Liquid-Liquid Phase Separation in Oligomeric Peptide Solutions.

Oligomeric peptides exist widely in living organisms and play a role in a broad range of biological functions. We report the first observation of liquid-liquid phase separation (LLPS) in peptide solutions, in particular, solutions of peptides consisting of noncovalent oligomers. We determined the binary phase boundary of the oligomeric peptide solution and compared the result to the well-established phase diagram of globular proteins. We also provide simple theoretical interpretations of the similarities and differences between the phase diagrams of peptides and proteins. Finally, by tuning inter-oligomer interactions using a crowding agent, we demonstrated that LLPS is a universal phenomenon that can be observed under different solution conditions for a variety of peptides.

Transformation of oligomers of lipidated peptide induced by change in pH.

Oligomerization of lipidated peptides is of general scientific interest and is important in biomedical and pharmaceutical applications. We investigated the solution properties of a lipidated peptide, Liraglutide, which is one of the glucagon-like peptide-1 (GLP-1) agonists used for the treatment of type II diabetes. Liraglutide can serve as a model system for studying biophysical and biochemical properties of micelle-like self-assemblies of the lipidated peptides. Here, we report a transformation induced in Liraglutide oligomers by changing pH in the vicinity of pH 7. This fully reversible transformation is characterized by changes in the size and aggregation number of the oligomer and an associated change in the secondary structure of the constituent peptides. This transformation has quite slow kinetics: the equilibrium is reached in a course of several days. Interestingly, while the transformation is induced by changing pH, its kinetics is essentially independent of the final pH. We interpreted these findings using a model in which desorption of the monomer from the oligomer is the rate-limiting step in the transformation, and we determined the rate constant of the monomer desorption.

Aß dimers differ from monomers in structural propensity, aggregation paths and population of synaptotoxic assemblies.

Dimers of Aß (amyloid ß-protein) are believed to play an important role in Alzheimer's disease. In the absence of sufficient brain-derived dimers, we studied one of the only possible dimers that could be produced in vivo, [Aß](DiY) (dityrosine cross-linked Aß). For comparison, we used the Aß monomer and a design dimer cross-linked by replacement of Ser²6 with cystine [AßS26C]2. We showed that similar to monomers, unaggregated dimers lack appreciable structure and fail to alter long-term potentiation. Importantly, dimers exhibit subtly different structural propensities from monomers and each other, and can self-associate to form larger assemblies. Although [Aß](DiY) and [AßS26C]2 have distinct aggregation pathways, they both populate bioactive soluble assemblies for longer durations than Aß monomers. Our results indicate that the link between Aß dimers and Alzheimer's disease results from the ability of dimers to further assemble and form synaptotoxic assemblies that persist for long periods of time.

Phase transitions in human IgG solutions.

Protein condensations, such as crystallization, liquid-liquid phase separation, aggregation, and gelation, have been observed in concentrated antibody solutions under various solution conditions. While most IgG antibodies are quite soluble, a few outliers can undergo condensation under physiological conditions. Condensation of IgGs can cause serious consequences in some human diseases and in biopharmaceutical formulations. The phase transitions underlying protein condensations in concentrated IgG solutions is also of fundamental interest for the understanding of the phase behavior of non-spherical protein molecules. Due to the high solubility of generic IgGs, the phase behavior of IgG solutions has not yet been well studied. In this work, we present an experimental approach to study IgG solutions in which the phase transitions are hidden below the freezing point of the solution. Using this method, we have investigated liquid-liquid phase separation of six human myeloma IgGs and two recombinant pharmaceutical human IgGs. We have also studied the relation between crystallization and liquid-liquid phase separation of two human cryoglobulin IgGs. Our experimental results reveal several important features of the generic phase behavior of IgG solutions: (1) the shape of the coexistence curve is similar for all IgGs but quite different from that of quasi-spherical proteins; (2) all IgGs have critical points located at roughly the same protein concentration at ~100 mg/ml while their critical temperatures vary significantly; and (3) the liquid-liquid phase separation in IgG solutions is metastable with respect to crystallization. These features of phase behavior of IgG solutions reflect the fact that all IgGs have nearly identical molecular geometry but quite diverse net inter-protein interaction energies. This work provides a foundation for further experimental and theoretical studies of the phase behavior of generic IgGs as well as outliers with large propensity to condense. The investigation of the phase diagram of IgG solutions is of great importance for the understanding of immunoglobulin deposition diseases as well as for the understanding of the colloidal stability of IgG pharmaceutical formulations.

Pathological crystallization of human immunoglobulins.

Condensation of Igs has been observed in pharmaceutical formulations and in vivo in cases of cryoglobulinemia. We report a study of monoclonal IgG cryoglobulins overexpressed by two patients with multiple myeloma. These cryoglobulins form crystals, and we measured their solubility lines. Depending on the supersaturation, we observed a variety of condensate morphologies consistent with those reported in clinical investigations. Remarkably, the crystallization can occur at quite low concentrations. This suggests that, even within the regular immune response to infections, cryoprecipitation of Ig can be possible.

Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins.

Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly, therefore, is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid ß-protein (Aß) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aß at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aß oligomers but were nontoxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of nontoxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.

Phase behavior of mixtures of human lens proteins Gamma D and Beta B1.

We have experimentally determined the coexistence surface characterizing the phase behavior of gammaD-betaB1-water ternary solutions. The coexistence surface fully describes the solution conditions, i.e., temperature, protein concentration, and protein composition, at which liquid-liquid phase separation occurs in a ternary solution. We have observed a significant demixing of gammaD and betaB1 i.e., large difference of composition in the two coexisting phases. This demixing suggests that the energy of the gammaD-betaB1 attractive interaction is significantly smaller than the energy of the gammaD-gammaD attractive interaction. We also observed the lowering of the phase separation temperature upon increasing of the fraction of betaB1 in solution. We provide a theoretical analysis of our experimental data, which enables a quantitative description of our principal experimental findings. In this way, we have evaluated the magnitude and temperature dependence of the relevant interprotein interaction energies. Our findings provide insight into the factors essential for maintaining lens proteins in a single homogeneous phase, thereby enabling lens transparency.

Despite its role in assembly, methionine 35 is not necessary for amyloid beta-protein toxicity.

An important component of the pathologic process underlying Alzheimer's disease is oxidative stress. Met(35) in amyloid beta-protein (A beta) is prone to participating in redox reactions promoting oxidative stress, and therefore is believed to contribute significantly A beta-induced toxicity. Thus, substitution of Met(35) by residues that do not participate in redox chemistry would be expected to decrease A beta toxicity. Indeed, substitution of Met(35) by norleucine (Nle) was reported to reduce A beta toxicity. Surprisingly, however, substitution of Met(35) by Val was reported to increase toxicity. A beta toxicity is known to be strongly related to its self-assembly. However, neither substitution is predicted to affect A beta assembly substantially. Thus, the effect of these substitutions on toxicity is difficult to explain. We revisited this issue and compared A beta 40 and A beta 42 with analogs containing Met(35)-->Nle or Met(35)-->Val substitutions using multiple biophysical and toxicity assays. We found that substitution of Met(35) by Nle or Val had moderate effects on A beta assembly. Surprisingly, despite these effects, neither substitution changed A beta neurotoxicity significantly in three different assays. These results suggest that the presence of Met(35) in A beta is not important for A beta toxicity, challenging to the prevailing paradigm, which suggests that redox reactions involving Met(35) contribute substantially to A beta-induced toxicity.

C-terminal peptides coassemble into Abeta42 oligomers and protect neurons against Abeta42-induced neurotoxicity.

Alzheimer's disease (AD) is an age-related disorder that threatens to become an epidemic as the world population ages. Neurotoxic oligomers of Abeta42 are believed to be the main cause of AD; therefore, disruption of Abeta oligomerization is a promising approach for developing therapeutics for AD. Formation of Abeta42 oligomers is mediated by intermolecular interactions in which the C terminus plays a central role. We hypothesized that peptides derived from the C terminus of Abeta42 may get incorporated into oligomers of Abeta42, disrupt their structure, and thereby inhibit their toxicity. We tested this hypothesis using Abeta fragments with the general formula Abeta(x-42) (x = 28-39). A cell viability screen identified Abeta(31-42) as the most potent inhibitor. In addition, the shortest peptide, Abeta(39-42), also had high activity. Both Abeta(31-42) and Abeta(39-42) inhibited Abeta-induced cell death and rescued disruption of synaptic activity by Abeta42 oligomers at micromolar concentrations. Biophysical characterization indicated that the action of these peptides likely involved stabilization of Abeta42 in nontoxic oligomers. Computer simulations suggested a mechanism by which the fragments coassembled with Abeta42 to form heterooligomers. Thus, Abeta(31-42) and Abeta(39-42) are leads for obtaining mechanism-based drugs for treatment of AD using a systematic structure-activity approach.

Oligomerization and phase transitions in aqueous solutions of native and truncated human beta B1-crystallin.

Human betaB1-crystallin is a major eye-lens protein that undergoes in vivo truncation at the N-terminus with aging. By studying native betaB1 and truncated betaB1DeltaN41, which mimics an age-related in vivo truncation, we have determined quantitatively the effect of truncation on the oligomerization and phase transition properties of betaB1 aqueous solutions. The oligomerization studies show that the energy of attraction between the betaB1DeltaN41 proteins is about 10% greater than that of the betaB1 proteins. We have found that betaB1DeltaN41 aqueous solutions undergo two distinct types of phase transitions. The first phase transition involves an initial formation of thin rodlike assemblies, which then evolve to form crystals. The induction time for the formation of rodlike assemblies is sensitive to oligomerization. The second phase transition can be described as liquid-liquid phase separation (LLPS) accompanied by gelation within the protein-rich phase. We refer to this process as heterogeneous gelation. These two phase transitions are not observed in the case of betaB1 aqueous solutions. However, upon the addition of poly(ethylene glycol) (PEG), we observe heterogeneous gelation also for betaB1. Our PEG experiments allow us to estimate the difference in phase separation temperatures between betaB1 and betaB1DeltaN41. This difference is consistent with the increase in energy of attraction found in our oligomerization studies. Our work suggests that truncation is a cataractogenic modification since it favors protein condensation and the consequent formation of light scattering elements, and highlights the importance of the N-terminus of betaB1 in maintaining lens transparency.