Project Summary/AbstractAlzheimer's disease (AD) is a fatal neurodegenerative disease affecting 5.5 million Americans. Despitemany decades of research there is still no known cure. AD is a protein misfolding disease where theAlzheimer's protein A aggregates from a random coil entity into fibrils which are highly organizedaggregates containing a cross- sheet structure. However the nature of the toxic species in Alzheimer'sdisease remains unknown. More and more attention has been given to the possibility that A aggregateswithin mitochondria rather than extracellular deposits of A may be responsible for the onset andprogression of the disease. Nature has developed mechanisms to prevent disease-associated proteinaggregation e.g. by the introduction of heat shock proteins (Hsp's) which are overexpressed when cellsundergo stress. The most important Hsp's in mitochondria are Hsp60 and Hsp70 whereas Hsp60 is theonly essential chaperone in bacteria yeast and mammals. It is known that Hsp60 iscytoprotective againstmany stressors in cells and is proposed to be directly protective against AD. However nothing is knownabout the mechanism of how this is achieved.Since almost nothing is known about the mitochondrial Hsp60 system including its co-chaperone Hsp10 Iwill use aims 1 and 2 of this proposal to explore this chaperone in absence of substrate protein. I seek tosolve the structure of different states of the chaperone during its protein folding cycle by cryo-electronmicroscopy (cryo-EM). Further using novel solution-state NMR methods to study sparsely-populatedstates I will investigate how the co-chaperone is involved in the reaction cycle. This will provide importantinformation on the role of Hsp10 in substrate encapsulation. Further during my independent phase I willuse the fundament knowledge obtained through aims 1 and 2 to move on to aim 3 where I will study theinteraction of Hsp60 with A. I will not only elucidate the structure of long-lived major states of A bound toHsp60 after substrate encapsulation by cryo-EM but also study the transient interaction of A with Hsp60during the initial phase of the substrate folding cycle prior to encapsulation by solution-state NMR. Thesestudies will provide information about which A species (monomer oligomer or fibril) interact with Hsp60and elucidate their structural features. Aims 1 and 2 will provide insights into fundamental questions aboutthe mechanisms chaperones use to efficiently fold proteins into their functional forms. Further the resultsfrom aim 3 will unveil details about how Hsp60 inhibits A aggregation and prevents neurodegenerativediseases and may open up novel therapeutic strategies against Alzheimer's disease.