DTT, b-ME and TCEP are commonly used in biochemistry for the reduction of cysteins. Recently, I investiged the effect of the oxidative state of polymer-binding peptides on their binding behaviour. I realized that cystein-containing as well as peptides lacking reducable cysteins responded comparably to reducing agents. Now I'm wondering whether the reducing agents simply compete with the polymer for their binding partner by adhering to the peptides, or chemical modifications of the peptide apart from cysteine reduction give rise to changes in binding behaviour.
Dear Hendrik Pütz
If you can tell us more details about the method you used for measuring the binding, it will probably be much easier to answer you question.
Dear Tomasz Fraczyk ,
Thank you for taking your time to answer!
Excuse me for not being precise enough.
My peptides are fused to fluorescence markers and binding is analyzed as function of the fluorescence of "immobilized" fusion-protein on the polymer-surface via MTP reader. I initially hypothesized that the fluorescence marker itself was compromised by reduction, rather than the binding. Control experiments where I exposed the markers to the reducing agents, however, showed that the fluoresence was not quenched. This brings me to the conclusion that the binding itself must be affected even though peptide sulfides are not necessarily involved.
Many thanks,
Hendrik
Maybe the reducing agents cause de-labeling of the peptides? What chemistry do you use for attaching the fluorescent markers?
The reducing agents could be affecting the structure of the immobilized fusion protein receptor for the peptide if it contains disulfide binds.
Dear Tomasz Fraczyk,
that is a very valid point. However, I did not click the marker to the peptide, but expressed the peptides together with GFP as fusion protein, so I think the delabeling is not the reason for a decrease in binding signals.
Dear Adam B Shapiro,
in my assay, the peptide is immobilized on different polymer surfaces such as polyethylene, which exclusively comprise C-C bonds. This makes me think the only effect affecting the binding might be interactions of the reductant with non-cysteine residues on the peptide itself.
Many thanks for your help!
Hendrik
Dear Hendrik Pütz
As far as I know, GFP contains two Cys residues. You mentioned that the reducing agents does not influence the fluorescence of GFP alone. However, maybe they influence the conformation of GFP in a way that the attached peptides are more or less available for the interaction with polymer. Maybe you can try changing the fluorescent marker to test that possibility.
DTT, b-ME and TCEP have quite different structures, so if they influence the fluorescence in your assay similarly, there is rather a low probability that it is because of their interaction with the peptides.
Dear Hendrik Pütz
I wonder if your peptides can bind transition metals. If so, and these metal ions are also present in your sample, the reducing agents may change the oxidation state of the metal ions instead of peptides. That may also influence the interaction.
In native GFP the two Cys are not disulfide linked, and attempts at reduction typically do not affect fluorescence. I doubt that is your problem. However, I suspect you are not removing the reducing agent after protein reduction, and residual agent is interacting transiently with your probe, or perhaps GFP. Try a desalting step between reduction and binding.
While reducing agents do not form strong bonds with non-cysteine amino acid side chains, thiolate definitely can associate with imidazolinium near neutral pH. So residual reducer may bind to His in your protein, forcing it into a cationic state. Peptides that bind plastic may do so via pi interaction (Article Characterization of phage that bind plastic from phage-displ...
). Histidine is known to participate in pi interactions important for protein structure, and it can do so in both protonated and neutral forms.
Dear Tomasz Fraczyk ,
you made some very good points here. I also think that the structural dissimilarity between reducing agents makes it more likely that the reduction itself must result in lowered fluorescence rather than electrostatic interactions of peptide and agent.
The GFP I'm using in fact has two cysteins in it, which however, should be in reduced (or atleast not in disulfide) state since they are not in range. The GFP itself still is a common feature of the constructs and worth to be investigated. Fortunately, I have an mCherry variant of one reductant-responsive, non-cystein-containing peptide at hand and will definitely see if I can reproduce the effect here.
Since I'm purifying via Ni-NTA some Ni(II) might come off the column together with the peptides for sure. I do excessive buffer exchange, but carryover (especially when we expect the TM to bind the peptide) might happen. I will think about an experiment to evaluate this. Maybe I can use some EDTA to strip potential Ni(II) after purification or add it to the assay-buffer itself.
Many thanks for your great effort to help me solve my problem!
Hendrik
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