Temperature-dependent sensitivity enhancement of solid-state NMR spectra of alpha-synuclein fibrils.
The protein alpha-synuclein (AS) is the primary fibrillar component of Lewy bodies, the pathological hallmark of Parkinson's disease. Wild-type human AS and the three mutant forms linked to Parkinson's disease (A53T, A30P, and E46K) all form fibrils through a nucleation-dependent pathway; however, the biophysical details of these fibrillation events are not yet well understood. Atomic-level structural insight is required in order to elucidate the potential role of AS fibrils in Parkinson's disease. Here we show that low temperature acquisition of magic-angle spinning NMR spectra of wild type AS fibrils-greatly enhances spectral sensitivity, enabling the detection of a substantially larger number of spin systems. At 0 +/- 3 degrees C sample temperature, cross polarization (CP) experiments yield weak signals. Lower temperature spectra (-40 +/- 3 degrees C) demonstrated several times greater signal intensity, an effect further amplified in 3D 15N-13C-13C experiments, which are required to perform backbone assignments on this sample. Thus 3D experiments enabled assignments of most amino acids in the rigid part of the fibril (approximately residues 64 to 94), as well as tentative site-specific assignments for T22, V26, A27, Y39, G41, S42, H50, V52, A53, T54, V55, V63, A107, I112, and S129. Most of these signals were not observed in 2D or 3D spectra at 0 +/- 3 degrees C. Spectra acquired at low temperatures therefore permitted more complete chemical shift assignments. Observation of the majority of residues in AS fibrils represents an important step towards solving the 3D structure.
Kloepper
Kathryn D
KD
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Zhou
Donghua H
DH
Li
Ying
Y
Winter
Kem A
KA
George
Julia M
JM
Rienstra
Chad M
CM
D002247:Carbon Isotopes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D017433:Protein Structure, Secondary; D013696:Temperature; D051844:alpha-Synuclein / Q000737:chemistry*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D051844:alpha-Synuclein