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.

Application of Amber Suppression To Study the Role of Tyr M210 in Electron Transfer in Photosynthetic Reaction Centers.

The initial light-induced electron transfer (ET) steps in the bacterial photosynthetic reaction center (RC) have been extensively studied and provide a paradigm for connecting structure and function. Although RCs have local pseudo- symmetry, ET only occurs along the A branch of chromophores. Tyrosine M210 is a key symmetry-breaking residue adjacent to bacteriochlorophyll B that bridges the primary electron donor P and the bacteriopheophytin acceptor H.

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.

Beyond the Vibrational Stark Effect: Unraveling the Large Redshifts of Alkyne C-H Bond in Solvation Environments.

C-H···O hydrogen bonds are formed in systems ranging from biomolecular complexes to small-molecule structures. Previous work has focused on the blueshifts in the C-H stretching frequency () induced by these hydrogen bonds and their chemical and biological roles. Here, we show that, in contrast, terminal alkyne C-H hydrogen bonds exhibit large redshifts (50-100 cm) upon hydrogen bonding with oxygen-containing solvents.

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.

Structure-function relationships in pure archaeal bipolar tetraether lipids.

Archaeal bipolar tetraether lipids (BTLs) are among the most unusual lipids occurring in nature because of their presumed ability to span the entire membrane to form a monolayer structure. It is believed that because of their unique structural organization and chemical stability, BTLs offer extraordinary adaptation to archaea to thrive in the most extreme milieus. BTLs have also received considerable attention for development of novel membrane-based materials.

A Fluorogenic Pseudoinfection Assay to Probe Transfer and Distribution of Influenza Viral Contents to Target Vesicles.

Fusion of enveloped viruses with endosomal membranes and subsequent release of the viral genome into the cytoplasm are crucial to the viral infection cycle. It is often modeled by performing fusion between virus particles and target lipid vesicles. We utilized fluorescence microscopy to characterize the kinetic aspects of the transfer of influenza viral ribonucleoprotein (vRNP) complexes to target vesicles and their spatial distribution within the fused volumes to gain deeper insight into the mechanistic aspects of endosomal escape.

Membrane localization accelerates association under conditions relevant to cellular signaling.

Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here, we directly compare an identical association reaction, the binding of complementary DNA strands, in solution and on supported membranes.

Critical Evaluation of Polarizable and Nonpolarizable Force Fields for Proteins Using Experimentally Derived Nitrile Electric Fields.

Molecular dynamics (MD) simulations are frequently carried out for proteins to investigate the role of electrostatics in their biological function. The choice of force field (FF) can significantly alter the MD results, as the simulated local electrostatic interactions lack benchmarking in the absence of appropriate experimental methods. We recently reported that the transition dipole moment (TDM) of the popular nitrile vibrational probe varies linearly with the environmental electric field, overcoming well-known hydrogen bonding (H-bonding) issues for the nitrile frequency and, thus, enabling the unambiguous measurement of electric fields in proteins ( , (17), 7562-7567).

Secondary Ion Mass Spectrometry of Single Giant Unilamellar Vesicles Reveals Compositional Variability.

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. It is generally assumed that the composition of individual GUVs is the same as the nominal stock composition; however, there may be significant compositional variability between individual GUVs. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified.

Simulation-guided engineering of split GFPs with efficient β-strand photodissociation.

Green fluorescent proteins (GFPs) are ubiquitous for protein tagging and live-cell imaging. Split-GFPs are widely used to study protein-protein interactions by fusing proteins of interest to split GFP fragments that create a fluorophore upon typically irreversible complementation. Thus, controlled dissociation of the fragments is desirable.

Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy.

The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore-environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations.

Optical control of ultrafast structural dynamics in a fluorescent protein.

The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters.

Enhanced active-site electric field accelerates enzyme catalysis.

The design and improvement of enzymes based on physical principles remain challenging. Here we demonstrate that the principle of electrostatic catalysis can be leveraged to substantially improve a natural enzyme's activity. We enhanced the active-site electric field in horse liver alcohol dehydrogenase by replacing the serine hydrogen-bond donor with threonine and replacing the catalytic Zn with Co.

Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism.

Chromophore photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket.

β-Lactamases Evolve against Antibiotics by Acquiring Large Active-Site Electric Fields.

A compound bound covalently to an enzyme active site can act either as a substrate if the covalent linkage is readily broken up by the enzyme or as an inhibitor if the bond dissociates slowly. We tracked the reactivity of such bonds associated with the rise of the resistance to penicillin G (PenG) in protein evolution from penicillin-binding proteins (PBPs) to TEM β-lactamases and with the development of avibactam (Avb) to overcome the resistance. We found that the ester linkage in PBP-PenG is resistant to hydrolysis mainly due to the small electric fields present in the protein active site.

Protein Electric Fields Enable Faster and Longer-Lasting Covalent Inhibition of β-Lactamases.

The widespread design of covalent drugs has focused on crafting reactive groups of proper electrophilicity and positioning toward targeted amino-acid nucleophiles. We found that environmental electric fields projected onto a reactive chemical bond, an overlooked design element, play essential roles in the covalent inhibition of TEM-1 β-lactamase by avibactam. Using the vibrational Stark effect, the magnitudes of the electric fields that are exerted by TEM active sites onto avibactam's reactive C═O were measured and demonstrate an electrostatic gating effect that promotes bond formation yet relatively suppresses the reverse dissociation.

Solvent Organization and Electrostatics Tuned by Solute Electronic Structure: Amide versus Non-Amide Carbonyls.

The ability to exploit carbonyl groups to measure electric fields in enzymes and other complex reactive environments by using the vibrational Stark effect has inspired growing interest in how these fields can be measured, tuned, and ultimately designed. Previous studies have concentrated on the role of the solvent in tuning the fields exerted on the solute. Here, we explore instead the role of the solute electronic structure in modifying the local solvent organization and electric field exerted on the solute.

Recombination between C and H to Form Acetylide (CH) Probes Nanoscale Interactions in Lipid Bilayers via Dynamic Secondary Ion Mass Spectrometry: Cholesterol and GM Clustering.

Although it is thought that there is lateral heterogeneity of lipid and protein components within biological membranes, probing this heterogeneity has proven challenging. The difficulty in such experiments is due to both the small length scale over which such heterogeneity can occur, and the significant perturbation resulting from fluorescent or spin labeling on the delicate interactions within bilayers. Atomic recombination during dynamic nanoscale secondary ion imaging mass spectrometry (NanoSIMS) is a non-perturbative method for examining nanoscale bilayer interactions.

A two-directional vibrational probe reveals different electric field orientations in solution and an enzyme active site.

The catalytic power of an electric field depends on its magnitude and orientation with respect to the reactive chemical species. Understanding and designing new catalysts for electrostatic catalysis thus requires methods to measure the electric field orientation and magnitude at the molecular scale. We demonstrate that electric field orientations can be extracted using a two-directional vibrational probe by exploiting the vibrational Stark effect of both the C=O and C-D stretches of a deuterated aldehyde.

Nitrile Infrared Intensities Characterize Electric Fields and Hydrogen Bonding in Protic, Aprotic, and Protein Environments.

Nitriles are widely used vibrational probes; however, the interpretation of their IR frequencies is complicated by hydrogen bonding (H-bonding) in protic environments. We report a new vibrational Stark effect (VSE) that correlates the electric field projected on the -C≡N bond to the transition dipole moment and, by extension, the nitrile peak area or integrated intensity. This linear VSE applies to H-bonding and non-H-bonding interactions.