Structural determination and dynamics of the mitochondrial import protein (MIM) by cryo-EM and magic-angle-spinning (MAS) nuclear magnetic resonance (NMR)

Mitochondria perform numerous key functions, from energy production to metabolic pathways, and are involved in ageing and cell death.

All these functions rely on about 1000 different proteins, of which 99% are synthesized outside the organelle and have to be imported and inserted into the right compartment.

These import and insertion are particularly demanding for the membrane proteins as they are inherently aggregation-prone.

How α-helical membrane proteins (MPs) are inserted into the mitochondrial outer membrane (MOM) is currently poorly understood.

In particular, the structure of the insertase responsible for inserting tens of these α-helical MPs into this membrane, called mitochondrial import protein (MIM) in yeast, is unknown.

My MitoMemInsert project addresses this important knowledge gap, by determining the atomic-resolution structure of MIM, by using cryo-EM combined with magic-angle-spinning nuclear magnetic resonance spectroscopy to resolve MIM’s structure in a lipid-bilayer environment.

This will open avenues to see how the MIM complex engages with polypeptides that shall be inserted into the membrane by MIM (so-called precursor proteins or ‘preproteins’).

Such a “static” structure determination would already be a big leap for the field: it will be the first structure of a MOM insertase of α-helical MPs.

As it is thought to have a novel architecture, this structure will provide new insights into MP insertion machinery in general.

In addition, I will use NMR to study the dynamics of this machinery, which I believe, will add important mechanistic knowledge of how the MIM complex engages with MPs that are being inserted in the MOM.

Overall, making use of my previous technical expertise, and adding new competences to my skill set, a combination of biophysical and structural approaches will allow me to resolve this important, and hitherto poorly addressed question in mitochondrial biogenesis and, more generally, biophysics.