Biology subjects

Smirnov, S. L.

Publications and source records attributed to Smirnov, S. L..

4 recordsLinked to original sources

bioRxiv2026

Actin filament bundling by the Arabidopsis VILLIN4 C-terminus involves the basic segment of the IDR linker and is sensitive to temperature and ionic strength

Plant villins are ubiquitous proteins involved in F-actin regulation in all plant organs. Villins consist of folded core and headpiece domains connected by an intrinsically disordered linker. In plant villins these linkers include up to 200 amino acid residues, which is substantially larger than their vertebrate counterparts (~40 residues). Moreover, the linkers in most types of plant villins exhibit a distinct charge partitioning pattern: a larger basic N-terminus and a smaller acidic C-terminus. The roles of these distinct, sizable and disordered linkers in plant villins are not well understood and are currently under active scrutiny. Here we demonstrate that the basic portion of the linker in Arabidopsis thaliana VILLIN4 (AtVLN4) is capable of actin filament bundling. These interactions are also affected by the presence of the adjacent headpiece domain and are altered by temperature and levels of potassium ions, major abiotic regulators that influence plant growth. Based on these findings, we propose that the F-actin bundling capacity of the basic segment in AtVLN4 is responsive to environmental changes and is a uniform property in closely related villins due to high sequence homology across multiple species.

biochemistry↗

bioRxiv2026

Undergraduate Biophysical Chemistry Series: Teaching through a Combination of a Purpose-built Textbook, Research-derived Biomolecular Samples and Computer Labs

Biophysics is a rapidly advancing field with an incredible breadth of topics. Thus, undergraduate biophysics instructors have to strategize and decide what topics they will cover in their courses. Educational institutions utilize a variety of biophysics textbooks. A common deficiency of each of the existing texts is that it serves well a given set of topics (theory, illustrations, practice problems) and leaves out other areas. A typical example includes good theory and problems for thermodynamics and kinetics while presenting molecular dynamics and various spectroscopic methods in a lacking or outdated way. The authors of this manuscript teach a capstone Biophysical Chemistry three-quarter series (Western Washington University/WWU, Bellingham, WA) which ideally should resonate with the general and major-specific courses the students take within their major at WWU. To achieve this goal and to enrich the traditional lecture-based delivery, the instructors have developed and brought together key pedagogical elements: purpose-built online textbook with a uniform structure of the academic content and practice problems, a study sample (oligopeptide) of biophysical significance with a growing set of experimental and computational data and student-centric in-class activities including computer labs. Our Biophysical series emphasizes concepts and methods of computational structural biology (Molecular Dynamics) and spectroscopic approaches (IR, UV and NMR). Here we describe the details of our integrative approach, summarize key outcomes and chart ways to advance the biophysical chemistry series further. Our textbook can be found through LibreText.

scientific communication and education↗

bioRxiv2026

Mutation-induced heterogeneity of the β7-β8 loop of the Staphylococcus aureus class A sortase leading to enhanced catalytic efficiency characterized by NMR and enzyme kinetics

Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino acid, and the second, an N-terminal glycine. The class A sortase from Staphylococcus aureus (saSrtA) was the first to be identified, and over 25 years later, the most widely used SML variants continue to be derivatives of a directed-evolution-identified pentamutant of saSrtA, or saSrtA5M. We previously characterized P94, a position mutated in saSrtA5M that interacts directly with a structurally conserved loop (the {beta}7-{beta}8 loop) near the active site of wild-type saSrtA only in the inactive conformation. This work revealed that the single P94X mutation dramatically affects relative saSrtA activity, as well as specificity for the P2 (or X) position in the LPXTG recognition motif. This is largely driven by Km effects. Here, we further interrogated P94 by probing structural changes in the active, apo state of saSrtA in the presence of the P94D mutation, as well as via mutations in Y187, the {beta}7-{beta}8 loop residue hypothesized to interact directly with P94. The saSrtA enzyme is allosterically activated by calcium; therefore, we were interested if P94D would induce structural changes in the calcium-bound apo enzyme. We used 1H-15N NMR experiments to compare spectra between enzymatically inactive variants of saSrtA with and without the P94D mutation. We also used NMR to calculate relative binding affinities for a pentapeptide substrate to these variants, as well as enzymatically inactive saSrtA5M. Our NMR data, in combination with enzymatic assays using active variants confirmed differences in the active, apo states of these enzymes. Overall, this work provides additional atomic detail regarding the importance of the P94 residue in saSrtA substrate recognition.

biochemistry↗

bioRxiv2023

Nine-residue low-complexity disordered peptide as a model system, an NMR/CD study

Disordered proteins and protein segments can be crucial for biological function. In this work we present a detailed biophysical characterization of the low-complexity nine-residue peptide with the sequence GGKGMGFGL. Based on proton solution NMR chemical shifts, circular dichroism measurements, as well as the analysis of concentration dependence of NMR linewidth, proton longitudinal relaxation times, hydrogen-deuterium exchange measurements, and 15N rotating frame NMR relaxation measurements, we conclude that the peptide is fully disordered and monomeric in solution. The peptide will serve as a model system for future structural and dynamics studies of biologically relevant disordered peptides in solution and solid states.

biochemistry↗