Megabody Scaffold Constructs as tools for Cryo-EM structure determination and novel purification strategies

Abstract

Cryo-EM structure determination currently faces several challenges when it comes to imaging and analysis of smaller proteins. Preferred orientation bias and lower SNR (Signal to Noise Ratio) make it difficult to image smaller proteins often resulting in skewed, misaligned and even incorrect images. Since many small proteins are of vital importance in biological systems, structural studies and therapeutics, methods are being developed to tackle this imaging obstacle. Megbody constructs are one such tool that has been developed to enable the structure determination of these proteins. These constructs have a large and rigid scaffold protein along with a linked nanobody. Our lab has designed such a megabody construct that uses the helical plasmid segregation protein ParM as its scaffold. ALFA tag is used as the antigen target of the linked nanobody NbALFA, with GST-ALFA used as the test protein.. The nanobody strongly and selectively binds the tagged target protein with the megabody. The helical nature of the scaffold allows for different viewing orientations of the target protein to be available due to the twisting of the helix. This allows all the obscured views from preferential bias to be cleared while the larger total structure prevents any adsorption and low SNR problems. We have shown in this thesis that our designed megabody is properly folded and functional. All its components work as intended with the design and the interaction of the megabody with the protein does not hinder any of its activities. Moreover this interaction is specific to the target protein tagged for nanobody in our construct. This makes our megabody construct capable of functioning as a proper tool to help image and analyse smaller proteins that can be bound to it. Alternate scaffold proteins and modifications to enhance megabody functions are also explored in this work.