Kuen-Phon Wu

Structural Reorganization of α-Synuclein at Low pH Observed by NMR and REMD Simulations

alpha-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinsons disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that alpha-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize alpha-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid beta component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation.

Characterization of conformational and dynamic properties of natively unfolded human and mouse α-synuclein ensembles by NMR: implication for aggregation

Conversion of human alpha-synuclein (aS) from the free soluble state to the insoluble fibrillar state has been implicated in the etiology of Parkinsons disease. Human aS is highly homologous in amino acid sequence to mouse aS, which contains seven substitutions including the A53T that has been linked to familial Parkinsons disease, and including five substitutions in the C-terminal region. It has been shown that the rate of fibrillation is highly dependent on the exact sequence of the protein, and mouse aS is reported to aggregate more rapidly than human aS in vitro. Nuclear magnetic resonance experiments of mouse and human aS at supercooled temperatures (263 K) are used to understand the effect of sequence on conformational fluctuations in the disordered ensembles and to relate these to differences in propensities to aggregate. We show that both aS are natively unfolded at low temperature with different propensities to secondary structure, backbone dynamics and long-range contacts across the protein. Mouse aS exhibits a higher propensity to helical conformation around the C-terminal substitutions as well as the loss of transient long-range contacts from the C- to the N-terminal end and hydrophobic central regions of the protein relative to human aS. Lack of back-folding from the C-terminal end of mouse aS exposes the N-terminal region, which is shown, by (15)N relaxation experiments, to be very restricted in mobility relative to human aS. We propose that the restricted mobility in the N-terminal region may arise from transient interchain interactions, suggesting that the N-terminal KTK(E/Q)GV repeats may serve as initiation sites for aggregation in mouse aS. These transient interchain interactions coupled with a non-A beta amyloid component (NAC) region that is both more exposed and has a higher propensity to beta structure may accelerate the rate of fibril formation of aS.

Detection of Transient Interchain Interactions in the Intrinsically Disordered Protein α-Synuclein by NMR Paramagnetic Relaxation Enhancement

NMR paramagnetic relaxation enhancement experiments were applied to the intrinsically disordered protein alpha-synuclein, the primary protein in Parkinsons disease, to directly characterize transient intermolecular complexes at neutral and low pH. At neutral pH, we observed weak N- to C-terminal interchain contacts driven by electrostatic interactions, while at low pH, the C- to C-terminal interchain interactions are significantly stronger and driven by hydrophobic contacts. Characterization of these first interchain interactions will provide fundamental insight into the mechanism of amyloid formation.

The A53T mutation is key in defining the differences in the aggregation kinetics of human and mouse α-synuclein.

Despite a 95% sequence similarity, the aggregation of human and mouse α-synuclein is remarkably different, as the human form is slower than the mouse form in forming fibrils but is associated with Parkinsons disease in both humans and transgenic mice. Here, the amino acid code underlying these differences is investigated by comparing the lag times, growth rates, and secondary structure propensities of a systematic series of eight human-mouse chimeras. Fluorescence analysis of these variants shows that the A53T substitution dominates the growth kinetics, while the lag phase is affected by a combination of the A53T and S87N substitutions. The secondary structure propensities derived from an NMR chemical shift analysis of the monomeric forms of the human-mouse variants enable us to establish a link between the changes in the conformational properties in the region of position 53 upon mutation and the corresponding changes in growth rates. These results suggest that the presence of an alanine residue at position 53 may be an evolutionary adaptation to minimize Parkinsons disease in humans and indicates that effective drug development efforts may be directed to target this N-terminal region of the sequence.

Unveiling transient protein-protein interactions that modulate inhibition of alpha-synuclein aggregation by beta-synuclein, a pre-synaptic protein that co-localizes with alpha-synuclein

Pathology in Parkinson’s disease is linked to self-association of α-Synuclein (αS) into pathogenic oligomeric species and highly ordered amyloid fibrils. Developing effective therapeutic strategies against this debilitating disease is critical and βS, a pre-synaptic protein that co-localizes with αS, can act as an inhibitor of αS assembly. Despite the potential importance of βS as an inhibitor of αS, the nature, location and specificity of the molecular interactions between these two proteins is unknown. Here we use NMR paramagnetic relaxation enhancement experiments, to demonstrate that βS interacts directly with αS in a transient dimer complex with high specificity and weak affinity. Inhibition of αS by βS arises from transient αS/βS heterodimer species that exist primarily in head- to- tail configurations while αS aggregation arises from a more heterogeneous and weaker range of transient interactions that include both head-to-head and head-to-tail configurations. Our results highlight that intrinsically disordered proteins can interact directly with one another at low affinity and that the transient interactions that drive inhibition versus aggregation are distinct by virtue of their plasticity and specificity.

Fast hydrogen exchange affects 15N relaxation measurements in intrinsically disordered proteins

Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, 1H–15N HSQC, is used to measure the 15N transverse relaxation rate (R2), the measured R2 rate is convoluted with the HX rate (kHX) and has higher apparent R2 values. Since the 15N R2 measurement is important for analyzing protein backbone dynamics, the HX effect on the R2 measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing 15N R2CPMG experiments on α-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R2CPMG can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D2O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R2CPMG values be obtained by methods described herein.

Backbone NMR assignments of DFP-inhibited mature subtilisin E

Here we report the backbone chemical shifts of the DFP-inhibited mature subtilisin E, which was uniformly labeled by 13C, 15N with a supplement of excess calcium.

Transient Protein-Protein Interactions in the IDP Alpha-Synuclein Detected by NMR: Implications for Protein Aggregation

BioMaPS Institute for Quantitative Biology and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, 08854.NMR paramagnetic relaxation enhancement experiments (PREs) have been applied to the intrinsically disordered protein alpha-synuclein, the primary protein in Parkinsons disease, to directly characterize transient intermolecular complexes at neutral and low pH as well as ionic strength-dependent solutions at pH 6.0. At neutral pH, we observed weak N- to C-terminal inter-chain contacts that are driven by electrostatic interactions while at low pH, C- to C-terminal inter-chain interactions are significantly stronger and driven by hydrophobic contacts. In addition to the pH-dependent transient self-associated alpha-synuclein complex, we also detected the changes of transient protein-protein interactions of alpha-synyclein in solution at varied [NaCl] (0-500 mM). By using titration-based PRE experiments, we have calculated approximate 6% and 2% transient head-to-tail complexes of alpha-synuclein in solution without and with the addition of 100 mM NaCl, respectively. The results presented here show that 1H NMR paramagnetic relaxation experiments are a powerful tool for visualizing transient low-populated initial encounter complexes in intrinsically disordered proteins. Characterization of these first inter-chain interactions correlated to the aggregation kinetics will provide fundamental insight into the mechanism of amyloid formation.