From Feynman's ratchet to time crystalline molecular motors.

Cats have an instinctive ability to use the connection governing parallel transport in the space of shapes to land safely on their feet. Here, we argue that the concept of connection, which is extensively used in general relativity and other parts of theoretical physics, also explains the impressive performance of molecular motors by enabling molecules to evade the conclusions of Feynman's ratchet-and-pawl analysis. First, we demonstrate the emergence of directed rotational motion from shape changes, which is independent of angular momentum. Then, we computationally design knotted polyalanine molecules and demonstrate the organization of individual atom thermal vibrations into collective rotational motion, which is independent of angular momentum. The motion occurs effortlessly even in ambient water and can be further enhanced through spontaneous symmetry breaking, rendering the molecule an effective theory time crystal. Our findings can be experimentally verified via nuclear magnetic resonance measurements and hold practical potential for molecular motor design and engineering.

Excited-state dynamics of all-trans protonated retinal Schiff base in CRABPII-based rhodopsin mimics.

The rhodopsin mimic is a chemically synthetized complex with retinyl Schiff base (RSB) formed between protein and the retinal chromophore that can mimic the natural rhodopsin-like protein. The artificial rhodopsin mimic is more stable and designable than the natural protein and hence has wider uses in photon detection devices. The mimic structure RSB, like the case in the actual rhodopsin-like protein, undergoes isomerization and protonation throughout the photoreaction process. As a result, understanding the dynamics of the RSB in the photoreaction process is critical. In this study, the ultrafast transient absorption spectra of three mutants of the cellular retinoic acid-binding protein II-based rhodopsin mimic at acidic environment were recorded, from which the related excited-state dynamics of the all-trans protonated RSB (AT-PRSB) were investigated. The transient fluorescence spectra measurements are used to validate some of the dynamic features. We find that the excited-state dynamics of AT-PRSB in three mutants share a similar pattern that differs significantly from the dynamics of 15-cis PRSB of the rhodopsin mimic in neutral solution. By comparing the dynamics across the three mutants, we discovered that the aromatic residues near the ß-ionone ring structure of the retinal may help stabilize the AT-PRSB and hence slow down its isomerization rate. The experimental results provide implications on designing a rhodopsin-like protein with significant infrared fluorescence, which can be particularly useful in the applications in biosensing or bioimaging in deeper tissues.

Ultralow-threshold green fluorescent protein laser based on high Q microbubble resonators.

Biological lasers have attracted vast attention because of their potential medical application prospects, especially the low threshold biological laser that can be used for ultrasensitive biological detection while leaving the luminous gain medium undamaged by the pump light. By coupling the low concentration green fluorescent protein (GFP) solution with a high Q whispering gallery mode microbubble resonator, we managed to fabricate a miniature GFP laser with the lowest threshold and highest Q value compared to any known type of the GFP laser. The threshold energy is as low as 380 fJ, two orders of magnitude lower than any type of GFP laser at present. The Q value of the optical cavity in this biological laser is 5.3 × 107, two orders higher than the highest Q value of GFP lasers. We further confirmed the long-term stability of the working characteristics of GFP laser. It can work well nearly a month in temperature 3-4°C. Finally, we measured the effects of different concentrations of fluorescent protein on laser threshold. The data show that this biological laser can be used for highly sensitive detection of GFP concentration, which is particularly useful when the GFP is used as tracers.

In silico study on the effects of disulfide bonds in ORF8 of SARS-CoV-2.

The COVID-19 epidemic, caused by virus SARS-CoV-2, has turned into a pandemic and threatened everyone's health for the past two years. In SARS-CoV-2, ORF8 is one of the most important accessory proteins with a role in immune modulation. There are multiple disulfide bonds in the wild type (WT) ORF8. Here, we present an in silico study on the effects of the disulfide bonds in ORF8 on the aspects of the structural properties and binding properties with the human leukocyte antigen (HLA-A). We first define five possible states for ORF8 with different disulfide bond reduction schemes. For each state, we collect the conformational ensemble using molecular dynamics (MD) simulations in an explicit solvent. From the analysis of the structural properties, we find that the reduction of the disulfide bonds has small effects on the global properties but much larger effects on the ORF8-specific region that is located on the surface of the ORF8 dimer. Interestingly, we find that the dimer does not break into two monomers at room temperature even if all the disulfide bonds get reduced. Further, we investigate the role of the disulfide bonds in the interactions with the human leukocyte antigen (HLA) by performing docking between HLA-A and the conformational ensembles of ORF8 in different states. We give predictions on the preferred binding sites for each state and validate the predictions for the WT dimer with the experimental data on epitopes. In the end, we evaluate the stability of the complexes formed between HLA-A and ORF8 in each state using MD simulations. Our observations can provide inspiration for inhibitor/drug design against ORF8 based on the binding pathway with HLA-A.

In silico method for identifying the key residues in a knotted protein: with MJ0366 as an example.

The knotted proteins are a class of rare but biologically important proteins, due to the special topology of their native structure. Here we present a simple in silico method to identify the key residues for knotting and unknotting in a knotted protein, using the trefoil protein MJ0366 as an example. We first simulate the folding process via the annealing molecular dynamics (AMD) simulations in the coarse-grained "Go"-like model. From the folding trajectories, we monitor the knotting process using the quantity "length of knot tails". In the meantime, we analyze the evolution of the local geometry of the Cα trace with the help of the Discrete Frenet Frame (DFF). We identify the key residues by correlating the local geometry at each residue with the variable "length of knot tails" in the folding process, where a higher correlation coefficient indicates that the residue is more important for knotting. We validate our method by comparing with the experimental results in the literature. With the same method, we further predict the key residues for unknotting MJ0366 using the AMD simulations in both the coarse-grained "Go"-like model and all-atom (AA) force field model, respectively. We find that the key residues for unknotting are partially overlapped with those for knotting, indicating that the pathways for unknotting and knotting are generally similar except for the existence of some non-native contact interactions in the unknotting process. This in silico method can provide a new insight for understanding the knotting and unknotting processes of a knotted protein.

Improved model on fluorescence decay in singlet fission materials.

Singlet fission (SF) materials are a kind of promising material for breaking the solar cell efficiency limit. Here we rebuild the four-electron spin Hamiltonian under our coordinate system and present an improved model described by the population evolution equations on fluorescence decay (FD) dynamics that contain several detailed physical processes. The improved model for total random molecular orientation gives a more consistent fitting on the experimental data [G. B. Piland et al., J. Phys. Chem. C, 2013, 117, 1224] about time-resolved FD of amorphous rubrene thin films in the presence of a strong magnetic field. The fitting can reflect the relative rates of the real physical processes. Further on, our results show two kinds of magnetic field effect for the variety of two molecular relative orientations with respect to each other and the magnetic field by investigating the singlet projection and FD dynamics of the system.

Can the geometry of all-atom protein trajectories be reconstructed from the knowledge of Cα time evolution? A study of peptide plane O and side chain Cß atoms.

We inquire to what extent can the geometry of protein peptide plane and side chain atoms be reconstructed from the knowledge of Cα time evolution. Due to the lack of experimental data, we analyze all atom molecular dynamics trajectories from the Anton supercomputer, and for clarity, we limit our attention to the peptide plane O atoms and side chain Cß atoms. We reconstruct their positions using four different approaches. Three of these are the publicly available reconstruction programs Pulchra, Remo, and Scwrl4. The fourth, Statistical Method, builds entirely on the statistical analysis of Protein Data Bank structures. All four methods place the O and Cß atoms accurately along the Anton trajectories; the Statistical Method gives results that are closest to the Anton data. The results suggest that when a protein moves under physiological conditions, its all atom structures can be reconstructed with high accuracy from the knowledge of the Cα atom positions. This can help to better understand and improve all atom force fields, and advance reconstruction and refinement methods for reduced protein structures. The results provide impetus for the development of effective coarse grained force fields in terms of reduced coordinates.

Clustering and percolation in protein loop structures.

BACKGROUND: High precision protein loop modelling remains a challenge, both in template based and template independent approaches to protein structure prediction. METHOD: We introduce the concepts of protein loop clustering and percolation, to develop a quantitative approach to systematically classify the modular building blocks of loops in crystallographic folded proteins. These fragments are all different parameterisations of a unique kink solution to a generalised discrete nonlinear Schrödinger (DNLS) equation. Accordingly, the fragments are also local energy minima of the ensuing energy function. RESULTS: We show how the loop fragments cover practically all ultrahigh resolution crystallographic protein structures in Protein Data Bank (PDB), with a 0.2 Ångström root-mean-square (RMS) precision. We find that no more than 12 different loop fragments are needed, to describe around 38 % of ultrahigh resolution loops in PDB. But there is also a large number of loop fragments that are either unique, or very rare, and examples of unique fragments are found even in the structure of a myoglobin. CONCLUSIONS: Protein loops are built in a modular fashion. The loops are composed of fragments that can be modelled by the kink of the DNLS equation. The majority of loop fragments are also common, which are shared by many proteins. These common fragments are probably important for supporting the overall protein conformation. But there are also several fragments that are either unique to a given protein, or very rare. Such fragments are probably related to the function of the protein. Furthermore, we have found that the amino acid sequence does not determine the structure in a unique fashion. There are many examples of loop fragments with an identical amino acid sequence, but with a very different structure.

Aspects of structural landscape of human islet amyloid polypeptide.

The human islet amyloid polypeptide (hIAPP) co-operates with insulin to maintain glycemic balance. It also constitutes the amyloid plaques that aggregate in the pancreas of type-II diabetic patients. We have performed extensive in silico investigations to analyse the structural landscape of monomeric hIAPP, which is presumed to be intrinsically disordered. For this, we construct from first principles a highly predictive energy function that describes a monomeric hIAPP observed in a nuclear magnetic resonance experiment, as a local energy minimum. We subject our theoretical model of hIAPP to repeated heating and cooling simulations, back and forth between a high temperature regime where the conformation resembles a random walker and a low temperature limit where no thermal motions prevail. We find that the final low temperature conformations display a high level of degeneracy, in a manner which is fully in line with the presumed intrinsically disordered character of hIAPP. In particular, we identify an isolated family of α-helical conformations that might cause the transition to amyloidosis, by nucleation.

A three dimensional visualisation approach to protein heavy-atom structure reconstruction.

BACKGROUND: A commonly recurring problem in structural protein studies, is the determination of all heavy atom positions from the knowledge of the central α-carbon coordinates. RESULTS: We employ advances in virtual reality to address the problem. The outcome is a 3D visualisation based technique where all the heavy backbone and side chain atoms are treated on equal footing, in terms of the Cα coordinates. Each heavy atom is visualised on the surfaces of a different two-sphere, that is centered at another heavy backbone and side chain atoms. In particular, the rotamers are visible as clusters, that display a clear and strong dependence on the underlying backbone secondary structure. CONCLUSIONS: We demonstrate that there is a clear interdependence between rotameric states and secondary structure. Our method easily detects those atoms in a crystallographic protein structure which are either outliers or have been likely misplaced, possibly due to radiation damage. Our approach forms a basis for the development of a new generation, visualization based side chain construction, validation and refinement tools. The heavy atom positions are identified in a manner which accounts for the secondary structure environment, leading to improved accuracy.

Degradation protection and enhanced biocompatibility of Mg alloys pretreated with plasma proteins.

After implantation of the Mg alloy in the human body, the adsorption of plasma protein on surface will cause a series of cell reactions and affect the degradation of Mg alloys. Herein, in vitro biological reactions of the ZK60 and AZ31 Mg alloys are analyzed in plasma protein environment. Combined with mass spectrometry analysis of the type of adsorbed proteins, it is shown that proteins such as fibrinogen, vitronectin, fibronectin, and prothrombin are prone to get adsorbed on the surface of the alloys than other proteins, leading to the promotion of MG63 cell adhesion and proliferation. The effect of selected proteins (fibrinogen, fibronectin, and prothrombin) on degradation of ZK60 and AZ31 Mg alloys is investigated using immersion tests. The degradation of AZ31 Mg alloy is significantly restrained with the presence of proteins. This is due to the protein adsorption effect on the sample surface. The molecular dynamics simulation results indicate that both fibrinogen and fibronectin tend to adsorb onto the AZ31 rather than ZK60, forming a stable protein layer on the AZ31 Mg alloy retarding the degradation of the samples. As to ZK60 alloy, the addition of protein inhibits the degradation in the short term, however, the degradation increases after a long time of immersion. This phenomenon is particularly pronounced in fibronectin solution.

Excited-State Dynamics of a CRABPII-Based Microbial Rhodopsin Mimic.

Microbial rhodopsin, a pivotal photoreceptor protein, has garnered widespread application in diverse fields such as optogenetics, biotechnology, biodevices, etc. However, current microbial rhodopsins are all transmembrane proteins, which both complicates the investigation on the photoreaction mechanism and limits their further applications. Therefore, a specific mimic for microbial rhodopsin can not only provide a better model for understanding the mechanism but also can extend the applications. The human protein CRABPII turns out to be a good template for design mimics on rhodopsin due to the convenience in synthesis and the stability after mutations. Recently, Geiger et al. designed a new CRABPII-based mimic M1-L121E on microbial rhodopsin with the 13-cis, syn (13C) isomerization after irradiation. However, it still remains a question as to how similar it is compared with the natural microbial rhodopsin, in particular, in the aspect of the photoreaction dynamics. In this article, we investigate the excited-state dynamics of this mimic by measuring its transient absorption spectra. Our results reveal that there are two components in the solution of mimic M1-L121E at pH 8, known as protonated Schiff base (PSB) and unprotonated Schiff base (USB) states. In both states, the photoreaction process from 13-cis, syn(13C) to all-trans,anti (AT) is faster than that from the inverse direction. In addition, the photoreaction process in the PSB state is faster than that in the USB state. We compared the isomerization time of the PSB state to that of microbial rhodopsin. Our findings indicate that M1-L121E exhibits behaviors similar to those of microbial rhodopsins in the general pattern of PSB isomerization, where the isomerization from 13C to AT is much faster than its inverse direction. However, our results also reveal significant differences in the excited-state dynamics of the mimic relative to the native microbial rhodopsin, including the slower PSB isomerization rates as well as the unusual USB photoreaction dynamics at pH = 8. By elucidating the distinctive characteristics of mimics M1-L121E, this study enhances our understanding of microbial rhodopsin mimics and their potential applications.

On the role of thermal backbone fluctuations in myoglobin ligand gate dynamics.

We construct an energy function that describes the crystallographic structure of sperm whale myoglobin backbone. As a model in our construction, we use the Protein Data Bank entry 1ABS that has been measured at liquid helium temperature. Consequently, the thermal B-factor fluctuations are very small, which is an advantage in our construction. The energy function that we utilize resembles that of the discrete nonlinear Schrödinger equation. Likewise, ours supports topological solitons as local minimum energy configurations. We describe the 1ABS backbone in terms of topological solitons with a precision that deviates from 1ABS by an average root-mean-square distance, which is less than the experimentally observed Debye-Waller B-factor fluctuation distance. We then subject the topological multi-soliton solution to extensive numerical heating and cooling experiments, over a very wide range of temperatures. We concentrate in particular to temperatures above 300 K and below the Θ-point unfolding temperature, which is around 348 K. We confirm that the behavior of the topological multi-soliton is fully consistent with Anfinsen's thermodynamic principle, up to very high temperatures. We observe that the structure responds to an increase of temperature consistently in a very similar manner. This enables us to characterize the onset of thermally induced conformational changes in terms of three distinct backbone ligand gates. One of the gates is made of the helix F and the helix E. The two other gates are chosen similarly, when open they provide a direct access route for a ligand to reach the heme. We find that out of the three gates we investigate, the one which is formed by helices B and G is the most sensitive to thermally induced conformational changes. Our approach provides a novel perspective to the important problem of ligand entry and exit.

Unusual crystal structures of MHC class I complexes reveal the elusive intermediate conformations explored during peptide editing in antigen presentation.

Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between conformational states as they sample peptides for potential ligands. To date, MHC I intermediates are largely uncharacterized experimentally and remain elusive. We present x-ray crystal structures of HLA-B8 loaded with 20mer peptides that show significant conformational heterogeneity at the N-terminus of the groove. Long stretches of N-terminal residues were missing in the electron density maps creating an unstructured and widely open-ended groove. Our structures also revealed highly unusual features in MHC I and peptide conformations, and in MHC I-peptide interaction at the N-terminus of the groove. Molecular dynamics simulations showed that the complexes have varying degrees of flexibility in a manner consistent with the structures. We suggest that our structures represent transient substates explored by MHC I molecules during peptide editing. The visualization of peptide-dependent conformational flexibility in MHC I groove is a major step forward in our conceptual understanding of peptide repertoire development in antigen presentation. Our study also raises questions about the role of the N-terminus of the groove in peptide editing.

Crystal structures of MHC class I complexes reveal the elusive intermediate conformations explored during peptide editing.

Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between numerous conformational states and these motions support peptide sampling. To date, MHC I intermediates are largely uncharacterized experimentally and remain elusive. Here, we present x-ray crystal structures of HLA-B8 loaded with 20mer peptides that show pronounced distortions at the N-terminus of the groove. Long stretches of N-terminal amino acid residues are missing in the electron density maps creating an open-ended groove. Our structures also reveal highly unusual features in MHC I-peptide interaction at the N-terminus of the groove. Molecular dynamics simulations indicate that the complexes have varying degrees of conformational flexibility in a manner consistent with the structures. We suggest that our structures have captured the remarkable molecular dynamics of MHC I-peptide interaction. The visualization of peptide-dependent conformational motions in MHC I is a major step forward in our conceptual understanding of dynamics in high-affinity peptide selection.

Long-Time Dynamics of Selected Molecular-Motor Components Using a Physics-Based Coarse-Grained Approach.

Molecular motors are essential for the movement and transportation of macromolecules in living organisms. Among them, rotatory motors are particularly efficient. In this study, we investigated the long-term dynamics of the designed left-handed alpha/alpha toroid (PDB: 4YY2), the RBM2 flagellum protein ring from Salmonella (PDB: 6SD5), and the V-type Na+-ATPase rotor in Enterococcus hirae (PDB: 2BL2) using microcanonical and canonical molecular dynamics simulations with the coarse-grained UNRES force field, including a lipid-membrane model, on a millisecond laboratory time scale. Our results demonstrate that rotational motion can occur with zero total angular momentum in the microcanonical regime and that thermal motions can be converted into net rotation in the canonical regime, as previously observed in simulations of smaller cyclic molecules. For 6SD5 and 2BL2, net rotation (with a ratcheting pattern) occurring only about the pivot of the respective system was observed in canonical simulations. The extent and direction of the rotation depended on the initial conditions. This result suggests that rotatory molecular motors can convert thermal oscillations into net rotational motion. The energy from ATP hydrolysis is required probably to set the direction and extent of rotation. Our findings highlight the importance of molecular-motor structures in facilitating movement and transportation within living organisms.

De Novo Design of a ß-Helix Tau Protein Scaffold: An Oligomer-Selective Vaccine Immunogen Candidate for Alzheimer's Disease.

Tau pathology is associated with many neurodegenerative disorders, including Alzheimer's disease (AD), where the spatio-temporal pattern of tau neurofibrillary tangles strongly correlates with disease progression, which motivates therapeutics selective for misfolded tau. Here, we introduce a new avidity-enhanced, multi-epitope approach for protein-misfolding immunogen design, which is predicted to mimic the conformational state of an exposed epitope in toxic tau oligomers. A predicted oligomer-selective tau epitope 343KLDFK347 was scaffolded by designing a ß-helix structure that incorporated multiple instances of the 16-residue tau fragment 339VKSEKLDFKDRVQSKI354. Large-scale conformational ensemble analyses involving Jensen-Shannon Divergence and the embedding depth D showed that the multi-epitope scaffolding approach, employed in designing the ß-helix scaffold, was predicted to better discriminate toxic tau oligomers than other "monovalent" strategies utilizing a single instance of an epitope for vaccine immunogen design. Using Rosetta, 10,000 sequences were designed and screened for the linker portions of the ß-helix scaffold, along with a C-terminal stabilizing α-helix that interacts with the linkers, to optimize the folded structure and stability of the scaffold. Structures were ranked by energy, and the lowest 1% (82 unique sequences) were verified using AlphaFold. Several selection criteria involving AlphaFold are implemented to obtain a lead-designed sequence. The structure was further predicted to have free energetic stability by using Hamiltonian replica exchange molecular dynamics (MD) simulations. The synthesized ß-helix scaffold showed direct binding in surface plasmon resonance (SPR) experiments to several antibodies that were raised to the structured epitope using a designed cyclic peptide. Moreover, the strength of binding of these antibodies to in vitro tau oligomers correlated with the strength of binding to the ß-helix construct, suggesting that the construct presents an oligomer-like conformation and may thus constitute an effective oligomer-selective immunogen.

First Principles Calculation of Protein-Protein Dimer Affinities of ALS-Associated SOD1 Mutants.

Cu,Zn superoxide dismutase (SOD1) is a 32 kDa homodimer that converts toxic oxygen radicals in neurons to less harmful species. The dimerization of SOD1 is essential to the stability of the protein. Monomerization increases the likelihood of SOD1 misfolding into conformations associated with aggregation, cellular toxicity, and neuronal death in familial amyotrophic lateral sclerosis (fALS). The ubiquity of disease-associated mutations throughout the primary sequence of SOD1 suggests an important role of physicochemical processes, including monomerization of SOD1, in the pathology of the disease. Herein, we use a first-principles statistical mechanics method to systematically calculate the free energy of dimer binding for SOD1 using molecular dynamics, which involves sequentially computing conformational, orientational, and separation distance contributions to the binding free energy. We consider the effects of two ALS-associated mutations in SOD1 protein on dimer stability, A4V and D101N, as well as the role of metal binding and disulfide bond formation. We find that the penalty for dimer formation arising from the conformational entropy of disordered loops in SOD1 is significantly larger than that for other protein-protein interactions previously considered. In the case of the disulfide-reduced protein, this leads to a bound complex whose formation is energetically disfavored. Somewhat surprisingly, the loop free energy penalty upon dimerization is still significant for the holoprotein, despite the increased structural order induced by the bound metal cations. This resulted in a surprisingly modest increase in dimer binding free energy of only about 1.5 kcal/mol upon metalation of the protein, suggesting that the most significant stabilizing effects of metalation are on folding stability rather than dimer binding stability. The mutant A4V has an unstable dimer due to weakened monomer-monomer interactions, which are manifested in the calculation by a separation free energy surface with a lower barrier. The mutant D101N has a stable dimer partially due to an unusually rigid ß-barrel in the free monomer. D101N also exhibits anticooperativity in loop folding upon dimerization. These computational calculations are, to our knowledge, the most quantitatively accurate calculations of dimer binding stability in SOD1 to date.

Optimizing Epitope Conformational Ensembles Using α-Synuclein Cyclic Peptide "Glycindel" Scaffolds: A Customized Immunogen Method for Generating Oligomer-Selective Antibodies for Parkinson's Disease.

Effectively presenting epitopes on immunogens, in order to raise conformationally selective antibodies through active immunization, is a central problem in treating protein misfolding diseases, particularly neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. We seek to selectively target conformations enriched in toxic, oligomeric propagating species while sparing the healthy forms of the protein that are often more abundant. To this end, we computationally modeled scaffolded epitopes in cyclic peptides by inserting/deleting a variable number of flanking glycines ("glycindels") to best mimic a misfolding-specific conformation of an epitope of α-synuclein enriched in the oligomer ensemble, as characterized by a region most readily disordered and solvent-exposed in a stressed, partially denatured protofibril. We screen and rank the cyclic peptide scaffolds of α-synuclein in silico based on their ensemble overlap properties with the fibril, oligomer-model and isolated monomer ensembles. We present experimental data of seeded aggregation that support nucleation rates consistent with computationally predicted cyclic peptide conformational similarity. We also introduce a method for screening against structured off-pathway targets in the human proteome by selecting scaffolds with minimal conformational similarity between their epitope and the same solvent-exposed primary sequence in structured human proteins. Different cyclic peptide scaffolds with variable numbers of glycines are predicted computationally to have markedly different conformational ensembles. Ensemble comparison and overlap were quantified by the Jensen-Shannon divergence and a new measure introduced here, the embedding depth, which determines the extent to which a given ensemble is subsumed by another ensemble and which may be a more useful measure in developing immunogens that confer conformational selectivity to an antibody.

Rational Generation of Monoclonal Antibodies Selective for Pathogenic Forms of Alpha-Synuclein.

Misfolded toxic forms of alpha-synuclein (α-Syn) have been implicated in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The α-Syn oligomers and soluble fibrils have been shown to mediate neurotoxicity and cell-to-cell propagation of pathology. To generate antibodies capable of selectively targeting pathogenic forms of α-Syn, computational modeling was used to predict conformational epitopes likely to become exposed on oligomers and small soluble fibrils, but not on monomers or fully formed insoluble fibrils. Cyclic peptide scaffolds reproducing these conformational epitopes exhibited neurotoxicity and seeding activity, indicating their biological relevance. Immunization with the conformational epitopes gave rise to monoclonal antibodies (mAbs) with the desired binding profile showing selectivity for toxic α-Syn oligomers and soluble fibrils, with little or no reactivity with monomers, physiologic tetramers, or Lewy bodies. Recognition of naturally occurring soluble α-Syn aggregates in brain extracts from DLB and MSA patients was confirmed by surface plasmon resonance (SPR). In addition, the mAbs inhibited the seeding activity of sonicated pre-formed fibrils (PFFs) in a thioflavin-T fluorescence-based aggregation assay. In neuronal cultures, the mAbs protected primary rat neurons from toxic α-Syn oligomers, reduced the uptake of PFFs, and inhibited the induction of pathogenic phosphorylated aggregates of endogenous α-Syn. Protective antibodies selective for pathogenic species of α-Syn, as opposed to pan α-Syn reactivity, are expected to provide enhanced safety and therapeutic potency by preserving normal α-Syn function and minimizing the diversion of active antibody from the target by the more abundant non-toxic forms of α-Syn in the circulation and central nervous system.