Biology subjects

Weil-Ktorza, O.

Publications and source records attributed to Weil-Ktorza, O..

6 recordsLinked to original sources

bioRxiv2023

The Role of Electron Spin-Polarizability and Charge Dynamics in Protein Function

Primitive nucleic acids and peptides likely collaborated during the earliest stages of biochemistry. What forces drove their interactions, and how did these forces shape the properties of primitive complexes? We investigated the association of two model primordial polypeptides with DNA. Coupling the peptides to a ferromagnetic substrate results in a dependence of the association rate and the extent of DNA binding on the orientation of magnetic moment of the substrate. The DNA binding could be nearly abolished by inverting the orientation of the magnetic field, despite the two polymers having complementary net charges. Inverting the chirality of either the entire peptide or just the connecting cysteine residue inverted the effect of the magnetic moment orientation. These results are attributed to the chiral-induced spin selectivity (CISS) effect, in which molecular chirality and electron spin interact to alter the electric polarizability of the protein. The observation of CISS effects governing simple protein-DNA complexes, suggests that this phenomenon was plausibly operative and potentially significant for primitive biomolecules. A key consequence of the CISS effect is to increase the kinetic stability of primitive protein-nucleic acid complexes. Taken together, our results show how emergent phenomena due to chirality and spin enhance bio-association.

biophysics↗

bioRxiv2023

Se-Glargine I. Chemical Synthesis of a Basal Insulin Analog Stabilized by an Internal Diselenide Bridge

Insulin, a small globular protein, has long provided a model for studies of biophysical principles with therapeutic application. The safety and efficacy of insulin replacement therapy for the treatment for diabetes mellitus have been enhanced by protein engineering. Here, we describe the chemical synthesis of a basal insulin analog stabilized by the substitution of an internal cystine (A6-A11) by a diselenide bridge. The studies focused on insulin glargine, the active component of clinical products Lantus(R) and Toujeo(R) (Sanofi). Formulated in solution at pH 4 in the presence of zinc ions, insulin glargine exhibits a shifted isoelectric point (from pH 4.5 to neutral pH) due to a basic extension of the B chain (ArgB31-ArgB32). Subcutaneous injection of such an acidic formulation leads to pH-dependent precipitation of protein-zinc complexes to form a long-lived depot. Pairwise substitution of CysA6 and CysA11 by selenocysteine (Sec; the 21st encoded amino acid) was effected by solid-phase peptide synthesis. The modified A chain also contained substitution of AsnA21 by Gly, introduced in glargine to avoid acid-catalyzed deamidation of the A21 carboxamide group in the formulation. Although classical chain combination of the di-Arg-extended B chain and modified A chain exhibited lower yield than does wild-type chain combination, substantial product was obtained through repeated reactions and successive purification. This strategy exemplifies the rational optimization of protein stability and may be generalizable to diverse disulfide-stabilized proteins of therapeutic interest.

biochemistry↗

bioRxiv2023

Se-Glargine II. Native Function of a Basal Insulin Analog Stabilized by an Internal Diselenide Bridge

Insulin glargine, the active component of basal clinical pharmaceutical formulations Lantus(R) and Toujeo(R) (Sanofi), provides a model for principles of therapeutic protein design. Formulated in solution at pH 4, insulin glargine exhibits a shifted isoelectric point (from pH 5.3 to neutral pH) due to a basic dipeptide B-chain extension (ArgB31-ArgB32). In the first article in this series, we described pairwise substitution of CysA6 and CysA11 by seleno-cysteine (Sec; the 21st encoded amino acid) by solid-phase peptide synthesis. 1H-2H amide proton exchange, as monitored by 1H-NMR spectroscopy, provides evidence that substitution of internal cystine A6-A11 by a diselenide bridge stabilizes the protein and damps segmental conformational fluctuations. Further, this analog and its major metabolites M1 and M2 (respectively denoting proteolytic derivatives lacking ArgB31-ArgB32 or ThrB30-ArgB31-ArgB32) exhibit native hormonal activity in mammalian cell-based assays measuring dose-dependent autophosphorylation of the insulin receptor (pIR/IR ratio) and metabolic gene regulation in human liver-derived HepG2 cells. The internal diselenide bridge also did not alter respective baseline mitogenicities of insulin glargine or its proteolytic products as evaluated by a qPCR-based assay of the balance between proliferative and antiproliferative cyclin gene expression; the assays employed L6 myoblasts over-expressing mitogenic IR isoform A. Given such native function, shelf life--and hence global access to insulin in the developing world--may be enhanced by stabilizing diselenide chemistry.

biochemistry↗

bioRxiv2023

Functional Ambidexterity of an Ancient Nucleic Acid-Binding Domain

Homochirality of biopolymers emerged early in the history of life on Earth, nearly 4 billion years ago. Whether the establishment of homochirality was the result of abiotic physical and chemical processes, or biological selection, remains unknown. However, given that significant events in protein evolution predate the last universal common ancestor, the history of homochirality may have been written into some of the oldest protein folds. To test this hypothesis, the evolutionary trajectory of the ancient and ubiquitous helix-hairpin-helix (HhH) protein family was analyzed for functional robustness to total chiral inversion of just one binding partner. Against expectations, functional ambidexterity was observed across the entire trajectory, from phase separation of HhH peptides with RNA to dsDNA binding of the duplicated (HhH)2-Fold. Moreover, dissociation kinetics, mutational analysis, and molecular dynamics simulations revealed significant overlap between the binding modes of a natural and a mirror-image protein to natural dsDNA. These data suggest that the veil between worlds with alternative chiral preferences may not be as impenetrable as is often assumed, and that the HhH protein family is an intriguing exception to the dogma of reciprocal chiral substrate specificity proposed by Milton and Kent (Milton et al. Science 1992).

biophysics↗

bioRxiv2022

Peptide-RNA Coacervates as a Cradle for the Evolution of Folded Domains

Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance (EPR) spectroscopy to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron-electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif reveals the presence of dimers, even in the absence of RNA, and transient -helical character. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. The distance distributions between spin labels are consistent with the symmetric (HhH)2-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.

biophysics↗

bioRxiv2020

Primordial emergence of a nucleic acid binding protein via phase separation and statistical ornithine to arginine conversion

De novo emergence, and emergence of the earliest proteins specifically, demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can peptides confer evolutionary relevant functions, let alone with minimal abiotic amino acid alphabets? How can such polypeptides evolve into mature proteins? Specifically, while nucleic acids binding is presumed a primordial function, it demands basic amino acids that do not readily form abiotically. To address these questions, we describe an experimentally-validated trajectory from a phase-separating polypeptide to a dsDNA-binding protein. The intermediates comprise sequence-duplicated, functional proteins made of only 10 amino acid types, with ornithine, which can form abiotically, as the only basic amino acid. Statistical, chemical modification of ornithine sidechains to arginine promoted structure and function. The function concomitantly evolved - from phase separation with RNA (coacervates) to avid and specific dsDNA binding - thereby demonstrating a smooth, gradual peptide-to-protein transition with respect to sequence, structure, and function.

biochemistry↗