Biocurious is a weblog about biology, quantified.

A Protein Aggregation Puzzle

by Andre on 18 May 2006

I’ve posted data from experiments in the past, but it’s always been to illustrate the kinds of things I work on or to share a method. This time, I want advice. You can also consider this a test of using weblogs as a collaborative tool, something I’m told is common in the humanities but seems to be extremely rare in science (but not unheard of—see Evolgen and Science and Politics).

Here’s the story so far.

I’ve started doing some experiments on vimentin, a ~45 nm long helical protein that serves as an intermediate filament in some cells’ cytoskeletons. See the Wikipedia entry on vimentin for all the background you need about the protein.

The data I want to talk about are really a side product from the main experiments I was doing but I think they are neat and I would like to learn more about them.

Here’s how they were generated. First, I put a drop of the protein/water solution onto freshly cleaved mica, a common substrate for AFM experiments, and let it sit for a few minutes so that the protein could adsorb to the surface. Next, I added some buffer solution to increase the ionic strength of the sample. This should cause the vimentin to self-assemble into fibers. Finally, the sample was allowed to dry slowly in air. When I took an image the next morning I got the image shown at the top (this image is 40 microns on a side).
I did the same thing, but dried the sample more quickly by blowing some nitrogen over it and I got the image shown at the bottom (this image is 10 microns on a side):

This reminded me of the undergrad lab I did on diffusion limited aggregation. For that experiment, I used a flat circular electrochemical cell to aggregate zinc from solution. Different solute concentrations give different patterns. This “phase diagram” is discussed even in the earliest papers. Depending on the voltage and concentration of zinc sulfate you can form dendrites (like I saw with vimentin) or fractal “stringy” stuctures.

The last piece of information I would like to bring to your attention (purely for motivation) is that Alzheimer’s disease and other neurodegenerative disorders result from the formation of protein aggregates of beta amyloid, a protein that can also form fibrils.

So, I’ve laid out the puzzle and now I have some questions for you readers (especially any nonlinear science types… you know who you are). Is the aggregation of small fibers any different from the aggregation of little balls? Can you convince me to do some related experiments to flesh out the story? Will we cure Alzheimer’s or other protein aggregation related diseases by understanding this problem better? :)

  1. allan    3962 days ago    #

    To be completely unhelpful: I seem to think that the anisotropy in the free-energy at the interface (between crystal and solution) depends a lot on the crystal structure. Since this anisotropy is important in the formation of dendrites, one would expect the fibres and spheres to form “different” dendrites simply because of the different crystal forms.

  2. isaac    3956 days ago    #

    It looks like your images are of salt. Typically for AFM, you want to conduct your experiments in low salt conditions. Your first image arises because nucleation of crystals is a stochastic, concentration-dependent event—in the case of the first image, growth is more rapid than nucleation, yielding nice crystalline order. In the second image, rapid drying ramped up the nucleation so that it overtook growth and it resulted in nasty salt aggregates.

    In the future you might want to rinse your mica surface with ddw; you’ll have to hope that increasing the ionic strength is to overcome a kinetic, and not thermodynamic barrier, and that your fibrils associate with mica enough to stay put. Otherwise it might be wise to consider using another technique to study your protein.

    in any case I doubt that it’s protein because it takes a long time for proteins to crystallize into such well ordered structures in 1, and in both cases the scale of your objects are way too big to be proteins.

    Aside from the pedestrian problem of salts on mica, the problem of balls vs fibers is a real one. It seems like the aggregation of little balls of alzheimer’s protein is off-pathway from the fibril-forming one. Sort of a temporary kinetic trap. It turns out that almost none of the “amyloid” proteins and/or peptides actually form nice, canonical amyloids in vitro, except sometimes in some really extenuating situations (insulin at pH 2, for example—not biologically but certainly pharmaceutically relevant). They almost always have some sort of strange competing aggregation phenomenon.

    A few things to think about:

    1) amyloids/fibrils as a 1-dimensional crystal.

    2) is amyloid a byproduct of some other problem or the cause?

    3) if so many amyloids exist, why are they all pathological?

  3. Andre    3952 days ago    #

    Hi Allen and Isaac,

    Thanks for your responses. First of all, Isaac is right about the salt, so other considerations aren’t relevant for these images, but it’s interesting to hear your perspectives on protein aggregation. Maybe I’ll do some more careful experiments with vimentin in the future.

    Next time I’ll try to post something with a slightly broader appeal (maybe some movies?) to see if we can get a little more participation, but that will have to wait since I’ll be travelling for a little over a week.

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