of the RNA polymerase (RNAP) holoenzyme to a target promoter^1,2. The RNAP holoenzyme (RNAPholo) is comprised of a multi-subunit catalytic core (RNAPcore) and a σ factor, which is required for recognizing the promoter sequence. Transcription activators and repressors, collectively termed TFs, regulate the gene expression through the binding components of the RNAPcore, σ factors, and/or DNA. Depending on the organism, a significant portion of its genome may be devoted to TFs that regulate transcription in response to physiological needs and environmental cues³.

Chlamydia is an obligate intracellular bacterium responsible for a variety of diseases in humans and animals^4,5,6,7,8. For example, Chlamydia trachomatis is arguably the number one sexually transmitted pathogen in humans worldwide, and a leading cause of blindness in some underdeveloped countries^4,5. Chlamydia has a unique developmental cycle characterized by two alternating cellular forms termed the elementary body (EB) and reticulate body (RB)⁹. Whereas, EBs are capable of survival in an extracellular environment, they are incapable of proliferation. EBs enter host cells through endocytosis and differentiate into larger RBs in a vacuole in the host cytoplasm within hours post-inoculation. No longer infectious, RBs proliferate through binary fission. Around 20 h, they start to differentiate back to the EBs, which exit the host cells around 30-70 h.

Progression of the chlamydial developmental cycle is regulated by transcription. Whereas a supermajority of the nearly 1,000 chlamydial genes are expressed during the midcycle during which RBs are actively replicating, only a small number of genes are transcribed immediately after the entry of EBs into the host cells to initiate the conversion of EBs into RBs, and another small set of genes are transcribed or increasingly transcribed to enable the differentiation of RBs into EBs^10,11.

The chlamydial genome encodes three σ factors, namely σ⁶⁶, σ²⁸ and σ⁵⁴. σ⁶⁶, which is equivalent to the housekeeping σ⁷⁰ of E. coli and other bacteria, is responsible for recognizing promoters of early and mid-cycle genes as well as some late genes, whereas σ²⁸ and σ⁵⁴ are required for the transcription of certain late genes. Several genes are known to carry both a σ⁶⁶-dependent promoter and a σ²⁸-dependent promoter¹².

Despite a complicated developmental cycle, only a small number of TFs have been found in chlamydiae¹³. GrgA (previously annotated as a hypothetical protein CT504 in C. trachomatis serovar D and CTL0766 in C. trachomatis L2) is a Chlamydia-specific TF initially recognized as an activator of σ⁶⁶-dependent genes¹⁴. Affinity pulldown assays have demonstrated that GrgA activates their transcription by binding both σ⁶⁶ and DNA. Interestingly, it was later found with that GrgA also co-precipitates with σ²⁸, and activates transcription from σ²⁸-dependent promoters in vitro¹⁵. To investigate whether GrgA has similar or different affinities for σ⁶⁶ and σ²⁸, we resorted to using BLI. BLI assays have shown that GrgA interacts with σ⁶⁶ at a 30-fold higher affinity than with σ²⁸, suggesting that GrgA may play differential roles in σ⁶⁶-dependent transcription and σ²⁸-dependent transcription¹⁵.

BLI detects the interference pattern of white light that reflects from a layer of immobilized protein on the tip of a biosensor and compares it to that of an internal reference layer¹⁶. Through the analysis of these two interference patterns, BLI can provide valuable and real-time information about the amount of protein bound to the tip of the biosensor. The protein that is immobilized to the tip of the biosensor is referred to as the ligand, and is generally immobilized with the help of a common antibody or epitope tag (e.g., a poly-His- or biotin-tag) that has an affinity for an associated particle (such as NTA or Streptavidin) on the tip of the biosensor. The binding of a secondary protein, referred to as the analyte, with the ligand at the tip of the biosensor creates changes in the opacity of the biosensor and therefore results in changes in interference patterns. When repeated over different concentrations of the analyte, BLI can provide not only qualitative but also quantitative information about the affinity between the ligand and analyte¹⁶.

To the best of our knowledge, we were the first to employ BLI to characterize protein-protein interactions in transcription¹⁵. In this publication, we demonstrate that a GrgA fragment, which was previously shown to be required for σ²⁸-binding, indeed mediates the binding. This manuscript focuses on steps of the BLI assays, and generation of BLI graphs and parameters of binding kinetics. Methods for the production (and purification) of ligands and analytes are not covered here.

Protocol

1. Preparation of proteins

1. Use a dialysis bag (with an appropriate cut-off size) to dialyze each protein to be used for BLI assays (including both the His-tagged ligand and the analyte) against 1,000 volumes of the BLI buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, 10 mM MgCl₂, 0.1 mM DTT, pH 8.0, pre-chilled to 4 ˚C) at 4 ˚C for 4 h.
   NOTE: BLI assays require the ligand to be present at concentrations that saturate the binding sites on the biosensor and the analyte to be highly purified so that the molar concentrations of the analyte that react with the ligand is known. Methodologies for the expression and purification of His- and Strep-tagged proteins are not covered here, but can be found in previous publications^14,15. Although this system does not require the ligand to be in highly purified form, it is essential to dialyze even unpurified ligands to the BLI buffer in order to minimize shifts in white-light interference patterns caused by any buffer changes during the assay.
2. Switch to fresh BLI buffer and continue the dialysis for another 4 h.

2. Biosensor hydration and assay set-up

1. Approximately 10 min prior to the start of an assay, pipette 200 µL of the BLI buffer into a PCR tube.
2. Remove a Ni-NTA-biosensor from the original packaging by holding the wide portion of the biosensor using a gloved hand.
3. Place the biosensor over the PCR-tube such that only the glass tip of the biosensor is submerged in the BLI buffer.
4. Keep the biosensor tip submerged for at least 10 min to ensure full hydration.
   1. Verify that the glass tip of the Ni-NTA-biosensor does not touch anything other than the BLI buffer during the above step.
      NOTE: This protocol uses a Ni-NTA-biosensor in conjunction with a His-tagged ligand. If needed, a SA-Streptavidin-biosensor can be used in conjunction with a biotinylated ligand instead if: (i) both the ligand and analyte carry a His tag or (ii) neither of them does.
5. Turn the BLItz machine on.
6. Ensure that the machine is connected to the computer through a USB data output port at the back of the machine.
7. On the computer, open the associated software (e.g., BLItz Pro), and click on Advanced Kinetics on the left-hand side of the screen.
8. On the software, type out all appropriate information about the experiment (including the Experiment Name, Description, Sample ID, and Protein Concentration) under each respective heading.
9. Click on Biosensor Type and choose Ni-NTA from the drop-down menu.
   1. Under the Run Settings heading, verify that the Shaker is set to Enable.
   2. Under the Step Type List heading, verify that there are 5 items listed: Initial Baseline, Loading, Baseline, Association, and Dissociation.
      NOTE: The duration of each step can be changed from default as needed. For optimal results, use a minimum of 30 s for Initial Baseline and Baseline; and 120 s for Association and Dissociation. The duration of the Loading step (ranging from 120 to 240 s) will depend upon the concentration of the ligand and affinity of the His-epitope tag on the ligand to the Ni-NTA-biosensor.
10. Remove the hydrated Ni-NTA-biosensor from the PCR tube and affix it to the biosensor mount on the machine by sliding the wide portion of the biosensor onto the mount.
    NOTE: Do not let the biosensor dry out during the experiment.
11. Place a 0.5 mL black microcentrifuge tube into the tube holder of the machine and pipette 400 µL of the BLI buffer into it.
12. Verify that the slider of the machine is positioned such that the tube holder is situated in front of the black arrow on the machine.
13. Close the cover of the machine such that the biosensor tip becomes submerged in the buffer in the microcentrifuge tube.
14. Click Next on the software to begin recording the Initial Baseline.

3. Loading of ligand onto biosensor

1. After the Initial Baseline step has finished recording, open the cover of the machine.
2. Move the slider to the right such that the drop holder (instead of the tube holder) is situated in front of the black arrow.
3. Pipette 4 µL of a dialyzed His-tagged ligand (from Step 1.1) onto the drop holder and close the cover of the machine.
   NOTE: The optimal concentration of the ligand to be used may vary for each protein. A concentration between 1.0 to 2.0 mg/mL is usually adequate to saturate the NTA at the tip of the biosensor in 240 s.
4. On the software, click Next to begin Loading.

4. Washing away additional ligand

1. After the Loading step has finished recording, open the cover of the machine.
2. Move the slider to the left such that the tube holder is once again situated in front of the black arrow.
3. Close the lid of the machine and ensure that the biosensor tip is submerged into the BLI buffer of the tube in the tube holder.
4. Click Next once again on the software to begin recording the Baseline.

5. Association of analyte to ligand

1. After the Baseline step has finished recording, open the cover of the machine.
2. Remove the drop holder and clean it by pipetting out any protein and rinsing it with double-deionized water (ddH₂O) for a total of 5 times.
   1. Use a tissue wipe to clean the surface of the drop holder after the wash.
3. Replace the drop holder back onto the machine.
4. Move the slider on the machine to the right such that the drop holder is once again situated in front of the black arrow.
5. Pipette 4 µL of a dialyzed analyte (from Step 1.1) onto the drop holder and close the cover of the machine.
6. On the software, click Next to begin Association.

6. Dissociation of analyte from ligand

1. After the Association step has finished recording, open the cover of the machine.
2. Move the slider on the machine to the right such that the tube holder is once again situated in front of the black arrow.
3. On the software, click Next to begin Dissociation.
4. After the Dissociation step has finished recording, open the cover of the machine.
5. Remove the drop holder and tube holder.
6. Rinse both with ddH₂O thoroughly to wash away any protein.
7. Remove the biosensor and discard it safely.

7. Repeating interactions with different concentrations

1. Repeat Steps 2-7 for the same ligand-analyte pair using different analyte concentrations.
   NOTE: The concentration of the analyte may need to be adjusted across several runs before obtaining optimal results. In our experience, a ratio of 1:5:10 of analyte concentrations, starting with 75 nM, is usually adequate.

8. Analyzing the data using the software

1. Once all runs have finished, save the data on the software by clicking File and then Save Experiment As on the left side of the screen.
2. Under the Run Data heading, select Step Correction and Fitting (1:1) and click Analyze to generate kinetic data.
3. To extract the quantitative data into a worksheet and generate graphs, click on Export to CSV and save the recorded data as a .csv file. Open the .csv file using spreadsheet software.
   1. To most effectively show the Association and Dissociation kinetics, remove all plot points prior to the Baseline step, and normalize all subsequent plot points from the final Baseline value.

Results

Through BLI assays, we previously established that binding of GrgA to σ²⁸ is dependent on a 28 amino acid middle region (residues 138-165) of GrgA¹⁵. Accordingly, compared with N-terminally His-tagged full length GrgA (NH-GrgA), a GrgA deletion construct lacking this region (NH-GrgAΔ138-165) had a decreased association rate and an increased dissociation rate, leading to a 3 million-fold loss of overall affinity (Table 1). Here, we demonstrate that this middle region dir...

Discussion

Protein-protein interactions are crucial for the regulation of transcription and other biological processes. They are most commonly studied through pulldown assays. Although pulldown assays are relatively easy to perform, they are poorly quantitative and may fail to detect weak but biologically meaningful interactions. In comparison, by detecting real-time association and dissociation between a ligand and an analyte, BLI provides association and dissociation rate constants, as well as, overall affinity.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BLItz machine	ForteBio	45-5000
Dialysis tubing cellulose membrane	MilliporeSigma	D9652
Dip and Read Ni-NTA biosensor tray	ForteBio	18-5101	Ready-to-use Ni-NTA biosensors for poly-His-tagged Proteins
Drop holder	ForteBio	45-5004
PCR tubes (0.2 mL)	Thomas Scientific	CLS6571
Microcentrifuge tubes (black)	Thermo Fisher Scientific	03-391-166
Kimwipes	Thermo Fisher Scientific	06-666A
DTT	Thermo Fisher Scientific	R0861
EDTA	MilliporeSigma	E6758
MgCl2	MilliporeSigma	M8266
NaCl	MilliporeSigma	S9888
Tris-HCl	GoldBio	T095100