Covalent Drug Binding in Live Cells Monitored by Mid-IR Quantum Cascade Laser Spectroscopy: Photoactive Yellow Protein as a Model System.

The detection of drug-target interactions in live cells enables analysis of therapeutic compounds in a native cellular environment. Recent advances in spectroscopy and molecular biology have facilitated the development of genetically encoded vibrational probes like nitriles that can sensitively report on molecular interactions. Nitriles are powerful tools for measuring electrostatic environments within condensed media like proteins, but such measurements in live cells have been hindered by low signal-to-noise ratios.

Electrostatic atlas of non-covalent interactions built into metal-organic frameworks.

Non-covalent interactions are central to the organization of matter and molecular recognition processes, yet they are difficult to characterize. Here we devise a platform strategy to systematically build non-covalent interactions with selective chemical groups into precisely designed configurations by using metal-organic frameworks (MOFs) as the molecular scaffold. Using the vibrational Stark effect benchmarked against computer models, we find the electric field provides a unifying metric for quantifying diverse non-covalent interactions in MOFs and solvation environments.

GLAPAL-H: Global, Local, And Parts Aware Learner for Hydrocephalus Infection Diagnosis in Low-Field MRI.

Objective: The study aims to develop a method for differentiating between healthy, post-infectious hydrocephalus (PIH), and non-post-infectious hydrocephalus (NPIH) in infants using low-field MRI, which is a safer, low-cost alternative to CT scans. The study develops a custom approach that captures hydrocephalic etiology while simultaneously addressing quality issues encountered in low-field MRI.

Methods: Specifically, we propose GLAPAL-H, a Global, Local, And Parts Aware Learner, which develops a multi-task architecture with global, local, and parts segmentation branches.

GLAPAL-H: Global, Local, And Parts Aware Learner for Hydrocephalus Infection Diagnosis in Low-Field MRI.

Objective: The study aims to develop a method for differentiating between healthy, post-infectious hydrocephalus (PIH), and non-post-infectious hydrocephalus (NPIH) in infants using low-field MRI, which is a safer, low-cost alternative to CT scans. The study develops a custom approach that captures hydrocephalic etiology while simultaneously addressing quality issues encountered in low-field MRI.

Methods: Specifically, we propose GLAPAL-H, a Global, Local, And Parts Aware Learner, which develops a multi-task architecture with global, local, and parts segmentation branches.

Magnetic resonance control of reaction yields through genetically-encoded protein:flavin spin-correlated radicals in a live animal.

Radio-frequency (RF) magnetic fields can influence reactions involving spin-correlated radical pairs. This provides a mechanism by which RF fields can influence living systems at the biomolecular level. Here we report the modification of the emission of various red fluorescent proteins (RFPs), in the presence of a flavin cofactor, induced by a combination of static and RF magnetic fields.

Environment- and Conformation-Induced Frequency Shifts of C-D Vibrational Stark Probes in NAD(P)H Cofactors.

NAD(P)H cofactors are found in all forms of life and are essential for electron and hydrogen atom transfer. The linear response of a carbon-deuterium (C-D) vibration based on the vibrational Stark effect can facilitate measurements of electric fields for critical biological reactions including cofactor-mediated hydride transfer. We find both inter- and intramolecular electric fields influence the C-D frequency in NAD(P)H and nicotinamide-like models where the reactive C4-hydrogen has been deuterated.

Influence of Fluorescence Lifetime Selections and Conformational Flexibility on Brightness of FusionRed Variants.

Fluorescent proteins (FPs) for bioimaging are typically developed by screening mutant libraries for clones with improved photophysical properties. This approach has resulted in FPs with high brightness, but the mechanistic origins of the improvements are often unclear. We focused on improving the molecular brightness in the FusionRed family of FPs with fluorescence lifetime selections on targeted libraries, with the aim of reducing nonradiative decay rates.

Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness.

The 3-fold higher brightness of the recently developed mCherry-XL red fluorescent protein (FP) compared to its progenitor, mCherry, is due to a significant decrease in the nonradiative decay rate underlying its increased fluorescence quantum yield. To examine the structural and dynamic role of the four mutations that distinguish the two FPs and closely related variants, we employed microsecond time scale, all-atom molecular dynamics simulations. The simulations revealed that the I197R mutation leads to the formation of multiple hydrogen-bonded contacts and increased rigidity of the β-barrel.

Directed Evolution of a Bright Variant of mCherry: Suppression of Nonradiative Decay by Fluorescence Lifetime Selections.

The approximately linear scaling of fluorescence quantum yield (ϕ) with fluorescence lifetime (τ) in fluorescent proteins (FPs) has inspired engineering of brighter fluorophores based on screening for increased lifetimes. Several recently developed FPs such as mTurquoise2, mScarlet, and FusionRed-MQV which have become useful for live cell imaging are products of lifetime selection strategies. However, the underlying photophysical basis of the improved brightness has not been scrutinized.

Characterizing dark state kinetics and single molecule fluorescence of FusionRed and FusionRed-MQ at low irradiances.

The presence of dark states causes fluorescence intermittency of single molecules due to transitions between "on" and "off" states. Genetically encodable markers such as fluorescent proteins (FPs) exhibit dark states that make several super-resolved single-molecule localization microscopy (SMLM) methods possible. However, studies quantifying the timescales and nature of dark state behavior for commonly used FPs under conditions typical of widefield and total internal reflection fluorescence (TIRF) microscopy remain scarce and pre-date many new SMLM techniques.

Photophysical Engineering of Fluorescent Proteins: Accomplishments and Challenges of Physical Chemistry Strategies.

Fluorescent proteins (FPs) have become ubiquitous tools for biological research and concomitantly they are intriguing molecules that are amenable to study with a wide range of experimental and theoretical tools. This perspective explores the connection between the engineering of improved FPs and basic ideas from physical chemistry that explain their properties and drive the molecular design of brighter and more photostable variants. We highlight some of the progress and the many knowledge gaps in understanding the relationship between FP brightness and photostability.

Enrichment of rare events using a multi-parameter high throughput microfluidic droplet sorter.

High information content analysis, enrichment, and selection of rare events from a large population are of great importance in biological and biomedical research. The fluorescence lifetime of a fluorophore, a photophysical property which is independent of and complementary to fluorescence intensity, has been incorporated into various imaging and sensing techniques through microscopy, flow cytometry and droplet microfluidics. However, the throughput of fluorescence lifetime activated droplet sorting is orders of magnitude lower than that of fluorescence activated cell sorting, making it unattractive for applications such as directed evolution of enzymes, despite its highly effective compartmentalization of library members.

Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter.

Green fluorescent proteins (GFP) and their blue, cyan and red counterparts offer unprecedented advantages as biological markers owing to their genetic encodability and straightforward expression in different organisms. Although significant advancements have been made towards engineering the key photo-physical properties of red fluorescent proteins (RFPs), they continue to perform sub-optimally relative to GFP variants. Advanced engineering strategies are needed for further evolution of RFPs in the pursuit of improving their photo-physics.