Protein complexes in Pyrococcus furiosus

Background

Protein-complex assembly is one of the key drivers of the highly dynamic behavior between all molecules in a cell. An estimated 80% of proteins collaborate with other proteins to execute tasks of a much higher complexity then the sum of their parts would suggest, potentially giving rise to several hundreds of thousands of interactions occurring within the cell. Many unique protein complexes have been discovered through elaborate and detailed biochemical investigations, which have yielded groundbreaking biological insights. The study of protein complexes in an unbiased fashion is still challenging, but now timely as technology has sufficiently progressed to make it possible. In cooperation with the various experts at Utrecht University we want to investigate such protein complexes in our model hyperthermophile Pyrococcus furiosus.

Aim

The main goal of this proposal is to develop an integrated approach to characterize proteins and their complexes by a combination of analytical and biophysical techniques with integrative structural modelling strategies. Distinctively, our novel workflow will combine data from the rapidly developing fields of cryo-electron tomography (Cryo-ET), native MS, top-down proteomics and crosslinking mass spectrometry (XL-MS) to uncover protein complexes in an unbiased fashion, ideally in their native state and ultimately in situ. Currently we are developing a thermostable fluorescent protein-tag that can be fused to a protein of interest, to be able to identify the latter by fluorescence microscopy. Secondly, we are trying to unravel the function of a novel structural protein of Pyrococcus furiosus by making gene knockouts.

Techniques

Multiple techniques are used to achieve our targets including CRISPR-Cas genome editing, advanced microscopy, anaerobic hyperthermophilic culturing, HPLC, GC, FPLC and regular plasmid design and cloning.