Martina Havenith

An extended dynamical hydration shell around proteins

The focus in protein folding has been very much on the protein backbone and sidechains. However, hydration waters make comparable contributions to the structure and energy of proteins. The coupling between fast hydration dynamics and protein dynamics is considered to play an important role in protein folding. Fundamental questions of protein hydration include, how far out into the solvent does the influence of the biomolecule reach, how is the water affected, and how are the properties of the hydration water influenced by the separation between protein molecules in solution? We show here that Terahertz spectroscopy directly probes such solvation dynamics around proteins, and determines the width of the dynamical hydration layer. We also investigate the dependence of solvation dynamics on protein concentration. We observe an unexpected nonmonotonic trend in the measured terahertz absorbance of the five helix bundle protein λ6–85* as a function of the protein: water molar ratio. The trend can be explained by overlapping solvation layers around the proteins. Molecular dynamics simulations indicate water dynamics in the solvation layer around one protein to be distinct from bulk water out to ≈10 Å. At higher protein concentrations such that solvation layers overlap, the calculated absorption spectrum varies nonmonotonically, qualitatively consistent with the experimental observations. The experimental data suggest an influence on the correlated water network motion beyond 20 Å, greater than the pure structural correlation length usually observed.

Solute-induced retardation of water dynamics probed directly by terahertz spectroscopy

The dynamics of water surrounding a solute is of fundamental importance in chemistry and biology. The properties of water molecules near the surface of a bio-molecule have been the subject of numerous, sometimes controversial experimental and theoretical studies, with some suggesting the existence of rather rigid water structures around carbohydrates and proteins [Pal, S. K., Peon, J., Bagchi, B. & Zewail A. H. (2002) J. Phys. Chem. B 106, 12376–12395]. Hydrogen bond rearrangement in water occurs on the picosecond time scale, so relevant experiments must access these times. Here, we show that terahertz spectroscopy can directly investigate hydration layers. By a precise measurement of absorption coefficients between 2.3 THz and 2.9 THz we could determine the size and the characteristics of the hydration shell. The hydration layer around a carbohydrate (lactose) is determined to extend to 5.13 ± 0.24 Å from the surface corresponding to ≈123 water molecules beyond the first solvation shell. Accompanying molecular modeling calculations support this result and provide a microscopic visualization. Terahertz spectroscopy is shown to probe the collective modes in the water network. The observed increase of the terahertz absorption of the water in the hydration layer is explained in terms of coherent oscillations of the hydration water and solute. Simulations also reveal a slowing down of the hydrogen bond rearrangement dynamics for water molecules near lactose, which occur on the picosecond time scale. The present study demonstrates that terahertz spectroscopy is a sensitive tool to detect solute-induced changes in the water network.

Dissecting the THz spectrum of liquid water from first principles via correlations in time and space

Solvation of molecules in water is at the heart of a myriad of molecular phenomena and of crucial importance to understanding such diverse issues as chemical reactivity or biomolecular function. Complementing well-established approaches, it has been shown that laser spectroscopy in the THz frequency domain offers new insights into hydration from small solutes to proteins. Upon introducing spatially-resolved analyses of the absorption cross section by simulations, the sensitivity of THz spectroscopy is traced back to characteristic distance-dependent modulations of absorption intensities for bulk water. The prominent peak at ≈200 cm-1 is dominated by first-shell dynamics, whereas a concerted motion involving the second solvation shell contributes most significantly to the absorption at about 80 cm-1 ≈2.4 THz. The latter can be understood in terms of an umbrella-like motion of two hydrogen-bonded tetrahedra along the connecting hydrogen bond axis. Thus, a modification of the hydrogen bond network, e.g., due to the presence of a solute, is expected to affect vibrational motion and THz absorption intensity at least on a length scale that corresponds to two layers of solvating water molecules. This result provides a molecular mechanism explaining the experimentally determined sensitivity of absorption changes in the THz domain in terms of distinct, solute-induced dynamical properties in solvation shells of (bio)molecules—even in the absence of well-defined resonances.

Long-Range Influence of Carbohydrates on the Solvation Dynamics of Water-Answers from Terahertz Absorption Measurements and Molecular Modeling Simulations

We present new terahertz (THz) spectroscopic measurements of solvated sugars and compare the effect of two disaccharides (trehalose and lactose) and one monosaccharide (glucose) with respect to the solute-induced changes in the sub-picosecond network dynamics of the hydration water. We found that the solute affects the fast collective network motions of the solvent, even beyond the first solvation layer. For all three carbohydrates, we find an increase of 2-4% in the THz absorption coefficient of the hydration water in comparison to bulk water. Concentration-dependent changes in the THz absorption between 2.1 and 2.8 THz of the solute-water mixture were measured with a precision better than 1% and were used to deduce a dynamical hydration shell, which extends from the surface up to 5.7 +/- 0.4 and 6.5 +/- 0.9 A for the disaccharides lactose and trehalose, respectively, and 3.7 +/- 0.9 A for the glucose. This exceeds the values for the static hydration shell as determined, for example, by scattering, where the long-range structure was found to be not significantly affected by the solute beyond the first hydration shell. When comparing all three carbohydrates, we found that the solute-induced change in the THz absorption depends on the product of molar concentration of the solute and the number of hydrogen bonds between the carbohydrate and water molecules. We can conclude that the long-range influence on the sub-picosecond collective water network motions of the hydration water is directly correlated with the average number of hydrogen bonds between the molecule and adjacent water molecules for carbohydrates. This implies that monosaccharides have a smaller influence on the surrounding water molecules than disaccharides. This could explain the bioprotection mechanism of sugar-water mixtures, which has been found to be more effective for disaccharides than for monosaccharides.

Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site.

Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

Label‐Free Imaging of Metal–Carbonyl Complexes in Live Cells by Raman Microspectroscopy

The search for novel metal complexes with therapeutic activity, in particular against cancer and infectious diseases, is an active and important area of research in medicinal inorganic chemistry. In addition to the well-studied platinumand ruthenium-based coordination complexes, organometallic compounds have gained considerable importance in recent years. Of those, metal–carbonyl compounds are steadily increasing in interest, with some exhibiting remarkable antitumor activity. The most prominent example is probably the use of such compounds as “solid storage forms” for carbon monoxide. These CO-releasing molecules (CORMs) allow the biological action of this important small molecule messenger to be investigated. To elucidate the biological mode of action of any drug candidate it is mandatory to obtain a detailed picture of the intracellular distribution of such substances and how it evolves over time. Until now, the localization of metal complexes inside cells has been studied using X-ray fluorescence (XRF) 14] and atomic absorption spectroscopy (AAS). While AAS offers high sensitivity but almost no spatial resolution, XRF requires intense X-ray sources such as synchrotrons which will cause damage to biological tissue and is also not routinely available as an analytical technique. Most cellular studies therefore use fluorescence microscopy. Furthermore, this technique requires the additional attachment of a fluorescent label, which might be difficult. Optical excitation can also cause additional problems such as the onset of photochemical reactions or photobleaching. Moreover, the label can alter the biodistribution and properties of the molecule of interest, as recently shown for ruthenium–bipyridyl complexes. Efforts have been made to overcome these limitations by identifying biologically active metal complexes which show inherent fluorescence in vivo, but this has only been possible for a small number of metal–ligand combinations. Thus, it is highly desirable to develop innovative and generally applicable imaging techniques for the study of the uptake and distribution of bioactive metal complexes which do not require any labeling or special photophysical properties but instead use the intrinsic spectroscopic signature of the compound of interest. Raman microspectroscopy is emerging as a powerful noninvasive method to assess and image cellular compartments and processes without further sample preparation or labeling. Since Puppels et al. first showed the feasibility of confocal Raman microspectroscopy for imaging cells, its ability to study whole cells and subcellular organelles such as the nucleus and chromatin, mitochondria, and lipid bodies has been demonstrated by various research groups. Apart from imaging subcellular features, Raman imaging has been used to follow the uptake of molecules by cells. So far, however, these investigations have been restricted to the incorporation of deuterated building blocks as sensitive and specific markers into bio(macro)molecules. Herein, we investigate the uptake and cellular distribution of the new manganese-based CORM [Mn(tpm)(CO)3]Cl (tpm = tris(1-pyrazolyl)methane), which has photoinduceable cytotoxic activity against cancer cells. Metal–carbonyl complexes such as [Mn(tpm)(CO)3]Cl show strong C O stretching vibrations between 1800 and 2200 cm , a region where vibrational signals from the constituents of the cell are negligible. We show that the C O vibrations of this compound can be used as an ideal marker for imaging these complexes in living cancer cells. Although the spectroscopic signature of metal–carbonyl compounds has already been used in bioanalytical techniques such as the carbonyl–metal immunoassay (CMIA), their use in cellular imaging is so far unprecedented, except for an investigation of osmium–carbonyl clusters in dried cells by using infrared microscopy. The IR and Raman spectra of solid [Mn(tpm)(CO)3]Cl show strong C O stretching vibrations at about 1944 and 2050 cm , as expected for local C3v symmetry (Figures S1 and S2 A in the Supporting Information). The different relative intensities of the two peaks can be explained by the distinct selection rules for Raman and IR spectroscopy. The O H stretching vibration localized at about 3400 cm 1 dominates the spectrum of a 2 mm aqueous solution of [Mn(tpm)[*] K. Meister, Dr. D. A. Schmidt, Prof. Dr. M. Havenith Lehrstuhl f r Physikalische Chemie II, Ruhr-Universit t Bochum Universit tsstrasse 150, 44801 Bochum (Germany) E-mail: martina.havenith@rub.de Homepage: www.rub.de/pc2

Water Determines the Structure and Dynamics of Proteins

Water is an essential participant in the stability, structure, dynamics, and function of proteins and other biomolecules. Thermodynamically, changes in the aqueous environment affect the stability of biomolecules. Structurally, water participates chemically in the catalytic function of proteins and nucleic acids and physically in the collapse of the protein chain during folding through hydrophobic collapse and mediates binding through the hydrogen bond in complex formation. Water is a partner that slaves the dynamics of proteins, and water interaction with proteins affect their dynamics. Here we provide a review of the experimental and computational advances over the past decade in understanding the role of water in the dynamics, structure, and function of proteins. We focus on the combination of X-ray and neutron crystallography, NMR, terahertz spectroscopy, mass spectroscopy, thermodynamics, and computer simulations to reveal how water assist proteins in their function. The recent advances in computer simulations and the enhanced sensitivity of experimental tools promise major advances in the understanding of protein dynamics, and water surely will be a protagonist.

High resolution spectroscopy of carboxylic acid in the gas phase: Observation of proton transfer in (DCOOH)2

We report the first fully analyzed high resolution spectrum of a carboxylic acid dimer in the gas phase. High resolution spectra in the region of the C–O stretch 1241.7–1250.7 cm−1 have been recorded for (DCOOH)2. The data could be fit within experimental uncertainty to a rigid rotor Watson S reduced Hamiltonian. The vibrational frequency of the C–O stretch in (DCOOH)2 is determined to be 1244.8461 (2) cm−1. Our spectra are the first direct experimental evidence for proton transfer tunneling in formic acid dimer (FAD), with FAD serving as a prototype for double hydrogen bonded organic complexes. Previous theoretical studies predicted proton transfer times covering a range of several orders of magnitude. Our measurements on (DCOOH)2 established a proton transfer time of 5.8 ns [tunneling splitting of 0.00286(25) cm−1]. The proton transfer was found to accelerate upon vibrational excitation of the skeleton motion (the vibrational C–O mode), corresponding to a tunneling splitting of 0.00999(21) cm−1. For thi...

Long-range protein-water dynamics in hyperactive insect antifreeze proteins.

Antifreeze proteins (AFPs) are specific proteins that are able to lower the freezing point of aqueous solutions relative to the melting point. Hyperactive AFPs, identified in insects, have an especially high ability to depress the freezing point by far exceeding the abilities of other AFPs. In previous studies, we postulated that the activity of AFPs can be attributed to two distinct molecular mechanisms: (i) short-range direct interaction of the protein surface with the growing ice face and (ii) long-range interaction by protein-induced water dynamics extending up to 20 Å from the protein surface. In the present paper, we combine terahertz spectroscopy and molecular simulations to prove that long-range protein–water interactions make essential contributions to the high antifreeze activity of insect AFPs from the beetle Dendroides canadensis. We also support our hypothesis by studying the effect of the addition of the osmolyte sodium citrate.

Aggregation-Induced Dissociation of HCl(H2O)4 Below 1 K: The Smallest Droplet of Acid

Minimally Acidic Acidity is usually construed in the context of a bulk liquid solvent: billions of trillions of molecules such as HCl, added to hundreds of billions of trillions of water molecules. What happens under sparser conditions, for example, in atmospheric or interstellar environments, when a single HCl molecule might interact with just three or four water molecules? Gutberlet et al. (p. 1545; see the Perspective by Zwier) explored this question using theoretical simulations together with vibrational spectroscopy in ultracold helium droplets that effectively isolated small aqueous HCl clusters. HCl remained intact upon solvation by one, two, or three water molecules. Dissociation into an ion pair, as occurs in bulk water, required the approach of a fourth water molecule and was facilitated by the geometry of the existing (H2O)3 cluster. Just four water molecules are sufficient to dissolve the acid HCl into a charged ion pair of proton and chloride. Acid dissociation and the subsequent solvation of the charged fragments at ultracold temperatures in nanoenvironments, as distinct from ambient bulk water, are relevant to atmospheric and interstellar chemistry but remain poorly understood. Here we report the experimental observation of a nanoscopic aqueous droplet of acid formed within a superfluid helium cluster at 0.37 kelvin. High-resolution mass-selective infrared laser spectroscopy reveals that successive aggregation of the acid HCl with water molecules, HCl(H2O)n, readily results in the formation of hydronium at n = 4. Accompanying ab initio simulations show that undissociated clusters assemble by stepwise water molecule addition in electrostatic steering arrangements up to n = 3. Adding a fourth water molecule to the ringlike undissociated HCl(H2O)3 then spontaneously yields the compact dissociated H3O+(H2O)3Cl− ion pair. This aggregation mechanism bypasses deep local energy minima on the n = 4 potential energy surface and offers a general paradigm for reactivity at ultracold temperatures.