In both my graduate work and during my postodoctoral work I’ve taken advantage of the strong interaction between streptavidin and biotin. Recall the last post we talked about how molecules can fit together into different positions to help stabilize each other. We often think of these in terms of how fast they switch positions from a together state “bound” to an “unbound” state. I’d like to expand upon more complicated systems in later posts but for now we’ll focus on this type of “two state” system. Biotin can position itself inside a small pocket in streptavidin and it loves to do so. Its ability to fit in or bind is known as the binding affinity and is often represented as its dissociation ability or dissociation constant (Kd). The Kd for biotin is about 1*10^-14. That means that for roughly every 100,000,000,000,000 biotin that get into the streptavidin binding pocket only 1 stays out. This makes it almost as strong as a chemical bond! One can do lots of amazing things with bonds these strong but they can also be very annoying.
On the positive side, the bond can be used for biochemical assays. In talking to me you may know that I developed an assay based on streptavidin and biotin binding (or in that case, biotin on a large dextran molecule). The streptavidin-biotin bond is strong enough that even small amounts of the molecules are able to find each other and bind together. This allows us to determine their ability to pass through various barriers like, for instance, a lipid membrane. The binds are tight and will hold together, which gives us a very reliable signal even in different conditions such as very acidic or high salt conditions. Though streptavidin and biotin can be used to work together for some assays, I’m not always happy when they stick together too much.
On the negative side, the binding between Streptavidin and Biotin can pose problems for other scientists. Currently I’m developing an assay to detect target molecules labeled with biotin. The idea is to use biotin to attach the molecule to a streptavidin surface because, again, it is a strong bond. The problem is that my samples might not be pure target-biotin and are contaminated with biotin. Because Streptavidin-biotin has a lower Kd than Streptavidin-target-biotin, the biotin fills the streptavidin pockets before the target-biotin. Also, if I use too little, my molecules no longer bind to the target, they bind to the streptavidin themselves! In this case it’s a bit of a vicious cycle of molecules not fitting into the correct spots! This is why I say that for some applications streptavidin-biotin are great but for others they make a bit of a sticky mess! As always, thanks for reading and come back to enjoy future posts talking about heat and kD and Molecular Yoga!
Molecules have been moving around and stretching in cramped spaces since the beginning of life on this planet. I just recently read a review paper by van der Gulik and Speijer, two researchers from the Netherlands about this topic. They described how the world may have developed the ability to create proteins from RNA and small peptides. RNA, as you may know, carry the molecular plans for the cell to build the proteins that more or less make up our cells’ structures. They do this by working together with another protein structure called a Ribosome to assemble amino acids, the building blocks of proteins. The plan RNA, called mRNA for “messenger” RNA has to get threaded through a tiny inner cavity in the ribosome. There mRNA meets up with a “transfer” RNA or tRNA carrying an amino acid. The mRNA and tRNA can then move and stretch to hook amino acids together to construct proteins! If you look at this process it seems pretty miraculous. How can all of these molecules can fit together in the proper positions at exactly the right time? Van der Guilk and Speijer have attempted to piece together a timeline of event to answer this question.
Though it may seem irreducibly complex, this process of protein production could have evolved over time from much simpler reactions in a similarly small space. To start, van der Guilk and Speijer not how certain types of RNA are known to be able to react with themselves. They can into a certain position and cut themselves like an enzyme. For this reason they are often called “Ribozymes”. Many people studying the early biochemistry of the Earth believe that “life” existed first in the form of an RNA-world of Ribozymes making and cutting other RNAs. The question then becomes when did proteins come into the mix? Some believe that Ribozymes eventually gained the ability to form RNA-Ribosomes on their own to assemble proteins. This is where van der Guilk and Speijer begin to diverge from some of the big names in the field. These two suggest that the process of going from RNAàProtein occurred with the help of small proteins, peptides, of two-five amino acids.
The central argument of van der Guilk and Speijer comes from two facts, 1: that early peptides could have assembled on their own and 2: that these peptides can stabilize RNA. Recall that we talked about in my previous post how molecular crowding can change the surroundings of a molecule and force it to stretch or react with other molecules. In our author’s minds, the RNAàProtein process developed not in the inner cavity of a Ribosome, but in the confined space of montmorillonite, which is like clay from a river or cave. The low water content conditions inside the layers of clay could have been enough to force two glycine amino acids to come together to form a peptide bond. These small peptides, the authors argue, could have been the first component of the “ribosome” that might have allowed RNA and Ribozymes to function. These proteins may have been able to wrap around RNA in a manner that protected it from wrapping around ions like Magnesium (as Mg2+) and cleaving itself. A peptide scaffold could have then developed specific amino acid side groups like carboxylic acid (COO- when ionized) to further protect RNA. Hypothetically functional ribosomes could have evolved from these very simple components.
I’ll leave out the rest of the paper for now which describes higher-order RNA structures and how codons might have developed. For this, I’ll leave you with the reference to the paper in case you are interested in looking more into it. Thanks for reading and please leave comment for discussion if you like!
Van der Guilk PTS and Speijer D, Life 2015, 5, 230-246.
For now I’ll continue with the food analogies to talk about one other source of uncertainty in biophysical and biomaterials measurements. That is the property of Molecular Crowding. When we think of a chemical reaction or forming materials our first thought is to considering it occurring in a vacuum, with infinite space around it. In nature, and especially inside of cells, this is not always the case. We need to take into account how much freedom the molecules in these places have to rotate and move. This might force them to undergo some molecular yoga!
Consider this curry I made for one lab potluck. In addition to the curry base itself it’s crowded full of spices and vegetables. If I were to cook the curry with just potatoes, for instance, each potato piece could cook and absorb the spices surrounding it. If I add some frozen veggies, however, the veggies near the potatoes would melt and dilute the curry around the potatoes with water. This would lead to a very slight change in flavor. To take our analogy to a cell, the cell’s cytoplasm can be like a veggie crowded curry. There are many many proteins floating around and some of them are changing the local concentration of molecules (like water!) in given parts of the cell. This can affect, say, the rate of enzymatic reaction by pushing molecules closer together so that the enzymes can grab more molecules. This is just another thing to consider when we’re thinking about how reactions inside of a vacuum can be very different than reactions in real life!
PS: I’m currently reading a very interesting paper about the origins of life and how molecules were forced to bend, twist, and react in weird ways to make up our modern system of biochemistry. I’ll post about it next time!