John B. Engen, PhD University of New Mexico jengen@hxms.com Phone:(505) 277-4226 Personal Website

Title: Effects of Tom20 and Tom22 on the Structure of Proteins

The unfolding of proteins in vivo is critically important for life but is not well understood. It is clear that the process is necessary and that it happens correctly, but the exact mechanisms and pathways are unknown. Although much has been done to study protein unfolding in vitro, the behavior of proteins in the test tube is most likely not the same as that inside cells. The primary reason for the difference is that in vivo, proteins are unfolded by cellular machinery and not by denaturants as is typical for in vitro experiments. One reason that so little is known about how proteins behave when inside cells is the lack of methods capable of probing the folding and unfolding of proteins in cellular contexts. Without knowledge of the in vivo unfolding of proteins, in vitro unfolding studies must be extrapolated to the context of cells, an endeavor that will most likely not provide information about what is really unknown: what proteins are doing in living cells.

The long-term goal of this research is to fully characterize the unfolding pathway of proteins in an in vivo context. The event we have chosen to focus on first is the unfolding of proteins prior to transport across the mitochondrial membrane, one of the best understood cellular unfolding events. The recent BRIN-supported project in my laboratory entitled "Protein unfolding / refolding during mitochondrial import" has addressed the early stages of protein unfolding prior to import. The objective of this application is the next logical step towards the long-term goal: understanding how proteins located in the outer membrane of mitochondria alter the normal, regularly folded conformation of proteins prior to import. The hypothesis is that Tom20 and/or Tom 22 induce some protein unfolding prior to mitochondrial import and that the protocols and techniques developed in the past two years will allow detailed analysis of the unfolding. The rationale for this work includes two things: 1) an understanding of how Tom proteins unfold proteins in the course of mitochondrial import and 2) the creation of new, improved methods for understanding how proteins structure is altered in real cellular contexts. Having dealt with these systems and techniques for the past three years, our research environment is very conducive for success.

To determine how Tom20 and Tom22 proteins alter the conformation of proteins that are imported into mitochondria, the following specific aim will be accomplished:

Understand what protein unfolding is catalyzed by the Tom20 & Tom22 proteins Based on literature reports, Tom20 and Tom22 interact with proteins just prior to transport. Binding is specific and may induce unfolding. We expect that our techniques will indicate if and where unfolding occurs in proteins interacting with Tom20/22.

With our innovative techniques of hydrogen exchange (HX) combined with mass spectrometry (MS), we expect to be able to see unfolding at a level of detail previously not possible. The outcome of this proposed research is expected to be detailed information about in vivo catalyzed unfolding by several proteins involved in early steps of mitochondrial import. This information is important if we are to understand the complex process catalyzed by import machinery. The results will contribute to basic knowledge about mitochondria-induced protein unfolding and cellular unfolding in general. Such information may ultimately be beneficial for understanding and treatment of diseases associated with improper protein transport. An additional benefit of successful completion is refinement of the techniques and protocols for probing protein unfolding and folding in vivo which could logically be extended to investigating other protein unfolding or folding events in vivo.