Synthetic Biophysics of Protein Disorder

Proteins are key to the molecular machinery of any living matter. Through developments in modern biophysical techniques, >100.000 three-dimensional protein structures were resolved to atomic detail. Currently, our understanding of how nature designs protein function is largely based on these static conformational snapshots of stable domain folds. In the past 20 years however, this view has been dramatically challenged by the discovery of intrinsically disordered proteins, protein domains or regions (here commonly referred to as IDPs), which are devoid of well-defined structure in their native state and best described as dynamic conformational ensembles. This structural adaptability and inherent malleability of IDPs imparts a functional versatility; i.e. enables them to adopt several motifs corresponding to different biological activities. Hence, IDPs are frequently hub proteins, central to the protein network of the cell that can form multiple different interactions in a highly dynamic fashion.

IDPs are of utmost importance: up to half of the human proteome appears to contain IDPs, which function in key biological processes such as cell-cycle, nucleocytoplasmic transport and transcriptional regulation. As such, IDPs are involved in a variety of diseases that pose an enormous societal challenge, such as cancers and neurodegenerative disease, like Parkinson’s, Huntington’s and Alzheimer’s diseases. The latter exemplifies that the (mis)folding of IDPs can have unfortunate consequences for the health of the affected individual. Also, viruses exploit the multi-functionality of IDPs to reprogram the regulatory mechanisms of the host by motif-mimicry of the eukaryotic proteins. All these biological scenarios are hallmarks of dynamic/disordered systems and their understanding opens many new opportunities for improving human health. Thus, there is a pressing need to reshape our classic deterministic view of structure to function relationships and to open new avenues towards targeting IDPs for basic and applied research, the latter including drug design as well as pharmaceutical investigations.

Investigation of IDPs

The dynamic nature of IDPs makes their experimental characterisation very difficult. Classical technologies in structural biology, like X-ray crystallography and electron microscopy (EM), are limited to largely static systems. Consequently, in the main, the disordered part of the proteome remains largely inaccessible to us, constituting what we refer here to as the “Dark Proteome”.

Fluorescence spectroscopy and microscopy are one of the few technologies that can, in principle, cope with the demands of studying protein dynamics in living cells. In addition, the sensitivity of fluorescence can even permit the observation of single molecules, which can yield direct insights into even heterogeneous molecular phenomena. However, as a labelling dependent technique, even the best fluorescence hardware is limited by the readout that can actually be obtained from the sample. By genetically attaching a fluorescent protein like GFP, that is permanently fused to the protein of interest, this roadblock was overcome and has dramatically impacted the way we work in life sciences. However, decades of GFP driven research have also revealed its limitations, some of which are inherent to the relatively large size of GFP (~25kDa). We develop technologies that permit manipulation of biomolecules and the custom design of new functionalities into biology using advanced chemical and synthetic biology tools. To detect even conformational changes, it is critical that the probe can be freely installed anywhere in a protein, anywhere inside the cell, in a site-specific fashion. Moreover, it is essential that the modification is as small and as minimally invasive as possible. 

A particularly important set of IDPs are those of the nucleocytoplasmic transport machinery, which is a system highly enriched in very long IDPs that is central to human health. It is mediated by probably the largest molecular machinery in the cell, the nuclear pore complexes (NPCs) (MW ~110 MDa). So-called scaffold nucleoporins (Nups) form the channel like structure of the NPC, while IDP type Nups (ID-Nups) constitute an actual permeability barrier and mediate transport. Owing to the exceptional plasticity of these very large IDPs, even the currently highest resolution EM tomograms, only successfully resolved the scaffold structure. The interior of the NPC was left as an empty central channel because the disordered nature of the barrier formed by IDPs in this moiety precludes subsequent structural analysis by EM. As one of the consequences of this lack of direct visualisation, the exact transport mechanism is still highly debated.  Since the NPC forms the central gateway to the nucleus, viruses have evolved different invasion strategies to pass the NPC, which are even less well understood.

In addition, ID-Nups also fulfil functions away from the NPC and directly engage with epigenetic mechanisms, gene regulation and transcriptional control.  Understanding how ID-Nups participate in target binding thus goes far beyond the relevance of “only” nucleocytoplasmic or viral transport. Owing to the highly heterogeneous sequence architecture of ID-Nups, their capacity to bind to their various partners is not well understood. 

To cope with these challenges, our team ranges from molecular/cellular biologists via chemists, spectroscopists to physicists and engineers.

For more about the Lemke lab click here for his personal lap webpage or here for his page at EMBL