Molecular-level secondary structure, polymorphism, and dynamics of full-length α-synuclein fibrils studied by solid-state NMR

Abstract

The 140-residue protein ␣-synuclein (AS) is able to form amyloid fibrils and as such is the main component of protein inclusions involved in Parkinson's disease. We have investigated the structure and dynamics of full-length AS fibrils by high-resolution solid-state NMR spectroscopy. Homonuclear and heteronuclear 2D and 3D spectra of fibrils grown from uniformly 13 C͞ 15 N-labeled AS and AS reverse-labeled for two of the most abundant amino acids, K and V, were analyzed. 13 C and 15 N signals exhibited linewidths of <0.7 ppm. Sequential assignments were obtained for 48 residues in the hydrophobic core region. We identified two different types of fibrils displaying chemical-shift differences of up to 13 ppm in the 15 N dimension and up to 5 ppm for backbone and side-chain 13 C chemical shifts. EM studies suggested that molecular structure is correlated with fibril morphology. Investigation of the secondary structure revealed that most amino acids of the core region belong to ␤-strands with similar torsion angles in both conformations. Selection of regions with different mobility indicated the existence of monomers in the sample and allowed the identification of mobile segments of the protein within the fibril in the presence of monomeric protein. At least 35 C-terminal residues were mobile and lacked a defined secondary structure, whereas the N terminus was rigid starting from residue 22. Our findings agree well with the overall picture obtained with other methods and provide insight into the amyloid fibril structure and dynamics with residue-specific resolution.EM ͉ protein structure ͉ amyloid ͉ Parkinson's disease ͉ protein aggregation T he ability of a protein to form amyloid fibrils is increasingly recognized as a general property of all polypeptide sequences (1). It is a phenomenon common to numerous neurodegenerative diseases such as Alzheimer's and Parkinson's diseases and spongiform encephalopathies (2, 3). Thus, the investigation of the mechanism(s) of protein misfolding as well as the detailed structures of amyloid fibrils is of paramount interest (4, 5). Parkinson's disease is defined by the presence of intracellular inclusions in dopaminergic neurons, the so-called Lewy bodies, which contain a high content of fibrils formed from the 140-aa cytoplasmic protein ␣-synuclein (AS) (6). The question of whether the mature fibrils themselves or rather protofilaments or folding intermediates are the neurotoxic species responsible for the cell death of dopaminergic neurons is still a subject of great controversy (7-9).AS belongs to the class of natively unfolded proteins, i.e., it lacks a well-defined secondary structure (10, 11), although long-range interactions have been shown to stabilize an aggregation-autoinhibited global protein architecture (12). Three regions of the protein can be classified. (i) The amphipathic N terminus (residues 1-60) consists of imperfect 11-mer repeats, with the consensus motif KTKEGV. (ii) The predominantly hydrophobic middle, also known as non-A␤ component region (residues 61-95...

250GHz CW gyrotron oscillator for dynamic nuclear polarization in biological solid state NMR

“…Two research areas in which high resolution MAS experiments have proved especially successful are studies of amyloid fibrils [37,[51][52][53][54][55][56][57] and membrane proteins [42,[58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75]. However, in both of these cases, low sensitivity currently limits the information that can be gleaned from the spectra.…”

Sensitivity enhancement in 13C solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing 1H T1 relaxation

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] Particularly, use of protein micro-/nano-crystals [17] has significantly improved resolution in high-resolution SSNMR of dilute spins such as 13 C and 15 N, permitting signal assignment and structural determination of various uniformly 13 C and/or 15 N-labeled proteins by SSNMR. [18][19][20][21][22][23][24][25] However, restricted sensitivity in 13 C and 15 N SSNMR has been still one of the major limiting factors in SSNMR analysis of proteins. In an experimental time required for 13 C SSNMR of proteins, more than 95 % is typically consumed for recycle delays to retrieve spin polarization by 1 H T 1 relaxation, to protect a probe from arcing due to RF irradiation, or to avoid sample degradation due to heating.…”

Charge neutralization and collapse of the C‐terminal tail of alpha‐synuclein at low pH

“…14 The aggregated, fibrillar form of aS displays the typical, highly ordered, cross-beta structure of amyloid fibrils, with the ordered fibril core containing residues $30-100. [15][16][17][18] Diverse oligomeric forms of the protein have also been observed, including short, ring-like protofibrils, short chain protofibrils, 19 and other more globular forms with different secondary structure contents. 20 Because some of the PD linked aS mutations accelerate fibril formation (A53T, E46K) [21][22][23][24][25] while others inhibit it (A30P), 19 but all increase oligomer formation, oligomers have been proposed to be the toxic forms of the protein.…”