The disruption of alpha-synuclein (alpha-syn) homeostasis in neurons is a potential cause of Parkinson's disease, which is manifested pathologically by the appearance of alpha-syn aggregates, or Lewy bodies. Treatments for neurological diseases are extremely limited. To study the potential use of gold nanoparticles (Au NPs) to limit alpha-syn misfolding, the binding and orientation of alpha-syn on Au NPs were investigated. alpha-Syn was determined to interact with 20 and 90 nm Au NPs via multilayered adsorption: a strong electrostatic interaction between alpha-syn and Au NPs in the hard corona and a weaker noncovalent protein protein interaction in the soft corona. Spectroscopic and light-scattering titrations led to the determinations of binding constants for the Au NP alpha-syn coronas: for the hard corona on 20 nm Au NPs, the equilibrium association constant was 2.9 +/- 1.1 X 10(9) M-1 (for 360 +/- 70 alpha-syn/NP), and on 90 nm Au NPs, the hard corona association constant was 9.5 +/- 0.8 X 10(10) M-1 (for 5300 +/- 700 alpha-syn/NP). The binding of the soft corona was thermodynamically unfavorable and kinetically driven and was in constant exchange with "free" alpha-syn in solution. A protease digestion method was used to deduce the alpha-syn orientation and structure on Au NPs, revealing that alpha-syn absorbs onto negatively charged Au NPs via its N-terminus while apparently retaining its natively unstructured conformation. These results suggest that Au NPs could be used to sequester and regulate alpha-syn homeostasis.