Vibrational spectroscopic determination of local solvent electric field, solute-solvent electrostatic interaction energy, and their fluctuation amplitudes

IR probes have been extensively used to monitor local electrostatic and solvation dynamics. Particularly, their vibrational frequencies are highly sensitive to local solvent electric field around an IR probe. Here, we show that the experimentally measured vibrational frequency shifts can be inversely used to determine local electric potential distribution and solute-solvent electrostatic interaction energy. In addition, the upper limits of their fluctuation amplitudes are estimated by using the vibrational bandwidths. Applying this method to fully deuterated N-methylacetamide (NMA) in D(2)O and examining the solvatochromic effects on the amide I' and II' mode frequencies, we found that the solvent electric potential difference between O(═C) and D(-N) atoms of the peptide bond is about 5.4 V, and thus, the approximate solvent electric field produced by surrounding water molecules on the NMA is 172 MV/cm on average if the molecular geometry is taken into account. The solute-solvent electrostatic interaction energy is estimated to be -137 kJ/mol, by considering electric dipole-electric field interaction. Furthermore, their root-mean-square fluctuation amplitudes are as large as 1.6 V, 52 MV/cm, and 41 kJ/mol, respectively. We found that the water electric potential on a peptide bond is spatially nonhomogeneous and that the fluctuation in the electrostatic peptide-water interaction energy is about 10 times larger than the thermal energy at room temperature. This indicates that the peptide-solvent interactions are indeed important for the activation of chemical reactions in aqueous solution.     

Vibrational solvatochromism and electrochromism of infrared probe molecules containing C≡O, C≡N, C=O, or C-F vibrational chromophore

Solvatochromic vibrational frequency shifts of a few different infrared (IR) probe molecules have been studied by carrying out quantum chemistry calculations for a number of their water clusters. We are particularly focused on the vibrational solvatochromic and electrochromic effects on the CO, CN, and CF stretch modes in carbon monoxide, acetone, 4-cyanopyridine, p-tolunitrile, fluorobenzene, and 3-fluoropyridine. Using multiple interaction site antenna model, we show that their solvatochromic vibrational frequency shifts can be successfully described by considering spatially nonuniform electrostatic potential generated by the surrounding water molecules. It turns out that the CO and CF stretch mode frequencies are approximately proportional to the solvent electric field projected onto the bond axes, whereas the vibrational frequencies of the nitrile stretch mode in 4-cyanopyridine and p-tolunitrile are not. Consequently, it is confirmed that the vibrational Stark tuning rates of the CO and CF stretching modes can be directly used to describe their solvatochromic frequency shifts in condensed phases. However, the nitrile stretch mode frequency shift induced by solvent electrostatic potential appears to be more complicated than its electrochromic phenomenon. To examine the validity of the distributed interaction site model for solvatochromic frequency shifts of these vibrational chromophores, we thus calculated the vibrational Stark tuning rates of the CO, CN, and CF stretch modes and found that they are in good agreement with the experimental results found in literatures. This confirms that a collection of properly chosen distributed interaction sites can be an excellent electric antenna sensing local electrostatics that affects on vibrational frequencies of IR probe modes.     

Optimized quantum mechanics/molecular mechanics strategies for nitrile vibrational probes: acetonitrile and para-tolunitrile in water and tetrahydrofuran

The nitrile (Ctriple bondN) group is a powerful probe of structure and dynamics because its vibrational frequency is extraordinarily sensitive to the electrostatic and chemical characteristics of its local environment. For example, site-specific nitrile labels are useful indicators of protein structure because their infrared (IR) absorption spectra can clearly distinguish between solvent-exposed residues and residues buried in the hydrophobic core of a protein. In this work, three variants of the optimized quantum mechanics/molecular mechanics (OQM/MM) technique for computing Ctriple bondN vibrational frequencies were developed and assessed for acetonitrile in water. For the most robust variant, the transferability of the OQM/MM methodology to different solutes and solvents was evaluated by simulating the IR absorption spectra of para-tolunitrile in water and tetrahydrofuran and comparing to experiment and density functional theory (DFT) calculations. The OQM/MM frequencies compared favorably to DFT for para-tolunitrile/water, and the calculated IR absorption spectra are in qualitative agreement with experiment. This suggests that a single parametrization of the OQM/MM technique is reasonable for the calculation of nitrile line shapes when the probe is attached to different chemical moieties and when the label experiences local environments of different polarity.     

Infrared Absorption Intensity Analysis as a New Tool for Investigation of Salt Effect on Proteins

The native protein structures in buffer solution are maintained by the electrostatic force as well as the hydrophobic force, salt ions play an important role in maintaining the protein native structures, and their effect on the protein stability has attracted tremendous interests. Infrared spectroscopy has been generally used in molecular structure analysis due to its fingerprint resolution for different species including macromolecules as proteins. However spectral intensities have received much less attention than the vibrational frequencies. Here we report that the spectral intensities of protein amide I band, the finger prints for the protein secondary structures, are very sensitive to the local electric field known as Onsager reaction field caused by salt ions. IR absorbance thermal titrations have been conducted for a series of samples including simple water soluble amino acids, water soluble monomeric protein cytochrome c and dimeric protein DsbC and its single-site mutant G49R. We found that at lower temperature range （10-20℃）, there exists a thermal activated salting-in process, where the IR intensity increases with a rise in the temperature, corresponding to the ions binding of the hydrophobic surface of protein. This process is absent for the amino acids. When further raising the temperature, the IR intensity decreases, this is interpreted as the thermal activated breaking of the ion-protein surface binding. Applying Van＇t Hoff plot to the thermal titration curves, the thermodynamic parameters such as AH and AS for salting-in and ion unbinding processes can be derived for various protein secondary structural components, revealing quantitatively the extent of hydrophobic interaction as well as the strength of the ion-protein binding.     

Phosphate vibrations probe local electric fields and hydration in biomolecules

The role of electric fields in important biological processes such as binding and catalysis has been studied almost exclusively by computational methods. Experimental measurements of the local electric field in macromolecules are possible using suitably calibrated vibrational probes. Here we demonstrate that the vibrational transitions of phosphate groups are highly sensitive to an electric field and show how that sensitivity can be quantified, allowing electric field measurements to be made in phosphate-containing biological systems without chemical modification.     

Hofmeister anionic effects on hydration electric fields around water and peptide

Specific ion effects on water dynamics and local solvation structure around a peptide are important in understanding the Hofmeister series of ions and their effects on protein stability in aqueous solution. Water dynamics is essentially governed by local hydrogen-bonding interactions with surrounding water molecules producing hydration electric field on each water molecule. Here, we show that the hydration electric field on the OD bond of HOD molecule in water can be directly estimated by measuring its OD stretch infrared (IR) radiation frequency shift upon increasing ion concentration. For a variety of electrolyte solutions containing Hofmeister anions, we measured the OD stretch IR bands and estimated the hydration electric field on the OD bond to be about a hundred MV∕cm with standard deviation of tens of MV∕cm. As anion concentration increases from 1 to 6 M, the hydration electric field on the OD bond decreases by about 10%, indicating that the local H-bond network is partially broken by dissolved ions. However, the measured hydration electric fields on the OD bond and its fluctuation amplitudes for varying anions are rather independent on whether the anion is a kosmotrope or a chaotrope. To further examine the Hofmeister effects on H-bond solvation structure around a peptide bond, we examined the amide I' and II' mode frequencies of N-methylacetamide in various electrolyte D(2)O solutions. It is found that the two amide vibrational frequencies are not affected by ions, indicating that the H-bond solvation structure in the vicinity of a peptide remains the same irrespective of the concentration and character of ions. The present experimental results suggest that the Hofmeister anionic effects are not caused by direct electrostatic interactions of ions with peptide bond or water molecules in its first solvation shell. Furthermore, even though the H-bond network of water is affected by ions, thus induced change of local hydration electric field on the OD bond of HOD is not in good correlation with the well-known Hofmeister series. We anticipate that the present experimental results provide an important clue about the Hofmeister effect on protein structure and present a discussion on possible alternative mechanisms.     

A polarizable force field for computing the infrared spectra of the polypeptide backbone

The shapes of the amide bands in the infrared (IR) spectra of proteins and peptides are caused by electrostatically coupled vibrations within the polypeptide backbone and code the structures of these biopolymers. A structural decoding of the amide bands has to resort to simplified models because the huge size of these macromolecules prevents the application of accurate quantum mechanical methods such as density functional theory (DFT). Previous models employed transition-dipole coupling methods that are of limited accuracy. Here we propose a concept for the computation of protein IR spectra, which describes the molecular mechanics (MM) of polypeptide backbones by a polarizable force field of "type II". By extending the concepts of conventional polarizable MM force fields, such a PMM/II approach employs field-dependent parameters not only for the electrostatic signatures of the molecular components but also for the local potentials modeling the stiffness of chemical bonds with respect to elongations, angle deformations, and torsions. Using a PMM/II force field, the IR spectra of the polypeptide backbone can be efficiently calculated from the time dependence of the backbone's dipole moment during a short (e.g., 100 ps) MD simulation by Fourier transformation. PMM/II parameters are derived for harmonic bonding potentials of amide groups in polypeptides from a series of DFT calculations on the model molecule N-methylacetamide (NMA) exposed to homogeneous external electric fields. The amide force constants are shown to vary by as much as 20% for relevant field strengths. As a proof of principle, it is shown that the large solvatochromic effects observed in the IR spectra of NMA upon transfer from the gas phase into aqueous solution are not only excellently reproduced by DFT/MM simulations but are also nicely modeled by the PMM/II approach. The tasks remaining for a proof of practice are specified.     

Structural disorder of the CD3zeta transmembrane domain studied with 2D IR spectroscopy and molecular dynamics simulations

In a recently reported study [Mukherjee, et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3528] we used 2D IR spectroscopy and 1-(13)C=(18)O isotope labeling to measure the vibrational dynamics of 11 amide I modes in the CD3zeta transmembrane domain. We found that the homogeneous line widths and population relaxation times were all nearly identical, but that the amount of inhomogeneous broadening correlated with the position of the amide group inside the membrane. In this study, we use molecular dynamics simulations to investigate the structural and dynamical origins of these experimental observations. We use two models to convert the simulations to frequency trajectories from which the mean frequencies, standard deviations, frequency correlation functions, and 2D IR spectra are calculated. Model 1 correlates the hydrogen-bond length to the amide I frequency, whereas model 2 uses an ab initio-based electrostatic model. We find that the structural distributions of the peptidic groups and their environment are reflected in the vibrational dynamics of the amide I modes. Environmental forces from the water and lipid headgroups partially denature the helices, shifting the infrared frequencies and creating larger inhomogeneous distributions for residues near the ends. The least inhomogeneously broadened residues are those located in the middle of the membrane where environmental electrostatic forces are weakest and the helices are most ordered. Comparison of the simulations to experiment confirms that the amide I modes near the C-terminal are larger than at the N-terminal because of the asymmetric structure of the peptide bundle in the membrane. The comparison also reveals that residues at a kink in the alpha-helices have broader line widths than more helical parts of the peptide because the peptide backbone at the kink exhibits a larger amount of structural disorder. Taken together, the simulations and experiments reveal that infrared line shapes are sensitive probes of membrane protein structural and environmental heterogeneity.     

Site-specific conversion of cysteine thiols into thiocyanate creates an IR probe for electric fields in proteins

The nitrile stretching mode of the thiocyanate moiety is a nearly ideal probe for measuring the local electric field arising from the organized environment of the interior of a protein. Nitriles were introduced into three proteins: ribonuclease S (RNase S), human aldose reductase (hALR2), and the reaction center (RC) of Rhodobacter capsulatus, through a facile synthetic scheme for the transformation of cysteine residues into thiocyanatoalanine. Vibrational Stark effect spectroscopy and Fourier transform infrared spectroscopy on the modified proteins demonstrated that thiocyanate residues are a highly general tool for probing electrostatic fields in proteins.     

Computational spectroscopy of ubiquitin: comparison between theory and experiments

Using the constrained molecular dynamics simulation method in combination with quantum chemistry calculation, Hessian matrix reconstruction, and fragmentation approximation methods, the authors have established computational schemes for numerical simulations of amide I IR absorption, vibrational circular dichroism (VCD), and two-dimensional (2D) IR photon echo spectra of the protein ubiquitin in water. Vibrational characteristic features of these spectra in the amide I vibration region are discussed. From the semiempirical quantum chemistry calculation results on an isolated ubiquitin, amide I local mode frequencies and vibrational coupling constants were fully determined. It turns out that the amide I local mode frequencies of ubiquitin in both gas phase and aqueous solution are highly heterogeneous and site dependent. To directly test the quantitative validity of thus obtained spectroscopic properties, they compared the experimentally measured amide I IR, 2D IR, and electronic circular dichroism spectra with experiments, and found good agreements between theory and experiments. However, the simulated VCD spectrum is just qualitatively similar to the experimentally measured one. This indicates that, due to delicate cancellations between the positive and negative VCD contributions, the prediction of protein VCD spectrum is critically relied on quantitative accuracy of the theoretical model for predicting amide I local mode frequencies. On the basis of the present comparative investigations, they found that the site dependency of amide I local mode frequency, i.e., diagonal heterogeneity of the vibrational Hamiltonian matrix in the amide I local mode basis, is important. It is believed that the present computational methods for simulating various vibrational and electronic spectra of proteins will be of use in further refining classical force fields and in addressing the structure-spectra relationships of proteins in solution.     

Theoretical study of internal field effects on peptide amide I modes

Charged terminal groups or polar side chains of amino acids create spatially nonuniform electrostatic potential around intramolecular peptide bonds and induce amide I mode frequency shifts in polypeptides. By carrying out a series of quantum chemistry calculation studies of various ionic di- and tripeptides as well as dipeptides of 20 different amino acids, these internal field effects on vibrational properties are theoretically investigated. The amide I local and normal mode frequencies and dipole and rotational strengths determining IR and vibrational circular dichroism intensities, respectively, are found to depend on the polar nature of side chains, whereas the vibrational coupling strength weakly does so. The empirical correction and fragment analysis methods were used to theoretically calculate the amide I local mode frequencies and dipole and rotational strengths. These values were directly compared with ab initio and density functional theory calculation results, and the agreements were found to be quantitative.     

Ultrafast vibrational spectroscopy of water and aqueous N-methylacetamide: Comparison of different electronic structure/molecular dynamics approaches

Kwac and Cho [J. Chem. Phys. 119, 2247 (2003)] have recently developed a combined electronic structure/molecular dynamics approach to vibrational spectroscopy in liquids. The method involves fitting ab initio vibrational frequencies for a solute in a cluster of solvent molecules to a linear combination of the electrostatic potentials on the solute atoms due to the charges on the solvent molecules. These authors applied their method to the N-methylacetamide-D/D(2)O system. We (S. A. Corcelli, C. P. Lawrence, and J. L. Skinner, [J. Chem. Phys. 120, 8107 (2004)]) have recently explored a closely related method, where instead of the electrostatic potential, the solute vibrational frequencies are fit to the components of the electric fields on the solute atoms due to the solvent molecules. We applied our method to the HOD/D(2)O and HOD/H(2)O systems. In order to make a direct comparison of these two approaches, in this paper we apply their method to the water system, and our method to the N-methylacetamide system. For the water system we find that the electric field method is superior to the potential approach, as judged by comparison with experiments for the absorption line shape. For the N-methylacetamide system the two methods are comparable.     

Azido Homoalanine is a Useful Infrared Probe for Monitoring Local Electrostatistics and Sidechain Solvation in Proteins

The use of IR probes to monitor protein structure, deduce local electric field, and investigate the mechanism of enzyme catalysis and protein folding has attracted increasing attention. Here, the azidohomoalanine (Aha) is considered as a useful IR probe. The intricate details of the distinct effects of backbone peptide bonds and H-bonded water molecules on the azido stretch mode of the IR probe Aha were revealed by carrying out QM/MM MD simulations of two variants of the protein NTL9, NTL9-Met1Aha and NTL9-Ile4Aha and comparing the resulting simulated IR spectra with experiments.     

Enhanced Sensitivity to Local Dynamics in Peptides by Use of Temperature-Jump IR Spectroscopy and Isotope Labeling

Site-specific isotopic labeling of molecules is a widely used approach in IR spectroscopy to resolve local contributions to vibrational modes. The induced frequency shift of the corresponding IR band depends on the substituted masses, as well as on hydrogen bonding and vibrational coupling. The impact of these different factors was analyzed with a designed three-stranded β-sheet peptide and by use of selected ¹³C isotope substitutions at multiple positions in the peptide backbone. Single-strand labels give rise to isotopically shifted bands at different frequencies, depending on the specific sites; this demonstrates sensitivity to the local environment. Cross-strand double- and triple-labeled peptides exhibited two resolved bands that could be uniquely assigned to specific residues, the equilibrium IR spectra of which indicated only weak local-mode coupling. Temperature-jump IR laser spectroscopy was applied to monitor structural dynamics and revealed an impressive enhancement of the isotope sensitivity to both local positions and coupling between them, relative to that of equilibrium FTIR spectroscopy. Site-specific relaxation rates were altered upon the introduction of additional cross-strand isotopes. Likewise, the rates for the global β-sheet dynamics were affected in a manner dependent on the distinct relaxation behavior of the labeled oscillator. This study reveals that isotope labels provide not only local structural probes, but rather sense the dynamic complexity of the molecular environment.     

Experimental and theoretical studies on the identification of p-biphenyloxycarbonylphenyl acrylate

This study presents the identification of a title compound, p-biphenyloxycarbonylphenyl acrylate by means of experimental and theoretical evidences. The spectroscopic properties of the compound were experimentally investigated by Fourier transformation-infrared spectra (in the region 400-4000 cm(-1)) and nuclear magnetic resonance (NMR) chemical shifts (with a frequency of 400 MHz). Moreover, the optimized molecular structures, vibrational frequencies including infrared intensities and Raman activities, corresponding vibrational spectra interpreted with the aid of normal coordinate analysis based on scaled density functional force field, thermodynamic properties, atomic charges and ultraviolet-visible (UV-vis) spectra were analyzed utilizing ab initio Hartree-Fock (HF) and Density Functional Theory (B3LYP) methods at 6-31G(d,p) calculation level. It was found that the vibrational frequencies and chemical shifts obtained were shown to have a good agreement with available experimental results. We not only simulated frontier molecular orbitals (FMO) and molecular electrostatic potential (MEP) but also evaluated the transition state and energy band gap clearly.     

Theoretical investigations of α,α,α-trifluoro-3, -p and o-nitrotoluene by means of density functional theory

This study reports the optimized molecular structures, vibrational frequencies including Infrared intensities and Raman activities, corresponding vibrational assignments, (1)H and (13)C NMR chemical shifts, the magnitudes of the JCH and JCC coupling constants, Ultraviolet-visible (UV-vis) spectra, thermodynamic properties and atomic charges of the title compounds, α,α,α-trifluoro-3, -p and o-nitrotoluene, in the ground state by means of the density functional theory (DFT) with the standard B3LYP/6-311++G(d,p) method and basis set combination for the first time. Theoretical vibrational spectra were interpreted by normal coordinate analysis based on scaled density functional force field. The results show that the vibrational frequencies and chemical shifts calculated were obtained to be in good agreement with the experimental data. Based on the comparison between experimental results and theoretical data, the calculation level chosen is powerful approach for understanding the identification of all the molecules studied. In addition, not only were frontier molecular orbitals (HOMO and LUMO), molecular electrostatic potential (MEP) and electrostatic potential (ESP) simulated but also the dipole moment, softness, electronegativity, chemical hardness, electrophilicity index, transition state and energy band gap values were predicted. According to the investigations, all compounds were found to be useful to bond metallically and interact intermolecularly; however, the thermodynamic properties confirm that the α,α,α-trifluoro-p-nitrotoluene was more reactive and more polar than the others.     

Nitrile and thiocyanate IR probes: quantum chemistry calculation studies and multivariate least-square fitting analysis

Hydration effects on the C[Triple Bond]N stretching mode frequencies of MeCN and MeSCN are investigated by carrying out ab initio calculations for a number of MeCN-water and MeSCN-water complexes with varying number of water molecules. It is found that the CN frequency shift induced by the hydrogen-bonding interactions with water molecules originate from two different ways to form hydrogen bonds with the nitrogen atom of the CN group. Considering the MeCN- and MeSCN-water cluster calculation results as databases, we first examined the validity of vibrational Stark effect relationship between the CN frequency and the electric field component parallel to the CN bond and found no strong correlation between the two. However, taking into account of additional electric field vector components is a simple way to generalize the vibrational Stark theory for the nitrile chromophore. Also, the electrostatic potential calculation method has been proposed and examined in detail. It turned out that the interactions of water molecules with nitrogen atom's lone pair orbital and with nitrile pi orbitals can be well described by the electrostatic potential calculation method. The present computational results will be of use to quantitatively simulate various linear and nonlinear vibrational spectra of nitrile compounds in solutions.     

Density functional theory study on the identification of 3-[(2-morpholinoethylimino)methyl]benzene-1,2-diol

This study deals with the identification of a title compound, 3-[(2-morpholinoethylimino)methyl]benzene-1,2-diol by means of quantum chemical calculations. The optimized molecular structures, vibrational frequencies and corresponding vibrational assignments, thermodynamic properties, charge analyses, nuclear magnetic resonance (NMR) chemical shifts and ultraviolet-visible (UV-vis) spectra of the title molecule in the ground state were evaluated using density functional theory (DFT) with the standard B3LYP/6-311++G(d,p) method and basis set combination for the first time. Theoretical vibrational spectra of the title compound were interpreted with the aid of normal coordinate analysis based on scaled density functional force field. The results show that the obtained optimized geometric parameters (bond lengths, bond angles and bond dihedrals) and vibrational frequencies were observed to be in good agreement with the available experimental results. Moreover, the calculations of the electronic spectra, (13)C and (1)H chemical shifts were compared with the experimental ones. Furthermore, we not only simulated the frontier molecular orbitals (FMO) and molecular electrostatic potential (MEP) but also determined the transition states and energy band gaps, as well. It was found that charge analyses supported the evidences of MEP. Infrared intensities and Raman activities were also reported.