Thermodynamic compensation to temperature extremes in B. subtilis vs T. maritima lysine riboswitches.

T. maritima and B. subtilis are bacteria that inhabit significantly different thermal environments, ∼80 vs.

High-resolution infrared spectroscopy of jet cooled cyclobutyl in the α-CH stretch region: large-amplitude puckering dynamics in a 4-membered ring radical.

Gas-phase cyclobutyl radical (c-CH) is generated at a rotational temperature of = 26(1) K in a slit-jet discharge mixture of 70% Ne/30% He and 0.5-0.6% cyclobromobutane (c-CHBr).

Ergodicity breaking in rapidly rotating C fullerenes.

Ergodicity, the central tenet of statistical mechanics, requires an isolated system to explore all available phase space constrained by energy and symmetry. Mechanisms for violating ergodicity are of interest for probing nonequilibrium matter and protecting quantum coherence in complex systems. Polyatomic molecules have long served as a platform for probing ergodicity breaking in vibrational energy transport.

Kinetic and Thermodynamic Control of G-Quadruplex Polymorphism by Na and K Cations.

G-Quadruplexes (G4s) are ubiquitous nucleic acid folding motifs that exhibit structural diversity that is dependent on cationic conditions. In this work, we exploit temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) to elucidate the kinetic and thermodynamic mechanisms by which monovalent cations (K and Na) impact folding topologies for a simple G-quadruplex sequence (5'-GGG-(TAAGGG)-3') with a three-state folding equilibrium. Kinetic measurements indicate that Na and K influence G4 formation in two distinctly different ways: the presence of Na modestly enhances an antiparallel G4 topology through an induced fit (IF) mechanism with a low affinity ( = 228 ± 26 mM), while K drives G4 into a parallel/hybrid topology via a conformational selection (CS) mechanism with much higher affinity ( = 1.

Energy-Resolved Femtosecond Hot Electron Dynamics in Single Plasmonic Nanoparticles.

Efficient excitation and harvesting of hot carriers from nanoscale metals is central to many emerging photochemical, photovoltaic, and ultrafast optoelectronic applications. Nevertheless, direct experimental evidence of the energy-dependent femtosecond dynamics in ubiquitous tens-of-nanometer gold structures remains elusive, despite the potentially rich interplay between interfacial and internal plasmonic fields, excitation distributions, and scattering processes. To explore the effects of nanoscale structure on these dynamics, we employ simultaneous time-, angle-, and energy-resolved photoemission spectroscopy of single plasmonic nanoparticles.

Non-equilibrium dynamics at the gas-liquid interface: State-resolved studies of NO evaporation from a benzyl alcohol liquid microjet.

First measurements of internal quantum-state distributions for nitric oxide (NO) evaporating from liquid benzyl alcohol are presented over a broad range of temperatures, performed by liquid-microjet techniques in an essentially collision-free regime, with rotational/spin-orbit populations in the Π manifolds measured by laser-induced fluorescence. The observed rotational distributions exhibit highly linear (i.e.

Breath analysis by ultra-sensitive broadband laser spectroscopy detects SARS-CoV-2 infection.

Rapid testing is essential to fighting pandemics such as coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Exhaled human breath contains multiple volatile molecules providing powerful potential for non-invasive diagnosis of diverse medical conditions. We investigated breath detection of SARS-CoV-2 infection using cavity-enhanced direct frequency comb spectroscopy (CE-DFCS), a state-of-the-art laser spectroscopic technique capable of a real-time massive collection of broadband molecular absorption features at ro-vibrational quantum state resolution and at parts-per-trillion volume detection sensitivity.

Ionic Cooperativity between Lysine and Potassium in the Lysine Riboswitch: Single-Molecule Kinetic and Thermodynamic Studies.

Functionality in many biological systems, including proteins and nucleic acid structures, including protein and nucleic acid riboswitch structures, can depend on cooperative kinetic behavior between multiple small molecule ligands. In this work, single-molecule FRET data on the lysine riboswitch reveals that affinity for the cognate lysine ligand increases significantly with K, providing evidence for synergism between lysine/K binding to the aptamer and successful folding of the riboswitch. To describe/interpret this more complex kinetic scenario, we explore the conventional 4-state ("square") model for aptamer binding as a function of K.

Nonequilibrium Scattering/Evaporation Dynamics at the Gas-Liquid Interface: Wetted Wheels, Self-Assembled Monolayers, and Liquid Microjets.

ConspectusWe often teach or are taught in our freshman courses that there are three phases of matter─gas, liquid and solid─where the ordering reflects increasing complexity and strength of interaction between the molecular constituents. But arguably there is also a fascinating additional "phase" of matter associated with the microscopically thin interface (<10 molecules thick) the gas and liquid, which is still poorly understood and yet plays a crucial role in fields ranging from chemistry of the marine boundary layer and atmospheric chemistry of aerosols to the passage of O and CO through alveolar sacs in our lungs. The work in this Account provides insights into three challenging new directions for the field, each embracing a rovibronically quantum-state-resolved perspective.

Ligand-Dependent Volumetric Characterization of Manganese Riboswitch Folding: A High-Pressure Single-Molecule Kinetic Study.

Nanoscopic differences in free volume result in pressure-dependent changes in free energies which can therefore impact folding/unfolding stability of biomolecules. Although such effects are typically insignificant under ambient pressure conditions, they are crucially important for deep ocean marine life, where the hydraulic pressure can be on the kilobar scale. In this work, single molecule FRET spectroscopy is used to study the effects of pressure on both the kinetics and overall thermodynamics for folding/unfolding of the manganese riboswitch.

Synergism in the Molecular Crowding of Ligand-Induced Riboswitch Folding: Kinetic/Thermodynamic Insights from Single-Molecule Spectroscopy.

Conformational dynamics in riboswitches involves ligand binding and folding of RNA, each of which can be influenced by excluded volume effects under "crowded" cellular conditions and thus incompletely characterized by studies under dilute buffer conditions. In this work, temperature-dependent single-molecule fluorescence resonance energy transfer (FRET) spectroscopy is used to characterize the thermodynamics of (i) cognate ligand and (ii) molecular crowders (PEG, polyethylene glycol) on folding of the lysine riboswitch. With the help of detailed kinetic analysis, we isolate and study the effects of PEG on lysine binding and riboswitch folding steps individually, from which we find that PEG crowding facilitates riboswitch folding primarily via a surprising .

High-resolution CH stretch spectroscopy of jet-cooled cyclopentyl radical: First insights into equilibrium structure, out-of-plane puckering, and IVR dynamics.

First, high-resolution sub-Doppler infrared spectroscopic results for cyclopentyl radical (CH) are reported on the α-CH stretch fundamental with suppression of spectral congestion achieved by adiabatic cooling to T ≈ 19(4) K in a slit jet expansion. Surprisingly, cyclopentyl radical exhibits a rotationally assignable infrared spectrum, despite 3N - 6 = 36 vibrational modes and an upper vibrational state density (ρ ≈ 40-90 #/cm) in the critical regime (ρ ≈ 100 #/cm) necessary for onset of intramolecular vibrational relaxation (IVR) dynamics. Such high-resolution data for cyclopentyl radical permit detailed fits to a rigid-rotor asymmetric top Hamiltonian, initial structural information for ground and vibrationally excited states, and opportunities for detailed comparison with theoretical predictions.

Formation and detection of metastable formic acid in a supersonic expansion: High resolution infrared spectroscopy of the jet-cooled cis-HCOOH conformer.

High-resolution direct absorption infrared spectra of metastable cis-formic acid (HCOOH) trapped in a cis-well resonance behind a 15 kcal/mol barrier are reported for the first time, with the energetically unstable conformer produced in a supersonic slit plasma expansion of trans-formic acid/H mixtures. We present a detailed high-resolution rovibrational analysis for cis-formic acid species in the OH stretch (ν) fundamental, providing first precision vibrational band origin, rotational constants, and term values, which in conjunction with ab initio calculations at the couple-cluster with single, double, and perturbative triple [CCSD(T)]/ANOn (n = 0, 1, 2) level support the experimental assignments and establish critical points on the potential energy surface for internal rotor trans-to-cis isomerization. Relative intensities for a- and b-type transitions observed in the spectra permit the transition dipole moment components to be determined in the body fixed frame and prove to be in good agreement with ab initio CCSD(T) theoretical estimates but in poor agreement with simple bond-dipole predictions.

High-resolution infrared spectroscopy of supersonically cooled singlet carbenes: Bromomethylene (HCBr) in the CH stretch region.

First high-resolution spectra of cold (∼35 K) singlet bromomethylene HCBr in the CH stretching (v) region from 2770 to 2850 cm are reported using near quantum shot-noise limited laser absorption methods in a slit jet supersonic discharge expansion source. Three rovibrational bands are identified at high S/N (20:1-40:1) and rotationally assigned to (i) the CH stretch fundamental (v) band X̃1,0,0←X̃0,0,0 and (ii) vibrational hot bands [X̃(1,1,0)←X̃(0,1,0) and X̃(1,0,1)←X̃(0,0,1)] arising from vibrationally excited HCBr populated in the discharge with single quanta in either the H-C-Br bend (v) or C-Br stretch (v) modes. Precision rotational constants are reported for a total of six states, with an experimentally determined CH stretch vibrational frequency (2799.

Regulating and Directionally Controlling Electron Emission from Gold Nanorods with Silica Coatings.

Dielectric coatings offer a versatile means of manipulating hot carrier emission from nanoplasmonic systems for emerging nanocatalysis and photocathode applications, with uniform coatings acting as regulators and nonuniform coatings providing directional photocurrent control. However, the mechanisms for electron emission through dense and mesoporous silica (SiO) coatings require further examination. Here, we present a systematic investigation of photoemission from single gold nanorods as a function of dense versus mesoporous silica coating thicknesses.

Lysine-Dependent Entropy Effects in the Lysine Riboswitch: Insights from Single-Molecule Thermodynamic Studies.

Riboswitches play an important role in RNA-based sensing/gene regulation control for many bacteria. In particular, the accessibility of multiple conformational states at physiological temperatures allows riboswitches to selectively bind a cognate ligand in the aptamer domain, which triggers secondary structural changes in the expression platform, and thereby "switching" between on or off transcriptional or translational states for the downstream RNA. The present work exploits temperature-controlled, single-molecule total internal reflection fluorescence (TIRF) microscopy to study the thermodynamic landscape of such ligand binding/folding processes, specifically for the lysine riboswitch.

Effects of Molecular Crowders on Single-Molecule Nucleic Acid Folding: Temperature-Dependent Studies Reveal True Crowding vs Enthalpic Interactions.

Biomolecular folding in cells can be strongly influenced by spatial overlap/excluded volume interactions (i.e., "crowding") with intracellular solutes.

Ultrasensitive multispecies spectroscopic breath analysis for real-time health monitoring and diagnostics.

Breath analysis enables rapid, noninvasive diagnostics, as well as long-term monitoring of human health, through the identification and quantification of exhaled biomarkers. Here, we demonstrate the remarkable capabilities of mid-infrared (mid-IR) cavity-enhanced direct-frequency comb spectroscopy (CE-DFCS) applied to breath analysis. We simultaneously detect and monitor as a function of time four breath biomarkers-[Formula: see text]OH, [Formula: see text], [Formula: see text]O, and HDO-as well as illustrate the feasibility of detecting at least six more ([Formula: see text]CO, [Formula: see text], OCS, [Formula: see text], [Formula: see text], and [Formula: see text]) without modifications to the experimental apparatus.

Measuring Excess Heat Capacities of Deoxyribonucleic Acid (DNA) Folding at the Single-Molecule Level.

Measurements of the thermodynamic properties of biomolecular folding (Δ°, Δ°, Δ°, etc.) provide a wealth of information on the folding process and have long played a central role in biophysical investigation. In particular, the excess heat capacity of folding (Δ) is crucial, as typically measured in bulk ensemble studies by differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC).

Pushing Camera-Based Single-Molecule Kinetic Measurements to the Frame Acquisition Limit with Stroboscopic smFRET.

Single-molecule fluorescence resonance energy transfer (smFRET) experiments permit detailed examination of microscopic dynamics. However, kinetic rate constants determined by smFRET are susceptible to systematic underestimation when the rate constants are comparable to the data acquisition rate. We demonstrate how such systematic errors in camera-based total internal reflection fluorescence microscopy experiments can be greatly reduced by using stroboscopic illumination/detection, allowing accurate rate constant determination up to the data sampling rate and yielding an order of magnitude increase in the dynamic range.

Controlling the Spatial and Momentum Distributions of Plasmonic Carriers: Volume Surface Effects.

Spatial and momentum distributions of excited charge carriers in nanoplasmonic systems depend sensitively on optical excitation parameters and nanoscale geometry, which therefore control the efficiency and functionality of plasmon-enhanced catalysts, photovoltaics, and nanocathodes. Growing appreciation over the past decade for the different roles of volume- surface-mediated excitation in such systems has underscored the need for explicit separation and quantification of these pathways. Toward these ends, we utilize angle-resolved photoelectron velocity map imaging to distinguish these processes in gold nanorods of different aspect ratios down to the spherical limit.

Chirality-Dependent Amino Acid Modulation of RNA Folding.

The preponderance of a specific d- or l-chirality in fats, sugars, amino acids, nucleic acids, and so on is ubiquitous in nature, yet the biological origin of such chiral dominance (i.e., with one enantiomer overwhelmingly present) remains an open question.

Single-molecule kinetic studies of DNA hybridization under extreme pressures.

Hydrostatic pressure can perturb biomolecular function by altering equilibrium structures and folding dynamics. Its influences are particularly important to deep sea organisms, as maximum pressures reach ≈1100 bar at the bottom of the ocean as a result of the rapid increase in hydraulic pressure (1 bar every 10 meters) under water. In this work, DNA hybridization kinetics has been studied at the single molecule level with external, tunable pressure control (P≈ 1500 bar), realized by incorporating a mechanical hydraulic capillary sample cell into a confocal fluorescence microscope.

Continuous angular control over anisotropic photoemission from isotropic gold nanoshells.

A variety of applications rely on the efficient generation of hot carriers within metal nanoparticles and charge transfer to surrounding molecules or materials. The optimization of such processes requires a detailed understanding of excited carrier spatial, temporal, and momentum distributions, which also leads to opportunities for active optical control over hot carrier dynamics on nanometer and femtosecond scales. Such capabilities are emerging in nanoplasmonic systems and typically rely on tuning optical polarization and/or frequency to selectively excite one or more discrete hot spots defined by the particle geometry.

Sequential Folding of the Nickel/Cobalt Riboswitch Is Facilitated by a Conformational Intermediate: Insights from Single-Molecule Kinetics and Thermodynamics.

The present work presents the first single-molecule fluorescence resonant energy transfer (smFRET) studies of the nickel/cobalt (NiCo) riboswitch, with temperature-dependent, single-molecule confocal microscopy to provide comprehensive kinetic and thermodynamic information on folding into a biochemically competent structure. The results indicate that the NiCo riboswitch first folds into a more compact "prefolded" conformation, with a preorganized binding pocket partially stabilized under physiological conditions by noncognate monovalent/divalent cations. Such a prefolded intermediate then has opportunity to fold further into a tightly ligand-bound structure, in response to the cognate ligands, Ni or Co, with submicromolar affinities.