Biology subjects

Pavlenok, M.

Publications and source records attributed to Pavlenok, M..

4 recordsLinked to original sources

bioRxiv2026

Improved Calreticulin Nanobody by Framework Engineering

Calreticulin is an emerging cancer biomarker, but current detection methods rely on expensive monoclonal antibodies that suffer from inefficient protein production, pharmacokinetic challenges and poor tissue penetration. Cal3, a calreticulin-specific nanobody, was constructed by replacing the complimentary determining region 2 (CDR2) of a soluble, clinically validated nanobody with a calreticulin-specific CDR2 isolated from a phage display library. However, the poor solubility and low yield of Cal3 limit its usefulness. In this study, we engineered CALR-Nb02 by adapting the core of Cal3 to a partial consensus framework sequence of stable nanobodies. CALR-Nb02 was purified with a 240-fold higher yield as a predominantly monomeric, soluble protein that exhibits an increased thermal stability and a higher calreticulin binding affinity (KD: 25-50 nM) compared with Cal3. These results reveal a strategy for quickly altering the specificity of a stable nanobody, and provide an improved calreticulin-binding reagent for future diagnostic, imaging, and therapeutic applications.

bioengineering↗

bioRxiv2025

Master control of protein secretion by Mycobacterium tuberculosis

Tuberculosis is the leading cause of death from a single infectious disease. Mycobacterium tuberculosis secretes proteins using five ESX systems with distinctive functions essential for its growth and virulence. Here we show that a non-canonical supercomplex of the EsxU-EsxT proteins, encoded in the esx-4 locus, with the orphan EsxE-EsxF proteins, encoded in the cpnT operon, is required for toxin secretion by M. tuberculosis. Surprisingly, the outer membrane localization of all Esx proteins and their secretion into the cytosol of infected macrophages also depend on the EsxEF-EsxUT supercomplex and ESX-4. These results not only demonstrate that the Esx proteins have dual functions as the long-sought outer membrane components of ESX systems and as secreted effector proteins, but also reveal a novel master control mechanism of protein secretion in M. tuberculosis. The mutual dependency of EsxEF and EsxUT on each other synchronizes ESX effector protein secretion, enabling M. tuberculosis to block phagosomal maturation and to permeabilize the phagosomal membrane only when it is capable of killing host cells by toxin secretion. The requirement of the ESX-4 system for general protein secretion is a critical vulnerability which could be targeted by drugs and/or vaccines to simultaneously block many virulence factors of M. tuberculosis.

microbiology↗

bioRxiv2021

Control of subunit stoichiometry in single-chain MspA nanopores

Transmembrane protein channels enable fast and highly sensitive electrical detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level as compared to other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Asymmetric mutations within subunits of the channel protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, random subunit assembly does not allow control of the channel composition of MspA and other oligomeric protein pores. In this study, we showed that it is feasible to convert octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. We constructed single-chain MspA trimers, pentamers, hexamers and heptamers to demonstrate that it is feasible to alter the subunit stoichiometry and the MspA pore diameter. All single-chain MspA proteins formed functional channels in lipid bilayer experiments. Importantly, we demonstrated that single-chain MspA discriminated all four nucleotides identical to MspA produced from monomers. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications. STATEMENT OF SIGNFICANCENanopore sequencing of DNA is a fast and cheap technology that uniquely delivers multi-kilobase reads. It is currently used world-wide in many applications such as genome sequencing, epigenetics, and surveillance of viral and bacterial pathogens and has started to revolutionize human lives in medicine, agriculture and environmental studies. However, the high base-calling error rates prevent nanopore DNA sequencing from reaching its full potential. In this study, we converted octameric MspA into a single-chain pore enabling asymmetric mutations to fine-tune the pore geometry and chemistry and address the shortcomings of nanopores. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

biophysics↗

bioRxiv2020

Stable Polymer Bilayers for Protein Channel Recordings at High Guanidinium Chloride Concentrations

Use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leak-free at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers which exhibit deleterious leakage under similar conditions. Further, insertion and functional analysis of channels such as -hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that -hemolysin channels maintain their structural integrity at 2M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.

biophysics↗