Studying the Structure and Function of Large Molecular Machines

Proteins carry out most cellular processes as members of dynamic multi-protein assemblies. Although progress has been made cataloging the constituents of specific complexes, we have only limited knowledge of how proteins assemble into macromolecular machines and how these machines perform their cellular functions. We are using yeast genetics, biochemistry and single particle cryo-electron microscopy (EM) to explore the structural and functional organization of complexes involved in pre-mRNA splicing and complexes involved in the protein ubiquitination pathway.

Single Particle Cryo-Electron Microscopy

Single particle cryo-EM is a powerful technique for determining the structures of large, dynamic complexes that are too difficult to crystallize. In this structural approach, purified complexes are applied to grids covered with holey carbon film and quickly frozen by plunging the grids into liquid ethane. The rapid freezing prevents water from forming ice crystals and embeds the molecules in a layer of vitrified (or amorphous) ice preserving the specimen in a near-native environment.  Images of the preserved particles are taken using an electron microscope. Digital image processing methods are then used to produce three-dimensional (3D) models from the images of particles trapped in vitrified ice. Using cryo-EM in combination with biological, biochemical, and biophysical techniques the Ohi lab pursues biological questions.

The Spliceosome

Although the human genome contains approx. 25,000 genes, it is estimated that we make over 90,000 proteins. The disparity between our genome and our proteome can be explained by the activity of the spliceosome, a large macromolecular machine composed of RNA and protein components. This complex catalyzes the excision of non-coding introns from a pre-messenger RNA (pre-mRNA) to create a mature message (mRNA). Although the composition of the spliceosome is known, it remains a mystery how this dynamic machine functions as a coherent unit. To understand spliceosome function and regulation it will be essential to develop 3D pictures of how the numerous spliceosomal proteins and RNA components organize into one machine. A number of projects in the lab focus on the functional and structural characterization of stable spliceosomal complexes isolated from the fission yeast S. pombe.

Bacterial Pathogenesis

There are a number of projects in the lab that focus on studying the structure and function of molecular machines required for bacterial pathogenesis. These include pore forming toxins and protein translocation machinery. We work with a multidisciplinary team of researchers — including Tim Cover and Borden Lacy at Vanderbilt University — to understand how these toxins function as both soluble and membrane inserted proteins. Our main goal is to gain a mechanistic understanding of how these toxins and translocation complexes contribute to pathogenesis. 

Understanding the Role of PMP22 in the Peripheral Nervous System Myelin

Peripheral myelin protein 22 (PMP22) is a tetraspan integral membrane protein that is abundant in the myelin of the peripheral nervous system (PNS). Mutations that encode changes in the amino acid sequence of PMP22 are one of the causes of Charcot-Marie-Tooth Disease (CMTD) and related peripheral neuropathies. CMTD afflicts 1:3000 people (2 million worldwide) and mutations that impact the PMP22 gene are the most common cause of this disorder. Pmp22 is a critical component of myelin; however, the exact function(s) of PMP22 in in myelin remain poorly understood. In a close collaboration with Dr. Chuck Sanders (Vanderbilt University), using a combination of biochemistry and EM, we have found that PMP22 promotes the formation of Myelin-Like Assemblies (MLAs) following reconstitution in lipid vesicles. We are exploring the hypotheses that the role of PMP22 in MLA formation is closely related to one of its native functions and also that at some CMTD mutants are likely to disrupt MLA formation in a manner that reflects how mutations promote dysmyelination in PNS tissue.