Biology subjects

Levitus, M.

Publications and source records attributed to Levitus, M..

6 recordsLinked to original sources

bioRxiv2026

A practical framework for measuring protein oligomerization equilibria by fluorescence correlation spectroscopy

Protein oligomerization equilibria are central to many biological processes and are often highly sensitive to environmental conditions such as ionic strength, pH, and ligand binding. Quantitative characterization of these equilibria remains experimentally challenging because stable protein complexes frequently dissociate only at concentrations that are difficult to access with conventional biophysical methods. Fluorescence correlation spectroscopy (FCS) is uniquely suited to this problem, as it provides direct access to diffusion coefficients of fluorescently labeled proteins at nanomolar concentrations. However, the quantitative interpretation of FCS data from oligomeric systems requires a rigorous mathematical framework and careful experimental practice that have not previously been described in sufficient detail to guide implementation. Here, we provide a comprehensive description of the experimental workflow and analytical framework for determining dissociation equilibrium constants by FCS, covering instrument calibration, sample preparation, data quality control, after-pulse correction, and nonlinear least-squares fitting. We discuss common sources of error and provide practical guidance on critical experimental considerations including surface passivation, buffer preparation, equilibration time, and the role of labeling efficiency. Using the homotrimeric sliding clamp PCNA as a model system, we demonstrate the complete workflow under a range of KCl concentrations and show that moderate ionic strength stabilizes the PCNA trimer while very high salt partially destabilizes the complex. The approach is general and applicable to any reversible protein self-association reaction accessible by fluorescence detection at low protein concentrations.

biophysics↗

bioRxiv2025

TRPM8 protein dynamics correlates with ligand structure and cellular function

Protein dynamics have emerged as a key feature associated with function in various systems. Here, NMR-based studies coupled with computational cheminformatics and cellular function are leveraged to identify a relationship between the human cold and menthol receptor TRPM8 dynamics, chemical structure, and cellular potency. TRPM8 is a validated target for a variety of pain indications but generally has been clinically limited by on-target side effects impacting thermosensing and thermoregulation. This study shows that cheminformatic analysis of a TRPM8 regulating small molecule ligand library correlates with cellular function. Electrophysiology studies further validate the relationship and show a correlation between chemical structure and functional features such as compound potency. Solution NMR studies of the TRPM8 voltage sensing-like domain, which houses the canonical menthol ligand binding site, show that ligand binding conformationally selects NMR-detected TRPM8 dynamics in a manner that quantitatively correlates with chemical structure. The relationship between chemical structure and protein dynamics can be used predictively, where a chemical structure is predictive of dynamics in a latent reduced dimensionality space. Moreover, the robustness of the conformational selection of the dynamic ensemble is evaluated by varying related and divergent chemotypes, signal-to-noise sensitivity, and sample bias. Taken together, this study identifies that protein dynamics can serve as a quantifiable bridge between chemical structure and cellular function, which has implications for drug discovery in difficult systems.

biophysics↗

bioRxiv2022

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity

Uracil DNA-glycosylase (UNG) is a base excision repair enzyme that removes the highly mutagenic uracil lesion from DNA by a base flipping mechanism. UNG excision efficiency depends on DNA sequence, yet the underlying principles that dictate UNG substrate specificity have remained elusive. Here, we show that UNG efficiency is dictated by the intrinsic local deformability of the substrate sequence around the uracil. UNG specificity constants (kcat/KM) and DNA flexibilities were measured for an engineered set of DNA substrates containing AUT, TUA, AUA, and TUA motifs. Time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations of the bare DNA indicated significant differences in substrate flexibilities. A strong correlation between UNG efficiency and substrate flexibility was observed, with higher kcat/KM values measured for more flexible strands. DNA bending and base flipping were observed in simulations, with more frequent uracil flipping observed for the more bendable sequences. Experiments show that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and resultant UNG activity. The finding that substrate flexibility controls UNG efficiency has implications in diverse fields, including the genesis of mutation hotspots, molecular evolution, and understanding sequence preferences of emerging base editors.

biophysics↗

bioRxiv2020

Potassium Glutamate and Glycine Betaine Induce Self-Assembly of Sliding Clamps into Higher Order Oligomers

Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli {beta}-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of {beta} is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the S. cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to {beta}. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.

biophysics↗

bioRxiv2019

Pitching single focus confocal data analysis one photon at a time with Bayesian nonparametrics

Fluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.

biophysics↗

bioRxiv2019

Evidence that the TRPV1 S1-S4 Membrane Domain Contributes to Thermosensing

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. However, the role of the S1-S4 domain in thermosensing is unclear. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, Far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

biochemistry↗