Biocurious is a weblog about biology, quantified.

Microreview: Single Molecule AFM - Focus on Spectrin

by Andre on 21 June 2005

BACKGROUND An important part of most (all?) cells is the protein network, appropriately named the cytoskeleton, that supports their structure like a springy scaffold and also plays an important role in intercellular transport, cell division, and cell motility.

In red blood cells, the main component of the cytoskeleton (about 25% of membrane associated proteins) is spectrin, a rod-like protein made of two subunits (the alpha and beta chains) that associate as a side-by-side dimer. Each of these chains has multiple domains that consist of bundles of three anti-parallel alpha helices. You can see a picture of the structure here, but I strongly recommend downloading a free program for viewing molecular structures like RasMol or VMD and then going to the protein data bank to get the atomic coordinates for spectrin. This way you’ll be able to rotate the molecule however you like and also adjust the viewing settings to emphasize features of interest or even setup a stereoscopic version that appears three dimensional. Getting used to browsing the protein data bank also allows you to quickly visualize any other proteins that may be of interest when you read about them in a paper or hear about their role in some disease in the news.

THE TECHNIQUE The advent of single molecule techniques in biophysics has given researchers an unprecedented ability to probe living systems and their components directly at their fundamental molecular scale. In particular, atomic force microscopy (AFM) has proven useful in monitoring, among other things, the unfolding and refolding trajectories of single protein molecules by applying piconewton forces over nanometer distances. The first time that humans used minute silicon nitride diving boards to pull on individual protein molecules was in 1997 when Rief et al. unfolded individual titin immunoglobulin (Ig) domains. The first time that the technique was applied to spectrin was in 1999, again by Rief and friends.

PAST RESEARCH So there’s a protein that is interesting to biologists and to physicists, it is possible to unfold it by force applied at the single molecule level, there are some preliminary results, but some interesting questions remain, and AFMs are commercially available to answer them. Needless to say, spectrin was pulled again to much avail. In the intervening years, various spectrin constructs were engineered and probed to learn about their structure and response to force. Basically, we know the following:

1. Single spectrin domains unfold in a two-state manner: at the experimental resolution, domains are either folded or unfolded, with nothing observed in between. [1]

2. The domains are facile: the unfolding happens at around 30 pN when pulled at 300 nm/s. By contrast, the Ig domains of titin unfold at forces that are ten times larger. This difference is attributed to the hydrogen bonds that stabilize the beta-barrel structure of the Ig domains compared to the hydrogen bonding in spectrin which is within, but not between, the alpha-helices. Since they are held together by weaker hydrophobic forces, it is not altogether unexpected that the alpha helices are easier to pull apart. [1]

3. In about 20-30% of events, tandem repeat unfolding is observed in short (2-4 domain) constructs of beta-spectrin. Tandem unfolding is identified in sawtooth patterns by double-length unfolding events occurring at the same force as single repeat events. By comparing traces with similar total unfolding lengths (e.g. one tandem event compared to two single repeat events), Law et al. [2] find that tandem and single repeat events are roughly equally probable.

4. A large number of unfolding events is less likely than a small number in any given trace. That is, Npk distributions decay with Npk as m-Npk. [2]

5. 15-30% of traces show parallel-chain or loop unfolding in short (2-4 domain) constructs of beta-spectrin. Parallel-chain and loop unfolding is identified in sawtooth patterns by double-force unfolding events with the same unfolding length as single repeat events. Loop formation is suggested to be more likely in longer constructs. [2]

6. Unfolding lengths are only ~two thirds of contour lengths suggesting some repeats are not fully extended (and are perhaps even still partially folded) during the experiment. This may have implications for nucleation of refolding in vivo. [2]

7. Tandem repeat unfolding becomes less likely at higher temperatures. This is attributed to partial unfolding of the helical linker between domains at higher temperatures that prevents the propagation of a cooperative transition to adjacent domains. Circular dichroism measurements show that a few percent of the helical content is lost during a temperature change from 10-23oC consistent with partial melting. Also, the variance in unfolding force does not increase with temperature suggesting that all the repeats are equally affected by elevated temperatures. [3]

8. Unfolding is more complicated in anti-parallel heterodimers potentially due to the small shearing force required to separate the dimers in addition to unfolding the subunits’ domains. [4]

9. Molecular dynamics simulations are consistent with unfolding beginning with the helical linker between domains. The linker is slightly separated from adjacent helices in one of the domains and subsequently unwinds while increasing backbone hydration. [5]

10. Recent detailed solutions studies by Batey et al. show many subtleties in the cooperativity between two beta-spectrin domains. For example, changing the linker regions destroys cooperativity as does introducing stabilizing salts. Mutations in one of the domains (R17) eliminated cooperativity while mutations in the other (R16) had no effect. A careful analysis of mutational studies showed that R16 and R17 stabilize each other whether folded or unfolded except for the case of R17 with unfolded R16. [6]

FUTURE WORK This July I’ll be traveling to Munich to start what will hopefully develop into a fruitful long-term collaboration with Matthias Rief, a pioneer in single molecule AFM who has continued to push the envelope technically and scientifically. We will be looking in more detail at the linker’s effect on cooperativity with force spectroscopy applied to two-repeat spectrin constructs with and without alpha-helical linkers present. This approach will complement the solution studies of Batey et al. [6] It will also be exciting to get the opportunity to use their recently developed equipment to look for unfolding intermediates and their relation to cooperativity.

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