Distinguishing Different Conformations of Membrane‐Bound Alpha‐Synuclein through Distance Measurements

Abstract

There is an increasing prevalence of neurodegenerative diseases such as Parkinson's Disease (PD). According to the Parkinson's Foundation, more than 10 million people live with PD around the world with 60,000 Americans being diagnosed each year (Parkinson's Foundation; Marras et al., 2018). Alpha-synuclein (α-syn) is a membrane binding protein whose function is not completely understood but has been identified with an increased risk of developing PD. Intrinsically disordered in solution, membrane-bound α-syn has distinct pathological and physiological states. Pathologically, it has been observed to aggregate, leading to the formation of Lewy bodies, plaque build-up, and eventually the development of PD (Spinelli et al., 2014) Physiologically, α-syn is believed to be involved in the transport of neurotransmitters (Ramakrishnan et al., 2012). To better understand the pathological state of α-syn, a deeper understanding of the physiological structure was needed. Previous studies had found two different conformations of α-syn: a horseshoe and an extended helix (Ulmer et al., 2005; Trexler and Rhoades, 2009). In this proposal, distance measurements will be performed to distinguish between the two conformations using an electron paramagnetic resonance spectroscopy experiment known as double electron-electron resonance (DEER). DEER measures the dipolar interaction between two unpaired electrons in a system. To accomplish this, α-syn was mutated in order to introduce unpaired electrons. Site-directed mutagenesis and site-directed spin labeling were employed to prepare double spin-labeled mutants of α-syn for DEER experiments. The ultimate goal of this proposal is to model and generate double mutants of α-syn to perform distance measurements on using DEER. With carefully designed placement of the spin labeled sites, the distance constraints should be able to be used to predict if the horseshoe or extended conformation is being sampled as α-syn binds to lipid vesicles of different sizes. Marras, C.; Beck, J.C.; Bower, J.H.; Roberts, E.; Ritz, B.; Ross, G.W.; Abbott, R.D.; Savica, R.; Van Den Eeden, S.K.; Willis, A.W.; Tanner, C.M. Prevalence of Parkinson's Disease across North America. npj Parkinson's Disease 2018, 4(21). Parkinson's Foundation. “Statistics.” Parkinson's Foundation. Ramakrishnan, N.A.; Drescher, M.J.; Drescher, D.G. The SNARE Complex in Neuronal and Sensory Cells. Mol Cell Neurosci. 2012, 50(1), 58-69. Trexler, A.J. and Elizabeth Rhoades. &[alpha]-Synuclein Binds Large Unilamellar Vesicles as an Extended Helix. Biochemistry 2009, 48, 2304-2306. Ulmer, T.S.; Bax, A.; Cole, N.B.; Nussbaum, R.L. Structure and Dynamics of Micelle-Bound Human Alpha-Synuclein. J Biol Chem 2005, 280(10), 9595-9603. Spinelli, K.J.; Taylor, J.K.; Osterberg, V.R.; Churchill, M.J.; Pollock, E.; Moore, C.; Meshul, C.K.; Unni, V.K. Presynaptic Alpha-Synuclein Aggregation in a Mouse Model of Parkinson's Disease. Journal of Neuroscience 2014, 34(6), 2037-2050.