Karol Wróblewski

New, universal tools for targeted protein degradation in bacteria can help to accelerate protein function studies and antimicrobial research. We describe a new method for degrading bacterial proteins using plasmid-encoded degrader peptides which deliver target proteins for degradation by a highly conserved ClpXP protease. We demonstrate the mode of action of the degraders on a challenging essential target, GroEL. The studies in bacteria are complemented by in vitro binding and structural studies. Expression of degrader peptides results in a temperature-dependent growth inhibition and depletion of GroEL levels over time. The reduction of GroEL levels is accompanied by dramatic proteome alterations. The presented method offers a new alternative approach for regulating protein levels in bacteria without genomic modifications or tag fusions. Our studies demonstrate that ClpXP is an attractive protease for the future use in bacterial-targeted protein degradation.

Simulating protein structure flexibility using classical methods is computationally demanding, especially for large proteins. To address this challenge, we have been developing the CABS-flex method, which enables fast simulations of protein structural flexibility by combining a coarse-grained simulation approach with all-atom detail. Previously available as the CABS-flex 2.0 web server, the method has now undergone a major upgrade with the release of CABS-flex 3.0. Key improvements include the introduction of intuitive flexibility modes that simplify the control of distance restraints and allow users to reflect known or expected dynamic regions; improved all-atom reconstruction for higher-quality model generation; a new feature for de novo peptide structure prediction, supporting both linear and cyclic peptides along with their conformational flexibility; and new tools for result analysis and visualization, facilitating deeper insights into structural flexibility. Additionally, AlphaFold pLDDT-derived restraints can be used as optional input for guiding simulations. The method accepts input as either a PDB/mmCIF structure or a sequence (for peptide modeling). Advanced options allow users to incorporate experimental or computational restraints. The CABS-flex 3.0 web server is available at https://lcbio.pl/cabsflex3. This website is free and open to all users, with no login requirement.

Understanding protein function often necessitates characterizing the flexibility of protein structures. However, simulating protein flexibility poses significant challenges due to the complex dynamics of protein systems, requiring extensive computational resources and accurate modeling techniques. In response to these challenges, the CABS‐flex method has been developed as an efficient modeling tool that combines coarse‐grained simulations with all‐atom detail. Available both as a web server and a standalone package, CABS‐flex is dedicated to a wide range of users. The web server version offers an accessible interface for straightforward tasks, while the standalone command‐line program is designed for advanced users, providing additional features, analytical tools, and support for handling large systems. This paper examines the application of CABS‐flex across various structure–function studies, facilitating investigations into the interplay among protein structure, dynamics, and function in diverse research fields. We present an overview of the current status of the CABS‐flex methodology, highlighting its recent advancements, practical applications, and forthcoming challenges.

New, universal tools for targeted protein degradation in bacteria can help to accelerate protein function studies and antimicrobial research. We have created a new method for degrading bacterial proteins using plasmid-encoded degrader peptides which deliver target proteins for degradation by a highly conserved ClpXP protease. We demonstrated the mode-of-action of the degraders on a challenging essential target GroEL. The studies in bacteria were complemented by in vitro binding and structural studies. Expression of degrader peptides resulted in a temperature-dependent growth inhibition and depletion of GroEL levels over time. The reduction of GroEL levels was accompanied by dramatic proteome alterations. The presented method offers a new alternative approach for regulating protein levels in bacteria without genomic modifications or tag fusions. Our studies demonstrate that ClpXP is an attractive protease for the future use in bacterial targeted protein degradation.

The structural modeling of peptides can be a useful aid in the discovery of new drugs and a deeper understanding of the molecular mechanisms of life. Here we present a novel multiscale protocol for the structure prediction of linear and cyclic peptides. The protocol combines two main stages: coarse-grained simulations using the CABS-flex standalone package and an all-atom reconstruction-optimization process using the Modeller program. We evaluated the protocol on a set of linear peptides and two sets of cyclic peptides, with cyclization through the backbone and disulfide bonds. A comparison with other state-of-the-art tools (APPTEST, PEP-FOLD, ESMFold and AlphaFold implementation in ColabFold) shows that for most cases, AlphaFold offers the highest resolution. However, CABS-flex is competitive, particularly when it comes to short linear peptides. As demonstrated, the protocol performance can be further improved by combination with the residue–residue contact prediction method or more efficient scoring. The protocol is included in the CABS-flex standalone package along with online documentation to aid users in predicting the structure of peptides and mini-proteins.