Oliver Zerbe

The number of internal repeat modules remains a major limiting factor in the construction and functional exploration of long repeat proteins. As a solution, we present High-Utility Recursive Directional Ligation for Extended Repeat proteins (HURDLER)—a modular cloning strategy combining codon optimization, seamless insertion of restriction sites, and modified recursive directional ligation. It presents a very robust technique to clone DNA for large repeat proteins, which may also contain non-repetitive parts, with high fidelity requiring a minimal number of individual cloning steps. Using HURDLER, we cloned designed armadillo repeat proteins with up to 85 internal modules, corresponding to >3700 residues. The method also generates intermediate variants with defined numbers of internal repeats, enabling systematic investigation of how the number of modules influences protein structure and function. HURDLER thereby provides a reliable solution to overcome restrictions in the overall number of internal modules used in the construction of very long repeat proteins. Such proteins are increasingly utilized as building blocks in macromolecular protein assemblies with defined structures.

Lipopolysaccharides (LPS) are the principal chemical component of the outer leaflet of Gram‐negative bacteria and constitute the first barrier of defense against foreign molecules. Inhibition of LPS transport presents a novel concept for antibiotic discovery, and components of the transport bridge are targets of antimicrobial peptides. LptD, a β‐barrel outer membrane protein, the terminal module of the Lpt transport bridge, however, remains largely unexplored as a drug target as its biosynthesis is complicated and screens against membrane proteins are challenging. Herein, we report a computationally designed, soluble E. coli LptD periplasmic epitope mimic, LptDm. We describe an efficient in silico design pipeline that includes verification of interactions of LptD mimics with the cognate ligands LptA and thanatin using nuclear magnetic resonance (NMR) and size‐exclusions chromatography (SEC) techniques. A small peptide library demonstrates that LptDm allows for selection of high‐affinity binders against LptD, rendering LptD accessible to modern drug discovery approaches.

Metallothioneins (MTs) are small proteins that coordinate d‐block metal ions in sulfur‐metal clusters to control metal ion concentrations within the cell. Here we study metal cluster formation in the MT of the periwinkle Littorina littorea (LlMT) by nuclear magnetic resonance (NMR). We demonstrate that the three Cd²⁺ ions in each domain are taken up highly cooperatively, that is, in an all‐or‐none fashion, with a four‐ to six‐fold higher affinity for the C‐terminal domain. During the transfer of metal ions from Cd²⁺‐loaded MT to apo MT, Cd²⁺ is most efficiently transferred from the metalated protein to the apo C‐terminal domain. This behavior might be connected to unique structural motifs in the C‐terminal domain, such as two double‐CXC motifs and an increased proportion of positively charged residues. In Cd²⁺/Zn²⁺ metal exchange experiments, the N‐terminal domain displayed the most efficient inter‐molecular metal exchange. Amide hydrogen exchange reveals fewer protected amides for the N‐terminal domain, suggesting the structure might more easily “open up” to facilitate metal exchange. Experiments with a physical separation of donor and acceptor species demonstrate that metal exchange and transfer require protein–protein contacts. These findings provide insights into the mechanism of metal uptake and metal transfer, which are important processes during metal detoxification in snail MTs.

Many peptidic natural products, such as lasso peptides, cyclic peptides, and cyclotides, are conformationally constrained and show biological stability, making them attractive scaffolds for drug development. Although many peptides can be synthesized and modified through chemical methods, knot-like lasso peptides such as microcin J25 (MccJ25) and their analogues remain elusive. As the chemical space of MccJ25 analogues accessible through purely biological methods is also limited, we proposed a hybrid approach: flow-based chemical synthesis of non-natural precursor peptides, followed by in vitro transformation with recombinant maturation enzymes, to yield a more diverse array of lasso peptides. Herein, we established the rapid, flow-based synthesis of chemically modified MccJ25 precursor peptides (57 amino acids). Heterologous expression of enzymes McjB and McjC was extensively optimized to improve yields and facilitate the synthesis of multiple analogues of MccJ25, including the incorporation of non-canonical tyrosine and histidine derivatives into the lasso scaffold. Finally, using our chemoenzymatic strategy, we produced a biologically active analogue containing three d-amino acids in the loop region and incorporated backbone N-methylations. Our method provides rapid access to chemically modified lasso peptides that could be used to investigate structure-activity relationships, epitope grafting, and the improvement of therapeutic properties.

Protein–protein interactions of c-Myc (MYC) are often regulated by post-translational modifications (PTMs), such as phosphorylation, and crosstalk thereof. Studying these interactions requires proteins with unique PTM patterns, which are challenging to obtain by recombinant methods. Standard peptide synthesis and native chemical ligation can produce such modified proteins, but are time-consuming and therefore typically limited to the study of individual PTMs. Herein, we report the development of flow-based methods for the rapid synthesis of phosphorylated MYC sequences (up to 84 AA), and demonstrate the versatility of this approach for the incorporation of other PTMs (N^ε-methylation, sulfation, acetylation, glycosylation) and combinations thereof. Peptides containing up to seven PTMs and phosphorylation at up to five sites were successfully prepared and isolated in high yield and purity. We further produced ten PTM-decorated analogues of the MYC Transactivation Domain (TAD) to screen for binding to the tumor suppressor protein, Bin1, using heteronuclear NMR and native mass spectrometry. We determined the effects of phosphorylation and glycosylation on the strength of the MYC:Bin1 interaction, and reveal an influence of MYC sequence length on binding. Our platform for the rapid synthesis of MYC sequences up to 84 AA with distinct PTM patterns thus enables the systematic study of PTM function at a molecular level, and offers a convenient way for expedited screening of constructs.

Many peptidic natural products—such as lasso peptides, cyclic peptides, and cyclotides—are conformationally constrained and show biological stability, making them attractive scaffolds for drug development. Although many peptides can be synthesized and modified through chemical methods, knot-like lasso peptides such as microcin J25 (MccJ25) and their analogs remain elusive. As the chemical space of MccJ25 analogs accessible through purely biological methods is also limited, we proposed a hybrid approach: flow-based chemical synthesis of non-natural precursor peptides followed by in vitro transformation with recombinant maturation enzymes to yield a more diverse array of lasso peptides. Herein, we established the rapid, flow-based synthesis of chemically modified MccJ25 precursor peptides (57 amino acids). Heterologous expression of enzymes McjB and McjC was extensively optimized to improve yields and facilitate the synthesis of multiple analogs of MccJ25, including the incorporation of non-canonical tyrosine and histidine derivatives into the lasso scaffold. Finally, using our chemoenzymatic strategy, we produced a biologically active analog containing three D-amino acids in the loop region and incorporated backbone N-methylations. Our method provides rapid access to chemically modified lasso peptides that could be used to investigate structure-activity relationships, epitope grafting, and improvement of therapeutic properties.

Protein-protein interactions of c-Myc (MYC) are often regulated by post-translational modifications (PTMs), such as phosphorylation, and crosstalk thereof. Studying these interactions requires proteins with unique PTM patterns, which are challenging to obtain by recombinant methods. Standard peptide synthesis and native chemical ligation can produce such modified proteins, but are time-consuming and therefore typically limited to the study of individual PTMs. Herein, we report the development of flow-based methods for the rapid synthesis of phosphorylated MYC sequences (up to 84 AA), and demonstrate the versatility of this approach for the incorporation of other PTMs (Nε methylation, sulfation, acetylation, glycosylation) and combinations thereof. Peptides containing up to seven PTMs and five phosphorylations were successfully prepared and isolated in high yield and purity. Our methodology was then applied in the production of ten PTM-decorated analogues of the MYC Transactivation Domain (TAD) to screen for binding to the tumor suppressor protein, Bin1, using heteronuclear NMR and native mass spectrometry. We determined the effects of phosphorylation and glycosylation on the strength of the MYC:Bin1 interaction, and reveal an influence of MYC sequence length on binding. Our platform for the rapid synthesis of MYC sequences up to 84 AA with distinct PTM patterns thereby enables the systematic study of PTM function at a molecular level, and offers a convenient way for an expedited screening of constructs.

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders.

G protein‐coupled receptors (GPCRs) are medically important membrane proteins that sample inactive, intermediate, and active conformational states characterized by relatively slow interconversions (~μs–ms). On a faster timescale (~ps–ns), the conformational landscape of GPCRs is governed by the rapid dynamics of amino acid side chains. Such dynamics are essential for protein functions such as ligand recognition and allostery. Unfortunately, technical challenges have almost entirely precluded the study of side‐chain dynamics for GPCRs. Here, we investigate the rapid side‐chain dynamics of a thermostabilized α _1B ‐adrenergic receptor (α _1B ‐AR) as probed by methyl relaxation. We determined order parameters for Ile, Leu, and Val methyl groups in the presence of inverse agonists that bind orthosterically (prazosin, tamsulosin) or allosterically (conopeptide ρ‐TIA). Despite the differences in the ligands, the receptor's overall side‐chain dynamics are very similar, including those of the apo form. However, ρ‐TIA increases the flexibility of Ile176 ^4×56 and possibly of Ile214 ^5×49 , adjacent to Pro215 ^5×50 of the highly conserved P ^5×50 I ^3×40 F ^6×44 motif crucial for receptor activation, suggesting differences in the mechanisms for orthosteric and allosteric receptor inactivation. Overall, increased Ile side‐chain rigidity was found for residues closer to the center of the membrane bilayer, correlating with denser packing and lower protein surface exposure. In contrast to two microbial membrane proteins, in α _1B ‐AR Leu exhibited higher flexibility than Ile side chains on average, correlating with the presence of Leu in less densely packed areas and with higher protein‐surface exposure than Ile. Our findings demonstrate the feasibility of studying receptor‐wide side‐chain dynamics in GPCRs to gain functional insights.

Metallothioneins (MTs) are small proteins present in all kingdoms of life. Their high cysteine content enables them to bind metal ions, such as Zn²⁺, Cd²⁺, and Cu⁺, providing means for detoxification and metal homeostasis. Three MT isoforms with distinct metal binding preferences are present in the Roman Snail Helix pomatia. Here, we use nuclear magnetic resonance (NMR) to follow the evolution of Cd²⁺ and Cu⁺ binding from the reconstructed ancestral Stylommatophora MT to the three H. pomatia MT (HpMT) isoforms. Information obtained from [¹⁵N,¹H]-HSQC spectra and T₂ relaxation times are combined to describe the conformational stability of the MT-metal complexes. A well-behaved MT-metal complex adopts a unique structure and does not undergo additional conformational exchange. The ancestor to all three HpMTs forms conformationally stable Cd²⁺ complexes and closely resembles the Cd²⁺-specific HpCdMT isoform, suggesting a role in Cd²⁺ detoxification for the ancestral protein. All Cu⁺-MT complexes, including the Cu⁺-specific HpCuMT isoform, undergo a considerable amount of conformational exchange. The unspecific HpCd/CuMT and the Cu⁺-specific HpCuMT isoforms form Cu⁺ complexes with comparable characteristics. It is possible to follow how Cd²⁺ and Cu⁺ binding changed throughout evolution. Interestingly, Cu⁺ binding improved independently in the lineages leading to the unspecific and the Cu⁺-specific HpMT isoforms. C-terminal domains are generally less capable of coordinating the non-cognate metal ion than N-terminal domains, indicating a higher level of specialization of the C-domain. Our findings provide new insights into snail MT evolution, helping to understand the interplay between biological function and structural features toward a comprehensive understanding of metal preference.