Aug 132007
 

Another feature of the protein society was a continued emphasis on trying to understand natively-disordered proteins, and by extension, the denatured state of natively ordered proteins. Because these two fields are highly related and use the same techniques, it seems to me best to lump them together for now. A couple of interesting points came up that I wanted to get down here for my own memory’s sake.

One point, and one that became a recurring theme in several talks at the symposium, was averaging bias. The first real discussion of this came from a really good talk by Michele Vendruscolo on the study of the natively-disordered 131-deletion mutant of staphylococcal nuclease. Some models that Dave Shortle had produced of the disordered state on the basis of paramagnetic relaxation enhancement had predicted ensembles that were too small with respect to the known radius of gyration. Michele pointed out that the PRE is an ensemble measurement, and many different ensembles can give rise to the same PRE. Additionally, the PRE is biased because below a certain threshold the effect is invisible. This means that the measurement ends up being biased towards closer approaches. Essentially his point was that the normal distribution cannot be assumed for the ensemble average of distance measurements in the denatured state (and it’s probably a questionable assumption in the native state as well).Kevin Plaxco gave a talk later on that really hammered this point home. He did a series of SAXS experiments to determine the radius of gyration for a ton of proteins, including several that had shown residual structure in NMR experiments. His results indicated that the experimentally determined radius of gyration matched that predicted for a random coil for all these proteins. As he pointed out, though, the Rg is totally insensitive to local structure, whereas because of anomalous averaging much of the NMR data is hypersensitive to local structure. This means that both results can be right — any given protein can have some percentage of its structure intact and as long as it’s a different piece for each protein and not too much, the ensemble can retain a random-coil-like Rg. If tertiary interactions are preserved this becomes a slightly more difficult proposition to swallow, though. Still, his work, and several other talks and posters presented during the symposium, made an excellent point. We simply cannot rely on the assumption of a normal distribution when we are analyzing NMR data from systems with so many degrees of freedom.

Another thread that showed up repeatedly was the ongoing attempt to understand exactly how disordered states interact and are regulated, especially by post-translational modifications such as phosphorylation. Most disordered regions have multiple binding partners, with affinity enhanced for a particular partner by a particular modification. In the simplest model for these interactions, the modification itself and some of the surrounding primary sequence is recognized. However, there’s an increasing amount of data, including a nice talk by a postdoc from Julie Forman-Kay’s group, that the post-translational modifications alter the structural characteristics of the disordered state itself. The Forman-Kay talk suggested that phosphorylation induced a condensation of the protein by attenuating a surplus of positive charge.

This could conceivably be taken further. Consider a bit of sequence like DKRSDKA, which could conceivably take the form of a β-strand if it weren’t for that concentration of positive charge on one side. A phosphate group on the serine could conceivably stabilize this structure and preorganize it for binding to a ligand, thus increasing affinity by reducing the energetic cost of binding.

It might even be possible to tune things more specifically. Take a sequence like GRDSSKAKSR. If you put this on a helix wheel you’ll see a huge blast of positive charge on one side, but also a pair of serines. Phosphorylate S5 and S9 and you could stabilize the helix. At the same time, this would make a β-strand conformation less likely because such a strand would have negative charges on one side and positive charges on the other. By contrast, if you phosphorylate S4 you’d do nothing to stabilize the unfavorable charge concentration on the helix, but the positive charge concentration on the strand would be attenuated (see cartoon). In this way phosphorylation might be used as a kind of conformational switch to preorganize the same sequence in different ways and thus reach different downstream effectors. We know that conformational rearrangements of the kind that lymphotactin undergoes give rise to different signals and protein behaviors. The role of differential preorganization in disordered proteins hasn’t been extensively studied yet, but may be equally important.

It’s increasingly clear that disordered regions are a major factor in cellular signaling. I’m not having much luck with the one I’m working on now, but I’m excited to see where the next few years lead this field.

 Posted by at 5:11 PM

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