Biology subjects

Chaudhari, A. S.

Publications and source records attributed to Chaudhari, A. S..

4 recordsLinked to original sources

bioRxiv2024

Light-dependent flavin redox and adduct states control the conformation and DNA binding activity of the transcription factor EL222

The activity of the transcription factor EL222 is regulated through protein-chromophore adduct formation, interdomain dynamics, oligomerization and protein-DNA interactions, all triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV/visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using non-canonical amino acids. The observation of only subtle atomic displacements between crystal structures of EL222 with and without blue-light back-illumination, was confirmed by NMR data indicating no major changes in secondary structure and fold compactness. Kinetic experiments in solution provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently-bound N5 protonated, and non-covalently-bound hydroquinone forms of FMN, respectively. Molecular modeling revealed differential dynamics and domain separation times arising from the three FMN states (oxidized, adduct, and reduced). Furthermore, while the dark state is largely monomeric, both lit states undergo slow monomer-dimer exchange. The photoinduced loss of -helicity, seen by infrared difference spectroscopy, was ascribed to dimeric EL222 species. Unexpectedly, NMR revealed that all three EL222 species (dark, lit1, lit2) can associate with DNA to some extent, but only under illumination a high population of stable complexes is obtained. Overall, we propose a refined model of EL222 photo-activation where photoinduced changes in the oxidation state of FMN and thioadduct formation shift the population equilibrium towards an open conformation that favors self-association and DNA-binding. Significance StatementFlavin-binding light-oxygen-voltage (LOV) proteins constitute a prominent example of highly evolved chromophore-containing proteins that convert light into biochemical changes in the cell. However, it is not well understood how blue-light orchestrates changes in LOV structure and function. Here we show that the dynamics, oligomerization and DNA-binding properties of the photocontrolled transcription factor EL222 are dependent on both the flavin redox state and thioadduct formation. In the dark, monomeric EL222 forms transient encounter complexes with DNA. Under illumination, two distinct lit states are sequentially generated, termed lit1 and lit2, that are both able to assemble into EL222:DNA (2:1) complexes. Our results reveal the coupling between flavin photochemistry (protonation and covalent linkage) and fold stability in EL222 and potentially other flavoproteins.

biochemistry↗

bioRxiv2024

Missing Wedge Completion via Unsupervised Learning with Coordinate Networks

Cryogenic electron tomography (cryoET) is a powerful tool in structural biology, enabling detailed 3D imaging of biological specimens at a resolution of nanometers. Despite its potential, cryoET faces challenges such as the missing wedge problem, which limits reconstruction quality due to incomplete data collection angles. Recently, supervised deep learning methods leveraging convolutional neural networks (CNNs) have considerably addressed this issue; however, their pretraining requirements render them susceptible to inaccuracies and artifacts, particularly when representative training data is scarce. To overcome these limitations, we introduce a proof-of-concept unsupervised learning approach using coordinate networks (CNs) that optimizes network weights directly against input projections. This eliminates the need for pretraining, reducing reconstruction runtime by 3 - 20x compared to supervised methods. Our in silico results show improved shape completion and reduction of missing wedge artifacts, assessed through several voxel-based image quality metrics in real space and a novel directional Fourier Shell Correlation (FSC) metric. Our study illuminates benefits and considerations of both supervised and unsupervised approaches, guiding the development of improved reconstruction strategies.

molecular biology↗

bioRxiv2022

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone and side-chains in EL222

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at forty-five positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/Visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of -helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A extension of the LOV domain in controlling EL222 photocycle length. SignificanceThe kinetics of fold switching between non-illuminated and blue-light-irradiated states in the transcription factor EL222 is important for understanding the signal transduction mechanism of LOV photoreceptors. Here we combine two native probes, the FMN chromophore (absorption bands in the UV/Visible region) and the protein backbone (amide bands in the infrared region), with genetically encoded cyano (C{equiv}N)-containing phenylalanine residues as infrared reporters of protein microenvironments. EL222 structural dynamics is more complex than expected if using a single type of probe. Local changes around residues 31 and 35 precede FMN-protein adduct rupture, which in turn precedes the global protein conformational relaxation. Our findings point the way forward to obtaining comprehensive descriptions of kinetic transitions in LOV and other photosensors.

biophysics↗

bioRxiv2022

OpenCap: 3D human movement dynamics from smartphone videos

Measures of human movement dynamics can predict outcomes like injury risk or musculoskeletal disease progression. However, these measures are rarely quantified in clinical practice due to the prohibitive cost, time, and expertise required. Here we present and validate OpenCap, an open-source platform for computing movement dynamics using videos captured from smartphones. OpenCaps web application enables users to collect synchronous videos and visualize movement data that is automatically processed in the cloud, thereby eliminating the need for specialized hardware, software, and expertise. We show that OpenCap accurately predicts dynamic measures, like muscle activations, joint loads, and joint moments, which can be used to screen for disease risk, evaluate intervention efficacy, assess between-group movement differences, and inform rehabilitation decisions. Additionally, we demonstrate OpenCaps practical utility through a 100-subject field study, where a clinician using OpenCap estimated movement dynamics 25 times faster than a laboratory-based approach at less than 1% of the cost. By democratizing access to human movement analysis, OpenCap can accelerate the incorporation of biomechanical metrics into large-scale research studies, clinical trials, and clinical practice.

bioengineering↗