Biology subjects

Hamer-Rogotner, S.

Publications and source records attributed to Hamer-Rogotner, S..

4 recordsLinked to original sources

bioRxiv2025

Systematic DNA nicking reveals the structural logic of protein recognition

Transcription factors (TFs) bind to specific genomic sites to regulate gene expression1,2. These interactions almost universally require DNA deformation and the accumulation of local mechanical strain within the double helix. As a result, TF-DNA recognition is determined not only by the linear base sequence but also by the spatial alignment of bases and phosphates, as well as their ability to adopt and retain structural deformations3. However, the sequence-centric focus of existing studies makes it challenging to directly probe DNA structural determinants and to decouple their impact from alterations in base sequences, limiting our ability to unravel the key factors influencing binding beyond the sequence identity and leaving significant gaps in our understanding of the principles governing TF-DNA recognition. Here, we introduce a high-throughput strategy to perturb TF binding sites without altering their base sequence, enabling systematic investigation of the structural features of DNA that govern TF binding. Our method, PIC-NIC, introduces single-strand breaks (SSBs) at every position within the binding site, selectively disrupting backbone continuity while preserving nucleotide identity, with the resulting effects on TF binding measured quantitatively. Applied to 15 human TFs spanning eight structural classes, and supported by seven high-resolution TF-DNA crystal structures and molecular dynamics simulations, PIC-NIC uncovers discrete backbone positions serving as structural anchor points where nicks can abolish binding, rewire sequence preferences, or even enhance affinity. By decoupling structural and chemical contributions, we demonstrate that DNA mechanics--encoded in backbone geometry and continuity--can independently shape binding specificity beyond the linear code of base identity. These findings shift the paradigm of TF-DNA recognition, establishing the backbone not as a passive scaffold, but as a functional determinant capable of directing regulatory mechanisms through its physical architecture.

biophysics↗

bioRxiv2025

High-efficiency Kemp eliminases by complete computational design

We present a fully computational workflow for de novo design of efficient enzymes using backbone fragments from natural proteins and without recourse to iterative experimental optimization. The best designed Kemp eliminase exhibits >140 mutations from any natural protein, high stability (>85 {degrees}C) and unprecedented catalytic efficiency (12,700 M-1s-1), surpassing previous computational designs by two orders of magnitude. We find that mutations both inside and outside the active site contribute synergistically to the high observed activity and stability. Mutation of an aromatic residue used in all prior Kemp eliminase designs increases efficiency to >105 M-1s-1. Our approach addresses critical limitations in design methodology, generating stable, high-efficiency, new-to-nature enzymes in complex folds and enables testing hypotheses on the fundamentals of biocatalysis through a limited experimental effort.

biochemistry↗

bioRxiv2023

Stable mammalian serum albumins designed for bacterial expression

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40{degrees}C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/534334v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@71ef93org.highwire.dtl.DTLVardef@27a975org.highwire.dtl.DTLVardef@1e657fforg.highwire.dtl.DTLVardef@19b3de3_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Computational design stabilized human and bovine serum albumins - Designs express solubly in E. coli and exhibit up to 40 {degrees}C increased thermostability - Some designs exhibit identical ligand binding properties - Crystal structure confirms design accuracy - Designs can be used in cell culture and in vitro applications

biochemistry↗

bioRxiv2019

Pore-modulating toxins exploit inherent slow inactivation to block K+ channels

Voltage dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified to \"pore-blockers\" that physically plug the ion conduction pathway and \"gating modifiers\" that disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here we show that Cs1, a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.

pharmacology and toxicology↗