Infrared Pump–Probe Study of Nanoconfined Water Structure in Reverse Micelle

The influence of nanoconfinement on water structure is studied with time- and frequency-resolved vibrational spectroscopy of hydrazoic acid (HN₃) encapsulated in reverse micelle. The azido stretch mode of HN₃ is found to be a promising infrared probe for studying the structure and local hydrogen-bond environment of confined and interfacial water in reverse micelle due to its narrow spectral bandwidth and large transition dipole moment. The results show a clear separation between the core and shell spectral components, making it advantageous over the previously studied infrared probes. The measured vibrational lifetimes appear to be substantially different for the interfacial and bulk-like environments but show no remarkable size dependency, which indicates that water structures around this IR probe are distinctively different in the core and shell regions. The influence of local hydrogen bond network in the first and higher solvation shells on the vibrational dynamics of HN₃ is further discussed

Distributed Multipolar Expansion Approach to Calculation of Excitation Energy Transfer Couplings

We propose a new approach for estimating the electrostatic part of the excitation energy transfer (EET) coupling between electronically excited chromophores based on the transition density-derived cumulative atomic multipole moments (TrCAMM). In this approach, the transition potential of a chromophore is expressed in terms of truncated distributed multipolar expansion and analytical formulas for the TrCAMMs are derived. The accuracy and computational feasibility of the proposed approach is tested against the exact Coulombic couplings, and various multipole expansion truncation schemes are analyzed. The results of preliminary calculations show that the TrCAMM approach is capable of reproducing the exact Coulombic EET couplings accurately and efficiently and is superior to other widely used schemes: the transition charges from electrostatic potential (TrESP) and the transition density cube (TDC) method

Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy

Conformational changes are central to protein function but challenging to characterize with both high spatial and temporal precision. The inherently fast time scale and small chromophores of infrared (IR) spectroscopy are well-suited for characterization of potentially rapidly fluctuating environments, and when frequency-resolved probes are incorporated to overcome spectral congestion, enable characterization of specific sites in proteins. We selectively incorporated <i>p</i>-cyanophenylalanine (CNF) as a vibrational probe at five distinct locations in the enzyme cytochrome P450cam and used IR spectroscopy to characterize the environments in substrate and/or ligand complexes reflecting those in the catalytic cycle. Molecular dynamics (MD) simulations were performed to provide a structural basis for spectral interpretation. Together the experimental and simulation data suggest that the CN frequencies are sensitive to both long-range influences, resulting from the particular location of a residue within the enzyme, as well as short-range influences from hydrogen bonding and packing interactions. The IR spectra demonstrate that the environments and effects of substrate and/or ligand binding are different at each position probed and also provide evidence that a single site can experience multiple environments. This study illustrates how IR spectroscopy, when combined with the spectral decongestion and spatial selectivity afforded by CNF incorporation, provides detailed information about protein structural changes that underlie function

Isonitrile as an Ultrasensitive Infrared Reporter of Hydrogen-Bonding Structure and Dynamics

Infrared (IR) probes based on terminally blocked β-isocyanoalanine (AlaNC) and <i>p</i>-isocyanophenylalanine (PheNC) amino acids were synthesized. These isonitrile (NC)-derivatized compounds were extensively characterized by FTIR and femtosecond IR pump–probe spectroscopies, and a direct comparison was made with popularly used nitrile (CN)- and azide (N₃)-derivatized analogs. It is shown that the isonitrile stretch frequency exhibits extremely high sensitivity to hydrogen-bonding interactions. In addition, the IR intensity of the isonitrile group is much higher than that of the nitrile group and almost as intense as that of the azido group. Furthermore, its vibrational lifetime is much longer than that of the nitrile and azido groups. To elucidate the origin of such a high H-bond sensitivity and IR intensity observed for isonitrile, extensive quantum chemical calculations were performed. It is shown that the Coulombic contributions to the vibrational frequency shifts of the isonitrile and nitrile stretch modes have opposite signs but similar magnitudes, whereas the contributions of exchange repulsion and charge delocalization to their frequency shifts are comparable. Therefore, the isonitrile stretch frequency is much more sensitive to H-bonding interactions because the blue-shifting exchange-repulsion effects are additionally enforced by such electrostatic effects. It is also shown that the much higher IR intensity of the isonitrile group compared to that of the nitrile group is due to the configuration reversal of the atomic electronegativity between the NC and CN groups. Owing to these features, we believe that isonitrile is a much better IR reporter of H-bonding structure and dynamics than the widely used nitrile and azide

Spectroscopic Signature for Stable β‑Amyloid Fibrils versus β‑Sheet-Rich Oligomers

We use two-dimensional IR (2D IR) spectroscopy to explore fibril formation for the two predominant isoforms of the β-amyloid (Aβ_1‑40 and Aβ_1‑42) protein associated with Alzheimer’s disease. Two-dimensional IR spectra resolve a transition at 1610 cm^–1 in Aβ fibrils that does not appear in other Aβ aggregates, even those with predominantly β-sheet-structure-like oligomers. This transition is not resolved in linear IR spectroscopy because it lies under the broad band centered at 1625 cm^–1, which is the traditional infrared signature for amyloid fibrils. The feature is prominent in 2D IR spectra because 2D lineshapes are narrower and scale nonlinearly with transition dipole strengths. Transmission electron microscopy measurements demonstrate that the 1610 cm^–1 band is a positive identification of amyloid fibrils. Sodium dodecyl sulfate micelles that solubilize and disaggregate preaggregated Aβ samples deplete the 1625 cm^–1 band but do not affect the 1610 cm^–1 band, demonstrating that the 1610 cm^–1 band is due to very stable fibrils. We demonstrate that the 1610 cm^–1 transition arises from amide I modes by mutating out the only side-chain residue that could give rise to this transition, and we explore the potential structural origins of the transition by simulating 2D IR spectra based on Aβ crystal structures. It was not previously possible to distinguish stable Aβ fibrils from the less stable β-sheet-rich oligomers with infrared light. This 2D IR signature will be useful for Alzheimer’s research on Aβ aggregation, fibril formation, and toxicity

Modulation of the Hydrogen Bonding Structure of Water by Renal Osmolytes

Osmolytes are an integral part of living organism, e.g., the kidney uses sorbitol, trimethylglycine, taurine and myo-inositol to counter the deleterious effects of urea and salt. Therefore, knowing that the osmolytes’ act either directly to the protein or mediated through water is of great importance. Our experimental and computational results show that protecting osmolytes, e.g., trimethylglycine and sorbitol, significantly modulate the water H-bonding network structure, although the magnitude and spatial extent of osmolyte-induced perturbation greatly vary. In contrast, urea behaves neutrally toward local water H-bonding network. Protecting osmolytes studied here show strong concentration-dependent behaviors (vibrational frequencies and lifetimes of two different infrared (IR) probes), while denaturant does not. The H-bond donor and/or acceptor (OH/NH) in a given osmolyte molecule play a critical role in defining their action. Our findings highlight the significance of the alteration of H-bonding network of water under biologically relevant environment, often encountered in real biological systems

Modulation of the Hydrogen Bonding Structure of Water by Renal Osmolytes

Osmolytes are an integral part of living organism, e.g., the kidney uses sorbitol, trimethylglycine, taurine and myo-inositol to counter the deleterious effects of urea and salt. Therefore, knowing that the osmolytes’ act either directly to the protein or mediated through water is of great importance. Our experimental and computational results show that protecting osmolytes, e.g., trimethylglycine and sorbitol, significantly modulate the water H-bonding network structure, although the magnitude and spatial extent of osmolyte-induced perturbation greatly vary. In contrast, urea behaves neutrally toward local water H-bonding network. Protecting osmolytes studied here show strong concentration-dependent behaviors (vibrational frequencies and lifetimes of two different infrared (IR) probes), while denaturant does not. The H-bond donor and/or acceptor (OH/NH) in a given osmolyte molecule play a critical role in defining their action. Our findings highlight the significance of the alteration of H-bonding network of water under biologically relevant environment, often encountered in real biological systems