Dear Madison,

You have a classic example of nucleic acid binding proteins. They are unstable at higher concentrations alone but perfectly fine with a partner. You may think about them as a chaperone for your protein.

There is nothing you can do about it except for changing buffers and adding stabilizers. A good example is a 20-50% glycerol to increase viscosity and maybe counterions to balance the positive charge of your protein. Glutamate may work as it is negatively charged. You may also add some detergent like TritonX-100 or Tween-20 to shield the hydrophobic patches on your protein. A removable detergent with a high CMC value would be better but they are expensive. Together, you may increase the protein concentration to above 1 mg/ml and keep it stable in a freezer. To help you with the work, please aliquot your sample into the optimized buffer and try not to reuse the same sample more than 2-3 times.

Good luck with your work,

Wes

Dear Madison,

in addition to Swietnicki comments....

I have a nucleic acid binding protein (basic pI) which is stable in high salt (300mM NaCl). The protein becomes cloudy and precipitates when the salt concentration is reduced.

I have another intrinsically disordered protein (not a nucleic acid binding protein) which was optimized for solubility by altering the pI of protein. Removing or addition of certain regions of protein makes it more acidic or less acidic. More acidic ones (pI 4 - 4.5) have good solubility (can be concentrated upto 50mg/mL) than the less acidic ones (pI 5, precipitates as I try to concentrate it). Here the solubility can be enhanced a bit by increasing NaCl concentration.

You could try by deleting some of the regions of your protein which are not involved in binding. Alternatively you could perform Thermofluor to have an idea about the optimum buffer composition.

The answers above are already quite useful. In addition, consider adding arginine to your buffer at say, 50mM - 100mM concentration to start. Arginine has worked well for me in the past for preventing aggregation of a wide range of proteins.

The above suggestions are all good ones in my opinion

Trehalose is helpful for stabilizing freeze-thaw cycles. If you can flash freeze your samples, all the better. Slow freezing can lead to inhomogeneous solutions with domains of high protein concentration. These domains will start the precipitation process.

Article Why is trehalose an exceptional protein stabilizer? An analy...

Thank you for your feedback! Here is what I have found out so far in case this helps anyone in the future.

Jeremy Pronchik I was flash freezing in liquid nitrogen, but I think perhaps some of my samples were not freezing fast enough (due to competition with other tubes). This could explain why some of my aliquots would crash upon thawing while others would not.

Wieslaw Swietnicki I added 25% glycerol this purification round, and it appeared to increase my precipitation during my concentration step, but perhaps the glycerol will improve the protein stability during storage. I have stored my samples at -20C to avoid the flash-freeze and thawing steps, so I hope this will prevent protein crashing.

After concentrating with the 25% glycerol, I aliquoted my sample (150 microliters each) and to each I added a total of 15 microliters to test a variety of conditions: control, 100 mM glycine, 50 mM arginine, 50 mM glutamate, 50 mM KCl, and 50 mM arginine + 50 mM glutamate.

I stored samples overnight and just checked them.

Tsepo Lebiletsa Tsekoa 50 mM arginine led almost the entire sample to crash, as did 50 mM glutamate, and the 50 mM arginine + 50 mM glutamate sample. I also noticed that any sample that contained 50 mM glutamate froze at -20C (despite having 25% glycerol), as such I had to thaw the sample and this could have caused further issues.

Vidhyadhar Nandana I have noticed that when bound to nucleic acid, my protein will crash with relatively low salt concentrations (~100 mM). After the overnight with the addition of only 50 mM KCl, my protein crashed as well. This indicates that my protein is also not stable with salt when not bound to nucleic acid.

The conditions that do not appear to have caused crashing are the control and the 100 mM glycine sample.

Any further insight you may have is appreciated! I find it odd that my protein crashes with such additives that the literature suggests improve protein stability and prevent aggregation. Additionally, most literature suggests a nucleic acid binding protein with a disordered region that is positively charged would be stable with higher salt concentrations, but I see crashing as low as 50 mM.

Dear Madison,

Thanks for the response. Each salt has a different effect on proteins. Please look up Hoffmeister's series to see if it is a crowding or a simple shielding.

The other aspect of your tests is the complete absense of detergents. It looks like hydrophobic interactions are critical for aggregation and you should try disrupting them by adding a detergent. Repeat the same with a detergent.

Best regards,

Wes

As I have said here many times any polysalt like your RNA binding protein can be dissociated and remain in solution by adding enough nonbinding simple salt like

NaCl.I suggest you forget the infinity of exotic cosolvents and just take water plus

increasing concentrations of NaCl.Above a certain concentration you will dissociate the RNA and both the RNA and the protein will coexist up to a certain higher concentration above which they will reform a complex .

Simplistic thinking is good for graduate students. At increased salt concentrations, you may not form a complex again if the salt interactions are critical for binding. Instead, the system will crash out on you because you will have only hydrophobic patches exposed instead of having them covered by RNA. Try it yourself and you may find a region where there is a complex but no agregation. Increasing protein concentration may lead to aggregation as before.

Dear Wieslaw.There is nothing simplistic in what I said.This is what is experimentally observed all the way from nucleic acid -small charged ligand complexes to nucleosomes ,chromatin and giant chromosomes as well as theoretically and computatiionally.When there are enough charges in ithe system

hydrophobicity,your favorite pet does not matter in most cases.Hydrophilic hydration and effective statistical ionic cloud interaction tip the balance towards

dissociation of binary charged complexes which maybe followed by reassociation

at higher concentrations when the water activity becomes too low because everybody who is charged loves water.I bet whith you that if our friend tries out my recipe, he will find a regime where the RNA is separated from the protein without using exotic solvent cocktails nobody understands and may also alter the

protein structure more than he would like .

Dear Dikeos,

Unfortunately, I have dealt with many difficult-to-handle proteins, the prions a prime example. Hoffmeister series is not an exotic system but a proven reality. If somebody wants to run a simple salt screen test, the discussion is beyond the point. Let him do it and be happy instead of engaging a group of other scientists.

In my opinion, the work should have been done in the first place before posting the problem. Knowing life, the opposite is true despite many examples.

Wieslaw Swietnicki Dikeos Mario Soumpasis

Yes, I tried a myriad of salt concentrations, and of different types of salts (such as NaCl, KCl, ammonium sulfate, etc. with my proteins prior to posting this question. All have resulted in the proteins crashing, even imidazole has been problematic, (if the protein is at the necessary concentration during a purification: >0.5 mg/mL). If I happen to come across a salt condition that works in the future, I will post it on here for others with the same question. I have been avoiding salts for some time now as they have always caused issues.

Dear Madison,

Thanks a lot for the explanation. Now you have to run a combination screen with salts and detergents.

The only option to avoid a futile work is to remove the DNA/RNA part or procure a synthetic part which may substitute for your DNA/RNA. Look up crystallography groups working on DNA/RNA-binding proteins and see what they use.

Best regards,

Wes

Dear Madison

Forget your myriad combinations of different salts at myriad supercritical protein

concentrations.I am speaking of a systematic step by step variation of the concentration of a non binding fully dissociated salt such as NaCl at fixed subcritical (for aggregation) protein concentration ,not of random trials with all

combinations of salts some of which e.g (ammonium sulfate) are known to promote aggregation vial preferential interactions with biopolymers and therefore widely used by crystallographers .I suggest an experiment in the range 0.1 to 1M

salt with any method capable to monitor the presence of protein-RNA complexes.

If what i suggest does not work I would like to look into your particular system and find out why.Is the structure of your intrinsically disordered protein known(Xrays,NMR)?

Dear Wieslaw

With exotic cocktails I did not mean the Hoffmeister series which is an empirical

fact useful in many cases particularly for anionic effects,but the various cosolvents and additives people use in their despair to crystallize proteins via induced aggregation.I have collaborated quite a few years with experimentalists in Saenger's Group in Berlin in order to understand these processes in aqueous solution and know what I am talking about.Unfortunately,only in the simplest case of protein+water+simple salt we can say something definite and semiquantitative.If you are interested ,please have a look at the papers I have uploaded here at RG.My suggestion is not a panacea but based on this work and the evidence provided in the field of nucleic-acid protein interactions.

Wieslaw Swietnicki

Dikeos Mario Soumpasis

Thank you for your responses.

I think there may be some confusion about my question. Just to clarify, I have been able to remove the majority of nucleic acid contamination from my protein, which is the reason (along with the tendency of disordered proteins to aggregate) that the protein was aggregating and I was trying to figure out if I could add something to stabilize the protein.

I use a 1M salt wash with the protein bound to affinity beads to disrupt the protein-RNA complexes. This has worked to remove the RNA. Now I am just looking to stabilize the purified protein in solution over longer periods of time. So far glycerol and storage at -20C to avoid freeze-thawing has helped. The first half of the structure is very similar (~70% sequence identity) to a paralegal protein in which the stuctured regions have been determined by X-ray or NMR, but the disordered domain, as expected, has never been resolved to my knowledge.

I hope my responses have helped to clarify my previous questions.

Dear Madison,

Thanks for the explanation. You have just made a reasonable protein much worse. Please remove the RNA completely if you wish but be prepared for the protein to aggregate. If you want to have a stable complex with RNA, use a synthetic replacement to stabilize it. To have have a disordered protein behaving fine in solution is hopeles. Please think over what you want before starting a new set of expermeinents.

There has been enough time spent on the problem. Please come back with a strategy what you need and what is the goal of your project. Otherwise, I am not going to waste my time.

Best regards,

Wes

Dear Madison

Thanks for your last answer which made things a bit clearer.After all in order to remove the RNA you have done just what I have suggested namely found a salt concentration that renders the RNA-protein complex thermodynamically unfavourable.Now what is your goal?Do you just want to preserve the protein in the freezer for later use and protect it from degradation(via microbes or radiation..etc.) then do what our forefathers were doing with their foods ,put it oil

,brine,honey ,glycerol,wax,etc.But if you want to have it in a dispersed aqueous phase or maybe weakly aggregated(gel like form) put back the simple salt and systematically lower the concentration until you get what you want.The diffuse ionic cloud around the protein will provide the charge compensation previously obtained via the bound RNA without affecting too drastically the hydration requirements of the protein

Thank you all for the helpful responses, I truly appreciate it. I believe I have been able to resolve my issues and achieve what I needed to. However, in reaching out to esteemed members of the scientific community, I felt at times some of the responses I received were disrespectful. As a woman in STEM, I wanted to express that these types of responses can often lead women to be hesitant to ask questions.

Only answers can be stupid. Questions are to be asked and answered.

Madison Edwards, I'm sorry you had to withstand the vitriol that was exchanged in this thread. I was also turned off by it every time I received a notification in my inbox that another answer was added only to find this. If it's any encouragement, it's the first I've seen anything like this on Researchgate.

Thanks for sharing your perspective on how you received it as a woman in STEM.