Biology subjects

Rosenblum, G.

Publications and source records attributed to Rosenblum, G..

6 recordsLinked to original sources

bioRxiv2026

Protein disorder controls allostery in DNA

Metabolism, gene expression, and signaling all require the adaptation of protein activity to the mixture of reactants and products in a cell. This trait to adapt, called allostery, is hardwired in the structure of proteins. Binding a ligand at one location in a protein can change distant locations, thus tuning protein activity. How allostery works has been subject of intense research since its discovery sixty years ago. The challenge is to understand the order of events that follow ligand-binding in the three-dimensional architecture of proteins. Here we simplify this task by studying allostery in DNA, a nearly one-dimensional system. DNA can transmit allosteric signals over many nanometers to generate cooperativity in the binding of transcription factors, an archetype of the long-range action of allostery. We found that binding of the transcription factor ComK amplifies intrinsic microsecond structural fluctuations in DNA many nanometers distant from the binding site. Yet, it is not protein binding per se, but the intrinsically disordered region (IDR) of the protein that amplifies these fluctuations. IDR removal does not only rigidify DNA, but it also abolishes allostery. The result is a structurally distorted protein-DNA complex that lost its function. These findings have important implications for our understanding of transcription activation and suggest a new functional role for IDRs in transcription factors.

biophysics↗

bioRxiv2024

Model-free photon analysis of diffusion-based single-molecule FRET experiments

Photon-by-photon analysis tools for diffusion-based single-molecule Forster resonance energy transfer (smFRET) experiments often describe protein dynamics with Markov models. However, FRET efficiencies are only projections of the conformational space such that the measured dynamics can appear non-Markovian. Model-free methods to quantify FRET efficiency fluctuations would be desirable in this case. Here, we present such an approach. We determine FRET efficiency correlation functions free of artifacts from the finite length of photon trajectories or the diffusion of molecules through the confocal volume. We show that these functions capture the dynamics of proteins from micro-to milliseconds both in simulation and experiment, which provides a rigorous validation of current model-based analysis approaches.

biophysics↗

bioRxiv2021

Diffusion of the disordered E-cadherin tail on β-catenin

Specific protein interactions typically require well-shaped binding interfaces. Here, we report a cunning exception. The disordered tail of the cell-adhesion protein E-cadherin dynamically samples a large surface area of the proto-oncogene {beta}-catenin. Single-molecule experiments and molecular simulations resolve these motions with high resolution in space and time. Contacts break and form within hundreds of microseconds without dissociation of the complex. A few persistent interactions provide specificity whereas unspecific contacts boost affinity. The energy landscape of this complex is rugged with many small barriers (3 - 4 kBT) and reconciles specificity, high affinity, and extreme disorder. Given the roles of {beta}-catenin in cell-adhesion, signalling, and cancer, this Velcro-like design has the potential to tune the stability of the complex without requiring dissociation.

biophysics↗

bioRxiv2020

Mechanistic basis for motor-driven membrane constriction by dynamin

Dynamin oligomerizes into helical filaments on tubular membrane templates and, through constriction, cleaves them in a GTPase-driven way. Structural observations of GTP-dependent cross-bridges between neighboring filament turns have led to the suggestion that dynamin operates as a molecular ratchet motor. However, the proof of such mechanism remains absent. Particularly, it is not known whether a powerful enough stroke is produced and how the motor modules would cooperate in the constriction process. Here, we characterized the dynamin motor modules by single molecule (sm) FRET and found strong nucleotide-dependent conformational changes. Integrating smFRET with molecular dynamics simulations allowed us to determine the forces generated in a power stroke. Subsequently, the quantitative force data and the measured kinetics of the GT-Pase cycle were incorporated into a model including both a dynamin filament, with explicit motor cross-bridges, and a realistic deformable membrane template. In our simulations, collective constriction of the membrane by dynamin motor modules, based on the ratchet mechanism, is directly reproduced and analyzed. Functional parallels between the dynamin system and actomyosin in the muscle are seen. Through concerted action of the motors, tight membrane constriction to the hemifission radius can be reached. Our experimental and computational study provides an example of how collective motor action in megadalton molecular assemblies can be approached and explicitly resolved.

biophysics↗

bioRxiv2020

Allostery through DNA drives phenotype switching

SummaryAllostery is a pervasive principle to regulate protein function. Here, we show that DNA also transmits allosteric signals over long distances to boost the binding cooperativity of transcription factors. Phenotype switching in Bacillus subtilis requires an all-or-none promoter binding of multiple ComK proteins. Using single-molecule FRET, we find that ComK-binding at one promoter site increases affinity at a distant site. Cryo-EM structures of the complex between ComK and its promoter demonstrate that this coupling is due to mechanical forces that alter DNA curvature. Modifications of the spacer between sites tune cooperativity and show how to control allostery, which paves new ways to design the dynamic properties of genetic circuits.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

The conformational distribution of a major facilitator superfamily peptide transporter is modulated by the membrane composition

While structural biology aims at explaining the biological function of membrane proteins with their structure, it is unclear how these proteins are modulated by the complex lipid composition of membranes. Here, we address this question by mapping the conformational distribution of the bacterial oligopeptide transporter DtpA using single-molecule fluorescence spectroscopy. We show that DtpA populates ensembles of conformers that respond sensitively to the environment. Detergents trap the transporter in an inward-open ensemble in which the substrate binding site faces the cytosol. However, re-constitution in Saposin nanoparticles with different lipid compositions, reveal a plethora of alternative conformations, including a fully inward-open ensemble whose existence had not been anticipated before. The relative abundance of these ensembles depends on the lipid composition of the nanoparticles. Our results therefore demonstrate that membranes sensitively affect the structural distribution of DtpA and we expect this to be a general property of membrane proteins.

biophysics↗