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 (J. Am. Chem. Soc. 2022, 144 (17), 7562-7567). Herein, we utilize this new strategy to enable comparisons of experimental and simulated electric fields in protein environments. Specifically, previously determined TDM electric fields exerted onto nitrile-containing o-cyanophenylalanine residues in photoactive yellow protein are compared with MD electric fields from the fixed-charge AMBER FF and the polarizable AMOEBA FF. We observe that the electric field distributions for H-bonding nitriles are substantially affected by the choice of FF. As such, AMBER underestimates electric fields for nitriles experiencing moderate field strengths; in contrast, AMOEBA robustly recapitulates the TDM electric fields. The FF dependence of the electric fields can be partly explained by the presence of additional negative charge density along the nitrile bond axis in AMOEBA, which is due to the inclusion of higher-order multipole parameters; this, in turn, begets more head-on nitrile H-bonds. We conclude by discussing the implications of the FF dependence for the simulation of nitriles and proteins in general.

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. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore-environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.

Exposure to lead-free frangible firing emissions containing copper and ultrafine particulates leads to increased oxidative stress in firing range instructors.

BACKGROUND: Since the introduction of copper based, lead-free frangible (LFF) ammunition to Air Force small arms firing ranges, instructors have reported symptoms including chest tightness, respiratory irritation, and metallic taste. These symptoms have been reported despite measurements determining that instructor exposure does not exceed established occupational exposure limits (OELs). The disconnect between reported symptoms and exposure limits may be due to a limited understanding of LFF firing byproducts and subsequent health effects. A comprehensive characterization of exposure to instructors was completed, including ventilation system evaluation, personal monitoring, symptom tracking, and biomarker analysis, at both a partially enclosed and fully enclosed range. RESULTS: Instructors reported symptoms more frequently after M4 rifle classes compared to classes firing only the M9 pistol. Ventilation measurements demonstrated that airflow velocities at the firing line were highly variable and often outside established standards at both ranges. Personal breathing zone air monitoring showed exposure to carbon monoxide, ultrafine particulate, and metals. In general, exposure to instructors was higher at the partially enclosed range compared to the fully enclosed range. Copper measured in the breathing zone of instructors, on rare occasions, approached OELs for copper fume (0.1 mg/m3). Peak carbon monoxide concentrations were 4-5 times higher at the partially enclosed range compared to the enclosed range and occasionally exceeded the ceiling limit (125 ppm). Biological monitoring showed that lung function was maintained in instructors despite respiratory symptoms. However, urinary oxidative stress biomarkers and urinary copper measurements were increased in instructors compared to control groups. CONCLUSIONS: Consistent with prior work, this study demonstrates that symptoms still occurred despite exposures below OELs. Routine monitoring of symptoms, urinary metals, and oxidative stress biomarkers can help identify instructors who are particularly affected by exposures. These results can assist in guiding protective measures to reduce exposure and protect instructor health. Further, a longitudinal study is needed to determine the long-term health consequences of LFF firing emissions exposure.

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 both H-bonding and non-H-bonding interactions. It can therefore be generally applied to determine electric fields in all environments. Additionally, it allows for semiempirical extraction of the H-bonding contribution to the blueshift of the nitrile frequency. Nitriles were incorporated at H-bonding and non-H-bonding protein sites using amber suppression, and each nitrile variant was structurally characterized at high resolution. We exploited the combined information available from variations in frequency and integrated intensity and demonstrate that nitriles are a generally useful probe for electric fields.

Comprehensive characterization of firing byproducts generated from small arms firing of lead-free frangible ammunition.

Following the introduction of lead-free frangible ammunition in United States Air Force small arms firing ranges, Combat Arms instructors have routinely reported experiencing adverse health symptoms during live fire training exercises, including sore throat, cough, and headache. Previous studies have found that these symptoms occur despite occupational exposure limits not being exceeded. To better characterize the potential source and mechanisms for health symptoms, a comprehensive characterization of the physicochemical properties of gases and aerosols emitted during the firing of the M9 pistol and M4 rifle using lead-free frangible ammunition was completed. Weapons were fired within a sealed chamber using a remote firing mechanism. A suite of direct-reading instruments and collection-based analytical methods were used to determine the composition of the emissions. Emissions were dominated by carbon monoxide and ultrafine particles. Other prevalent gases included carbon dioxide, ammonia, formaldehyde, hydrogen cyanide, and nitric oxide when measured using Fourier-transform infrared spectroscopy. An electrical, low-pressure impactor showed that, on average, the count median diameter immediately after firing was 36 ± 4 nm (n = 10 rounds) and 32 ± 3 nm (n = 14 rounds) for the M9 pistol and M4 rifle, respectively. Analytical methods were used to determine that emitted particles were primarily composed of soot, copper, and potassium, with trace amounts of calcium, silicon, sodium, sulfur, and zinc. Results from this research confirm prior work and expand upon the characterization of emissions generated from firing lead-free frangible ammunition. By employing multiple methods to measure and analyze data we were able to quantify both total and respirable particle fractions and determine particle morphology and composition. Characterization of the emissions provides insight into potential exposure risks that may lead to the development of adverse health symptoms allowing for the development of strategies for risk mitigation.

Kruppel-like factor 15 is a critical regulator of cardiac lipid metabolism.

The mammalian heart, the body's largest energy consumer, has evolved robust mechanisms to tightly couple fuel supply with energy demand across a wide range of physiologic and pathophysiologic states, yet, when compared with other organs, relatively little is known about the molecular machinery that directly governs metabolic plasticity in the heart. Although previous studies have defined Kruppel-like factor 15 (KLF15) as a transcriptional repressor of pathologic cardiac hypertrophy, a direct role for the KLF family in cardiac metabolism has not been previously established. We show in human heart samples that KLF15 is induced after birth and reduced in heart failure, a myocardial expression pattern that parallels reliance on lipid oxidation. Isolated working heart studies and unbiased transcriptomic profiling in Klf15-deficient hearts demonstrate that KLF15 is an essential regulator of lipid flux and metabolic homeostasis in the adult myocardium. An important mechanism by which KLF15 regulates its direct transcriptional targets is via interaction with p300 and recruitment of this critical co-activator to promoters. This study establishes KLF15 as a key regulator of myocardial lipid utilization and is the first to implicate the KLF transcription factor family in cardiac metabolism.