1. MPP OUTSIDE REQUEST FORM Review Date: 10/25/11 Requestor(s): Dennis Winge Institution: U. Utah Request Title: Yeast mitochondrial protein SDH5

2. Notes from Dennis Winge:This is great news. We are working hard on Sdh5, so this news comes at a great time. I modeled Sdh5 and got a working model with high confidence using Phyre. The PyMol model is enclosed. The model shows one helix atop two other packed helices oriented in parallel and this helix has many of the highly conserved residues in Sdh5 (shown in sticks and cyan). We are currently testing the functional significance of these conserved residues by mutagenesis. We are trying to understand how Sdh5 mediates flavination of Sdh1. We have mutants of Sdh1 that diminish interaction with Sdh5 and diminish Sdh1 flavination. We are trying to set up an in vitro flavination assay with recombinant Sdh5. We have a construct that expresses well, but we would be very open to a collaboration with you on this study. We want to understand whether Sdh5 associates with FAD independently of Sdh1 or not. The conserved Sdh5 residues are likely an interface for interaction with Sdh1 and we want to address that. We would appreciate some of the recombinant protein and we are open to pursuing a collaborative study with you folks. Concerning your structural pursuit on Sdh5, there is a structure of the bacterial Sdh5 homolog YgfY. Thus the structure by itself won’t be a high impact study. However, combining it with other studies would make a very nice story. I would attempt HSQC +/- FAD and try to get the Sdh1 molecule for filtered HSQC studies to probe the interaction. That would be hot. We are pleased that the progress has been swift with Sdh5. Since you are close on sequential assignments and NOEs, we would appreciate an attempt at HSQC + FAD. We are starting on mutagenesis of Sdh5, on the two highly conserved helical segments. We want to establish that these segments form an interface for Sdh1 interaction and function. That would make a nice story. We have a reasonable expression system of His-tagged Sdh1, but it is largely in inclusion bodies. We have done some limited work in refolding it. That would be the biggest hurdle. It may work to refold it in the present of Sdh5. That may work for the HSQC. If you sent some Sdh5, we could try the refolding. We would also like some pure Sdh5, to enable us to generate antisera.