SSB Wrapping Kinetics and Binding Mode Determination

11/6/2015

Students tested whether their SSB proteins wrapped DNA and if they have the two DNA binding modes similar to E.coli SSB. Experiments were done using a stop flow instrument where 1 uM SSB was rapidly mixed with 20 nM DNA containing a Cy3-Cy5 FRET pair. Wrapping = high FRET, formation of the differential binding modes is determined if there is a loss in the FRET signal.

One SSB protein from Bacillus anthracis does not the two binding modes!!

6 Comments

Madyson Riddell

11/17/2015 02:28:56 pm

B. anthracis does not copy the same binding mode that E.coli does! This is very cool. In my research there have been hypothesis about a monomeric SSB for B.A. instead of tetrameric like in E. coli. I think this is a very important difference and a potential target for antibiotics! Also is there any potential for the salt ions interfering with the fluorescent tags?

Dr. Antony

11/23/2015 05:37:02 am

Madyaon,
Indeed, the substitution observed in Ba compared to Ec-SSB lies at the subunit-subunit interface and known to affect tetramerization.

Sure, Salt can affect the fluorescent tags. You will have to follow changes in signal intensity as a function of salt concentration to study such effects. Here is an example of one such study on Cy3 fluorescence: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1265978/

Phil McFarland

11/19/2015 04:53:34 pm

I enjoyed this experiment because it gave us the ability to illustrate the dynamic binding modes of SSB. Using FRET to measure intermolecular tag distance on ssDNA is such a sophisticatedly simple method of determining binding orientation within SSB.
I did have a question regarding the determinants of SSB binding modes. I have read a few papers that show a correlation between binding mode transitions relative to salinity, protein-DNA ratio, pH, and C-terminus interaction. Is there a particular hierarchy among these variables to SSB orientation. In other words, what variable has the most influence on the binding mode state of SSB. Additionally, I am curious as to the cellular implications of this dynamic binding orientation. What is occurring within the cell to lead SSB to alter its interaction with ssDNA. I know you spoke briefly in class that a particular orientation is favorable during repair vs replication but I was hoping you could provide a little more detail.
As I look at my data for S. typhi, I am recognizing that the high salt FRET signal displays a slight dip after the initial spike in high FRET signal. This decrease in signal is subtle and significantly smaller than the low salt FRET signal drop. Does this decrease in signal signify that some S. typhi SSB are transitioning into the 35 binding mode at high salt concentrations?

Dr. Antony

11/23/2015 05:29:46 am

Phil,
You make several interesting observations and these are complex.

1. [NaCl], [SSB], [MgCl2] changes promote transition between the binding modes. Base on our recent chimera work between Ec-SSB and Pf-SSB, we believe the cause of the binding modes to be the intrinsic structure of the intervening disordered loop and the interaction of the C-terminus with the DNA binding domain. This interaction is affected by the various factors leading to the various binding mode. Look up - http://www.ncbi.nlm.nih.gov/pubmed/25562210

2. In vivo, these binding modes are probably controlled through protein-protein interactions. Its an assumption as we have not proved this.

3. The S.typhi wrapping (number of bases) might be different that Ec-SSB. We have performed the experiment under the assumption that its 65-70 bases. If it were less for example, then the FRET pairs are sitting too close and flopping around and can bind to another tetramer etc - leading the complex changes in signal. One needs to calculating the binding site-size for quantitative interpretation of this experiment.

Nicholas Callard

12/4/2015 07:09:39 pm

Even though I wasn't in lab the day this experiment was conducted I was still very intrigued by the results that my group observed from the experiment. Our high salt condition only produced moderate FRET. While this was probably due to experimental error (lagging strand) there’s a possibility that the difference in Yersinia pestis's C-terminal tails and loops could be the cause of this difference in binding from E. coli. All the differentiations in the genomic sequence from E.coli were within the C-terminal tails. Knowing this might be a little optimistic, it still would be interesting to research further.

Tai Lu

12/7/2015 09:29:36 pm

This lab was very interesting in my opinion because with my friend Mike (who also has Yersinia Pestis), when we did this the outcome was not expected because the high salt condition only produced moderate FRET like what Nicholas Callard said. I also thought that it may have been an error as well during the preparation of the samples. Mike and I decided to retry the samples and when we got the results, it was still the same thing. That meant that we knew that it was not an experimental error. Overall I really enjoyed doing this lab. It gave us the ability to show the binding modes of SSB.

SSB Wrapping Kinetics and Binding Mode Determination

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