Maximilian Fottner


Last Name

Fottner

First Name

Maximilian

ORCID

0000-0002-8628-183X

Organisational unit

09740 - Lang, Kathrin / Lang, Kathrin

Filters

2024 - 20254
Reset filters

Search Results

Publications 1 - 4 of 4
  • Hijacking a bacterial ABC transporter for genetic code expansion
    Item type: Journal Article
    Iype , Tarun; Fottner, Maximilian; Böhm, Paul; et al. (2025)
    Nature
    The site-specific encoding of non-canonical amino acids (ncAAs) provides a powerful tool for expanding the functional repertoire of proteins1, 2, 3–4. Its widespread use for basic research and biotechnological applications is, however, hampered by the low efficiencies of current ncAA incorporation strategies. Here we reveal poor cellular ncAA uptake as a main obstacle to efficient genetic code expansion and overcome this bottleneck by hijacking a bacterial ATP-binding cassette (ABC) transporter5 to actively import easily synthesizable isopeptide-linked tripeptides that are processed into ncAAs within the cell. Using this approach, we enable efficient encoding of a variety of previously inaccessible ncAAs, decorating proteins with bioorthogonal6 and crosslinker7 moieties, post-translational modifications8,9 and functionalities for chemoenzymatic conjugation. We then devise a high-throughput directed evolution platform to engineer tailored transporter systems for the import of ncAAs that were historically refractory to efficient uptake. Customized Escherichia coli strains expressing these evolved transporters facilitate single and multi-site ncAA incorporation with wild-type efficiencies. Additionally, we adapt the tripeptide scaffolds for the co-transport of two different ncAAs, enabling their efficient dual incorporation. Collectively, our study demonstrates that engineering of uptake systems is a powerful strategy for programmable import of chemically diverse building blocks.
  • Deciphering functional roles of protein succinylation and glutarylation using genetic code expansion
    Item type: Journal Article
    Weyh, Maria; Jokisch, Marie-Lena; Nguyen, Tuan-Anh; et al. (2024)
    Nature Chemistry
    Post-translational modifications (PTMs) dynamically regulate cellular processes. Lysine undergoes a range of acylations, including malonylation, succinylation (SucK) and glutarylation (GluK). These PTMs increase the size of the lysine side chain and reverse its charge from +1 to -1 under physiological conditions, probably impacting protein structure and function. To understand the functional roles of these PTMs, homogeneously modified proteins are required for biochemical studies. While the site-specific encoding of PTMs and their mimics via genetic code expansion has facilitated the characterization of the functional roles of many PTMs, negatively charged lysine acylations have defied this approach. Here we describe site-specific incorporation of SucK and GluK into proteins via temporarily masking their negative charge through thioester derivatives. We prepare succinylated and glutarylated bacterial and mammalian target proteins, including non-refoldable multidomain proteins. This allows us to study how succinylation and glutarylation impact enzymatic activity of metabolic enzymes and regulate protein-DNA and protein-protein interactions in biological processes from replication to ubiquitin signalling.
  • One-pot dual protein labeling for simultaneous mechanical and fluorescent readouts in optical tweezers
    Item type: Journal Article
    Silbermann, Laura-Marie; Fottner, Maximilian; van Der Meulen, Ronald; et al. (2025)
    Protein Science
    Optical tweezers are widely used in the study of biological macromolecules but are limited by their one-directional probing capability, potentially missing critical conformational changes. Combining fluorescence microscopy with optical tweezers, employing F & ouml;rster resonance energy transfer (FRET) pairs, addresses this issue. When integrating fluorescence microscopy with optical tweezers, orthogonal protein conjugation methods are needed to enable simultaneous, site-specific attachment of fluorophores and DNA handles, commonly used to apply force to molecules of interest. In this study, we utilized commercially available reagents for dual site-specific labeling of the homodimeric heat shock protein 90 (Hsp90) using thiol-maleimide and inverse electron demand Diels-Alder cycloaddition (IEDDAC) bioorthogonal reactions. In a one-pot approach, Hsp90 modified with a cysteine mutation and the non-canonical amino acid cyclopropene-L-lysine (CpK) was labeled with the FRET pair maleimide-Atto 550 and maleimide-Atto 647N, alongside single-stranded methyltetrazine-modified DNA oligonucleotide. Optical tweezers experiments with this labeled Hsp90 construct revealed structural transitions consistent with previous studies, validating the approach. Fluorescence measurements confirmed the proximity of FRET pairs in the N-terminally closed state of Hsp90 in this experimental setup. This integrative method provides a powerful tool for probing complex protein conformational dynamics beyond the limitations of traditional optical tweezers.
  • Genetic Code Expansion Approaches to Decipher the Ubiquitin Code
    Item type: Review Article
    Wanka, Vera; Fottner, Maximilian; Cigler, Marko; et al. (2024)
    Chemical Reviews
    The covalent attachment of Ub (ubiquitin) to target proteins (ubiquitylation) represents one of the most versatile PTMs (post-translational modifications) in eukaryotic cells. Substrate modifications range from a single Ub moiety being attached to a target protein to complex Ub chains that can also contain Ubls (Ub-like proteins). Ubiquitylation plays pivotal roles in most aspects of eukaryotic biology, and cells dedicate an orchestrated arsenal of enzymes to install, translate, and reverse these modifications. The entirety of this complex system is coined the Ub code. Deciphering the Ub code is challenging due to the difficulty in reconstituting enzymatic machineries and generating defined Ub/Ubl-protein conjugates. This Review provides a comprehensive overview of recent advances in using GCE (genetic code expansion) techniques to study the Ub code. We highlight strategies to site-specifically ubiquitylate target proteins and discuss their advantages and disadvantages, as well as their various applications. Additionally, we review the potential of small chemical PTMs targeting Ub/Ubls and present GCE-based approaches to study this additional layer of complexity. Furthermore, we explore methods that rely on GCE to develop tools to probe interactors of the Ub system and offer insights into how future GCE-based tools could help unravel the complexity of the Ub code.
Publications 1 - 4 of 4