Identifying intrinsically disordered proteins in crowds

Australian scientists from RMIT, ANSTO and CSIRO have revealed new insights into intrinsically disordered proteins and protein regions (IDPs)/(IDRs) and how they behave under various physiological processes.

IDPs carry out a range of important biological tasks and play a key role in several biological functions, including various metabolisms, cellular signalling, infections, illnesses and tissue repairs, as well as drug delivery. Unlike other functional proteins, they do not have a stable three-dimensional structure; rather, the same protein can rearrange in multiple pathways and may adapt to engage in different interactions with different consequences. The best example of IDPs/IDRs is the spike protein within the envelope of the SARS-CoV-2 virus: their adaptability not only enables them to latch onto a cell for viral entry, but also to evade immunity.

Professor Naba Dutta of RMIT said the new study provides previously unknown experimental evidence and a theoretical framework to predict how these proteins change shape in the complex, crowded environment of the cell. He noted, “They have the ability to transform from one shape to another, very fast, in response to the local environment. This makes it very challenging to analyse them using conventional techniques.”

Dutta and his collaborators conducted several high-tech experiments and provided evidence that Rec1-resilin, which is exceptionally elastic and commonly used to form various tough materials for biomedical applications by the group, is an IDP with unique characteristics; for instance, the resilin protein enables insects such as fleas with the ability to jump more than 100 times their own height in microseconds. The group meticulously described the protein’s structure and interface, and its evolution in a complex, crowded environment, in the journal Science Advances.

“We first got interested in these proteins when we were working on the development of materials for biomedical applications, such as hydrogels using silk and resilin,” said senior instrument scientist Dr Jitendra Mata, an expert in small angle scattering techniques at ANSTO.

“Silk is very tough and resilin is very elastic, so when you mix the two together you get a material that has exceptional properties and can be used for tissue repair.

“So, in developing these materials, we realised we did not have a good understanding of how these proteins function at a fundamental level, especially in a crowded environment. This knowledge is essential to develop materials for biomedical applications.”

Rec1-resilin responds to multiple stimuli and responds to changes in its environment, such as temperature, pH and the presence of ions and other substances, making it valuable as a responsive biomaterial — for performing tasks such as tissue repair or therapeutic delivery, for example. But as Dutta noted, “We all know that characterising an isolated protein in pristine conditions will not provide you the information you need, because it is not the environment where it normally operates.” It was only through deuteration, followed by ultra-small and small angle neutron scattering (USANS and SANS), that the researchers could identify the IDP and its metamorphosis in the soup of molecules that typically crowd the cellular environment.

The Rec1-resilin was produced by researchers at CSIRO. Deuteration of the protein, in which hydrogen is replaced by deuterium, was performed at ANSTO’s National Deuteration Facility (NDF). The deuterated protein was biosynthesised at the NDF before being used for SANS and USANS experiments at ANSTO’s Australian Centre for Neutron Scattering (ACNS). Experimentally identifying the shapes of the ensembles of IDPs and their metamorphosis under various crowding circumstances revealed the links between their sequences and functions.

By performing a series of contrast-matching experiments on deuterium-labelled proteins that enabled the scientists to ‘hide’ one component and study the other, the group examined the effect of crowding on the shape, size, stability and structure and the metamorphosis of an IDP. Theoretical modelling was also used to develop a framework that predicts the 3D structure of resilin in the presence of five different crowding agents at various concentrations.

To date, there have been very few studies of the impact of macromolecular crowding on the dynamic shapes of IDP ensembles, their stability and their transformation. With their study now completed, Dutta and the team look forward to seeing how their research will be taken to the next level.

Image caption: Simulations of deuterated Rec1-resilin under crowded macromolecular conditions. Image shared under CC BY-NC 4.0