Biology subjects

Serebryany, E.

Publications and source records attributed to Serebryany, E..

8 recordsLinked to original sources

bioRxiv2026

Disulfide engineering reveals unexpected pro- and anti-aggregation conformers of human α-synuclein

Intrinsically disordered proteins can aggregate in many distinct conformations (polymorphs). Polymorphs are a striking example of fold-switching: one primary structure able to form distinct tertiary structures. Distinct polymorphs can yield distinct molecular, cellular, and disease phenotypes. Disulfide crosslinks canalize disordered proteins into distinct regions of the conformational landscape, even if the monomer remains disordered. Human -synuclein is a disordered, natively Cys-free protein involved in synaptic transmission. Its amyloid aggregation is implicated in Parkinsons disease and other synucleinopathies. Recent cryo-EM fibril structures have revealed distinct classes of amyloid polymorphs that wild-type human -synuclein can adopt. We created three double-Cys -synuclein variants predicted to form intramolecular disulfide bridges compatible with known amyloid polymorphs, plus three other double-Cys variants at arbitrary positions as controls. We purified all six variants as intramolecularly disulfide-crosslinked monomers in solution. Two of the three predicted pro-aggregation variants formed amyloid fibrils as expected, and two of the three control variants did not. Surprisingly, one of the controls formed amyloid fibrils with a distinctive morphology by negative-stain TEM, suggesting the possibility of a previously unknown polymorph. Conversely, the crosslinked variant that failed to aggregate as expected showed sub-stoichiometric, dose-dependent anti-aggregation activity, strongly suppressing amyloid formation by wild-type -synuclein.

biophysics↗

bioRxiv2025

Native-state dimerization drives misfolding and aggregation of Cys-depleted variants of cataract-associated human lens γD-crystallin

Cataract, the leading cause of blindness worldwide, results from age-related misfolding and aggregation of long-lived crystallin proteins in the eye lens. The cytoplasm of fiber cells in the lens core becomes increasingly oxidizing with age, allowing non-native disulfides to drive light-scattering aggregation of {gamma}-crystallins. Despite this vulnerability to non-native disulfides, and despite lacking any native-state disulfides, {gamma}-crystallins are unexpectedly Cys-rich. To understand this paradox, we investigated how replacing all four Cys residues in the aggregation-prone N-terminal domain of {gamma}D-crystallin affects its stability and aggregation. Cys removal precludes the disulfide-driven aggregation pathway we reported previously. Here, we characterize two full-length human {gamma}D-crystallin variants: C18S/C32S/C41S/C78S ("NCS") and C18T/C32A/C41A/C78A ("NCA/T"). Thermodynamic and kinetic stability measurements indicate the N-terminal domain was greatly destabilized in both variants relative to WT, with NCS more destabilized than NCA/T. Upon mild heating or partial denaturation, both variants formed light-scattering aggregates, which were amorphous by transmission electron microscopy. Surprisingly, the aggregation proceeded exclusively from a dimer of natively folded molecules held together by a C-terminal disulfide bridge. These dimers form readily even in the WT protein, and evidence of them has been found in the lens. Aggregation was strongly suppressed by the lenss native chemical chaperone, myo-inositol. The aggregation rate depended linearly on protein concentration, indicating that the rate limiting step was a transformation of the natively-folded to misfolded molecules within the dimer. We propose that many age-related chemical modifications could destabilize the native fold of human {gamma}D-crystallin, favor misfolding within disulfide-bridged dimers, and thereby cause aggregation.

biochemistry↗

bioRxiv2025

Enzyme-free biochemical production of seamlessly N-to-C cyclized peptides from natural or recombinant proteins

Many naturally occurring or synthetic cyclic peptides are valuable as pharmaceuticals, but this stable and versatile class of molecules has not yet found applications beyond medicine. The main reason is the high cost of developing, producing, and altering these molecules via the gold-standard solid-phase synthesis methods. We focus on a class of cyclic peptides that have no disulfides, only canonical amino acids, and seamless peptide backbones. Known as orbitides or circular bacteriocins, such compounds are ribosomally synthesized and enzymatically cyclized by plants and bacteria. We report a simple method for producing them from naturally abundant proteins or from recombinantly expressed precursor polypeptides. The reaction proceeds under mild aqueous conditions, without the need for enzymes, and using only one chemical reagent, which is readily available. We demonstrate production of a 17-mer cyclic peptide from a wild-type human eye lens {gamma}-crystallin and of a set of 10-residue cyclic peptides from recombinantly expressed polypeptide precursors. We investigate the effects of reaction conditions and sequence changes on reaction efficiency, identify the products by their complex mass spectrometry fragmentation patterns, and chromatographically separate linear and cyclic peptide forms. Our methodology opens the way to large-scale, cost-effective production of stable yet biodegradable, easily designable cyclic peptides for applications not only in medicine, but in areas like biotechnology, materials, agriculture, and pest control. It may also enable production of diverse cyclic peptide libraries from arbitrarily chosen natural protein sources.

biochemistry↗

bioRxiv2022

Cotranslational formation of disulfides guides folding of the SARS CoV-2 receptor binding domain

Many secreted proteins contain multiple disulfide bonds. How disulfide formation is coupled to protein folding in the cell remains poorly understood at the molecular level. Here, we combine experiment and simulation to address this question as it pertains to the SARS-CoV-2 receptor binding domain (RBD). We show that, whereas RBD can refold reversibly when its disulfides are intact, their disruption causes misfolding into a nonnative molten-globule state that is highly prone to aggregation and disulfide scrambling. Thus, non-equilibrium mechanisms are needed to ensure disulfides form prior to folding in vivo. Our simulations suggest that co-translational folding may accomplish this, as native disulfide pairs are predicted to form with high probability at intermediate lengths, ultimately committing the RBD to its metastable native state and circumventing nonnative intermediates. This detailed molecular picture of the RBD folding landscape may shed light on SARS-CoV-2 pathology and molecular constraints governing SARS-CoV-2 evolution.

biophysics↗

bioRxiv2022

Systematic conformation-to-phenotype mapping via limited deep-sequencing of proteins

Non-native conformations drive protein misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well-suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.

biophysics↗

bioRxiv2020

A native chemical chaperone in the human eye lens

Cataract is one of the most prevalent protein aggregation disorders and still the most common cause of vision loss worldwide. The metabolically quiescent core region of the human lens lacks cellular or protein turnover; it has therefore evolved remarkable mechanisms to resist light-scattering protein aggregation for a lifetime. We now report that one such mechanism involves an unusually abundant lens metabolite, myo-inositol, suppressing aggregation of lens crystallins. We quantified aggregation suppression using our previously well-characterized in vitro aggregation assays of oxidation-mimicking human {gamma}D-crystallin variants and investigated myo-inositols molecular mechanism of action using solution NMR, negative-stain TEM, differential scanning fluorometry, thermal scanning Raman spectroscopy, turbidimetry in redox buffers, and free thiol quantitation. Unlike many known chemical chaperones, myo-inositols primary target was neither the native nor the unfolded state of the protein, nor the final aggregated state, but rather the rate-limiting bimolecular step on the aggregation pathway. Given recent metabolomic evidence that it is severely depleted in human cataractous lenses compared to age-matched controls, we suggest that maintaining or restoring healthy levels of myo-inositol in the lens may be a simple, safe, and globally accessible strategy to prevent or delay lens opacification due to age-onset cataract.

biophysics↗

bioRxiv2019

Conformational catalysis of cataract-associated aggregation by interacting intermediates in a human eye lens crystallin

Human {gamma}D-crystallin (H{gamma}D) is an abundant and highly stable two-domain protein in the core region of the eye lens. Destabilizing mutations and post-translational modifications in this protein are linked to onset of aggregation that causes cataract disease (lens turbidity). Wild-type H{gamma}D greatly accelerates aggregation of the cataract-related W42Q variant, without itself aggregating. The mechanism of this \"inverse prion\" catalysis of aggregation remained unknown. Here we provide evidence that an early unfolding intermediate with an opened domain interface enables transient dimerization of the C-terminal domains of wild-type and mutant, or mutant and mutant, H{gamma}D molecules, which deprives the mutants N-terminal domain of intramolecular stabilization by the native domain interface and thus accelerates its misfolding to a distinct, aggregation-prone intermediate. A detailed kinetic model predicts universal power-law scaling relationships for lag time and rate of the resulting aggregation, which are in excellent agreement with the data. The mechanism reported here, which we term interface stealing, can be generalized to explain how common domain-domain interactions can have surprising consequences, such as conformational catalysis of unfolding, in multidomain proteins.\n\nSignificanceMost known proteins in nature consist of multiple domains. Interactions between domains may lead to unexpected folding and misfolding phenomena. This study of human {gamma}D-crystallin, a two-domain protein in the eye lens, revealed one such surprise: conformational catalysis of misfolding via intermolecular domain interface \"stealing.\" An intermolecular interface between the more stable domains outcompetes the native intramolecular domain interface. Loss of the native interface in turn promotes misfolding and subsequent aggregation, especially in cataract-related {gamma}D-crystallin variants. This phenomenon is likely a contributing factor in the development of cataract disease, the leading worldwide cause of blindness. However, interface stealing likely occurs in many proteins composed of two or more interacting domains.

biophysics↗

bioRxiv2018

Catalysis of cataract-associated human γD crystallin aggregation via dynamic disulfide exchange

Several mutations in human {gamma}D-crystallin (H{gamma}D), a long-lived eye lens protein, cause misfolding and aggregation, leading to cataract. Surprisingly, wild-type H{gamma}D catalyzes aggregation of its cataract related W42Q variant while itself remaining soluble - the inverse of the classical prion-like scenario whereby misfolded polypeptides catalyze aggregation of natively folded ones. The search for a biochemical mechanism of catalysis of W42Q aggregation by WT has revealed that WT H{gamma}D can transfer a disulfide bond to the W42Q variant. The transferred disulfide kinetically traps an aggregation-prone intermediate made accessible by the W42Q mutation, facilitating light-scattering aggregation of the W42Q variant. The aggregating variant thus becomes a disulfide sink, removing the disulfides from solution. Such redox \"hot potato\" competitions among wild-type and mutant or modified polypeptides may be relevant for many long-lived proteins that function in oxidizing environments. In these cases aggregation may be forestalled by inhibiting disulfide flow toward damaged polypeptides.

biochemistry↗