Water and biological molecules probed by Terahertz spectroscopy

Water is a major constituent of living cells but cellular constituents such as proteins are so tightly packed that the space available for water is limited. To investigate water at the interface  and its interactions with biomolecules, Martina Havenith, with colleagues Martin Gruebele and David Leitner, applied to HFSP to fund a project to use the new method of Terahertz spectroscopy as a probe for water-biomolecule interaction. This Germany-USA collaboration was considered very high-risk but proved a success and has resulted in exciting findings about the nature of water surrounding proteins.

Martina Havenith is Chair of Physical Chemistry at the Ruhr-University, Bochum, Germany. With a background in physics, she has made major contributions to the development of new laser techniques for approaching problems at the interface between physics and biophysical chemistry. She is a member of the German Academy of Sciences, Leopoldina,  and has recently been appointed to the Austrian Science Council.

What are the properties of water in a living cell?  With a cytoplasmic packing density of up to 400 mg/ml of protein, nucleic acids, lipids, carbohydrates and small molecules or ionic compounds, there is little distance from any one molecule to its nearest neighbors - only about 20-30 Å, depending on the molecular size.  The roughly 10 layers of water molecules that can  fit into these spaces have entirely different properties from water in “bulk” due to its interactions with cellular components.  Water as we know it, that hydrogen-bonded bulk liquid melting at 0 °C and boiling at 100 °C, may not exist within cells.  Recently, a new technique has provided a means to observe water dynamics. i.e. the fast collective motion of water molecules, around biological molecules.  Terahertz light, at frequencies between microwaves and the infrared (1012 Hertz = 1 Terahertz), can excite collective motions of solvent molecules and of biomolecules whose time scales are on the order of a picosecond.  This corresponds to the important time scales for hydrogen bond rearrangement in water and collective, functionally important motions of large biomolecules such as proteins and nucleic acids.  In fact, the motions of the protein or nucleic acid and the hydrogen bond rearrangement of water are coupled.  Modern terahertz instrumentation, building on decades of progress in far infrared spectroscopy of solid samples and films, is now powerful enough to penetrate water layers and look at fully solvated proteins, carbohydrates, lipids, and nucleic acids. 

With THz technology and computer simulations now at a stage where spectra of solvated biomolecules could be obtained and interpreted, our groups sought support from the Human Frontier Science Program to initiate a series of studies on biomolecule-solvent interactions and dynamics.  We hoped that THz technology would shed light on motions on that critical time scale of hydrogen bond breaking and formation coupled to functionally important dynamics of biomolecules that cannot be accessed by other methods, such as nuclear magnetic resonance (NMR).  Since THz technology was new, there was considerable risk that the method would not be sensitive enough to provide the information we were seeking – others told us that it would be impossible or even hopeless. We were extremely thrilled that HFSP supported our high-risk endeavor in 2004.  With HFSP funding we have been able to establish THz spectroscopy as a now blossoming and widely used tool in the study of biomolecule-solvent dynamics and interactions.

At first glance, there appeared to be little information contained in the THz spectrum of a biomolecule.  The frequency dependence of the absorbance is largely featureless in the THz regime.  However, with the help of the HFSP support we soon discovered that measurements of the THz absorbance as a function of biomolecule concentration in solution provides clues about the extent of the hydration layer, i.e., the number of water molecules around the biomolecule that are dynamically distinct from bulk water, as well as giving information on the dynamic coupling between the biomolecule and water.  Two kinds of water, bulk water and hydration water, each with distinct absorption coefficients, were identified to account for the variation of the absorbance with biomolecule concentration.   By studying the change in absorbance with concentration we could deduce the size of the hydration layer.  In combination with molecular dynamics simulations modeling the same system, we explored at the molecular level the biomolecule-solvent dynamics underlying the THz spectra.

As our work developed, several novel key concepts in the Terahertz spectroscopy of biomolecules in water emerged, with major implications for our investigation of the interaction between water and biological macromolecules.  (a) “Terahertz defect”:  Biomolecules absorb less THz light than water over part of the frequency range, and when biomolecules are dissolved in water, the absorption coefficient of the solution often decreases at certain frequencies  (e.g. 2.5 THz for proteins in water).  (b) Another key concept is the “Terahertz excess”. Despite the fact that pure biomolecule solids or films generally absorb less than bulk water between 1 - 3 THz, there are still many situations where the biomolecule+water mixture absorbs more than either the biomolecule or a bulk water sample. This can be explained only by invoking a third substance: biological or hydration water.  If the presence of biomolecules perturbs nearby water molecules, this could have an effect on many of the measurable properties of water: density, relaxation rates, reorientation rates.

Water has a built-in probe of its orientation: its dipole moment, with a negative charge at the oxygen end and positive charge at the hydrogen end of the molecule.  The dynamical reorientation of the water dipole moment turns out to be affected over particularly long distances, up to several nanometers from the surface of a biomolecule.  This reorientation arises as water molecules within the hydrogen bonding network tumble around and diffuse, constantly making and breaking hydrogen bonds.  Couple that with the radius-squared increase of the number of water molecules as one moves outward to more remote solvation shells, huge numbers of water molecules can be affected, each a little bit, by a single biomolecule.


Dynamical hydration layer around a protein.  The five helix bundle protein known as  λ*6-85  is shown surrounded by 1000 water molecules in the dynamical hydration shell. All of them are shown to be affected by a single protein in their picosecond hydrogen bond dynamics

A simple picture of a biomolecule in water thus has to include the protein, nucleic acid or carbohydrate (causing a Terahertz defect), bulk water (if far enough away), and, in between, hydration water with new physical properties, including a propensity for enhanced Terahertz absorption – the Terahertz excess.  The hydration water defined in this way is not the same as the sterically bound water molecules probed by X-ray crystallography, NMR or neutron crystallography.  We have therefore detected a dynamical hydration shell, which includes all water molecules that show water network dynamics distinct from the bulk, and thus a distinct THz absorbance.  The influence of the biomolecule can reach much further than the static hydration radius since it involves only a change in the motions and not a fixed H-bond to the protein.

Our work has shown that this simple picture works quantitatively in some cases with a homogeneous surface, for instance water molecules surrounding small sugar molecules, but breaks down in other cases, for instance hydration water around crowded proteins. In the latter case, the effect of proteins on the surrounding water shell reaches out so far that water molecules begin to “see”more than one protein. In addition, the influence of the protein will now be governed by several key parameters such as hydrophobicity and hydrophilicity of the side chains and steric hindrance. In addition, many-body interactions become important, so the influence of the hydration bond dynamics of water in the hydration shell becomes more complex. Molecular dynamics simulations of the solvation water around biomolecules shows a retardation of the H-bond dynamics for hydrophilic as well as hydrophobic protein surface areas. Whereas the first is explained by H-bond with the proteins, the retarded hydrogen bond dynamics around hydrophobic residues is at first glance surprising and can be explained by the additional imposed steric constraints on the water molecules at hydrophobic sites.

A detailed analysis using ab initio molecular dynamics simulations revealed a fundamental mechanistic difference between correlated molecular dipole oscillations at infrared and THz frequencies. While at infrared frequencies beyond 1000 cm-1 (30 THz) the molecular dipoles of neighbouring water molecules are correlated purely due to electronic polarization effects, at THz frequencies the nuclear motion of neighbouring water molecules are responsible for the observed correlated oscillation of molecular dipoles. While the vibrational motion of atoms is strictly localized on single molecules at infrared frequencies, at frequencies below 1000 cm-1, the onset of truly correlated, and thus collective nuclear motion of neighbouring water molecules is observed. In particular at THz frequencies, below 200 cm-1 (6 THz), the collective vibrational motion included water molecules significantly beyond next neighbours.

What is the role of the dynamical hydration shell? In order to address this, kinetic studies are necessary on the interactions between protein and water detected by THz absorption. As a first proof of principle experiment we have now visualized the changes in THz absorption during the protein folding process and recorded changes in the THz absorption with millisecond time resolution. This new method has been called kinetic terahertz absorption (KITA) spectroscopy.



              Click on image for details of the KITA setup



KITA records the time-course of attenuation of the THz pulse by the sample - which is related to the THz absorption - after initiation of a biological process such as protein folding. To initiate protein folding we have mixed an unfolded (“denatured”) protein with a denaturant-free buffer and monitored the changes in THz absorption, which visualizes protein-water interaction. In order to compare the time constant of the protein-water interactions with other relevant kinetic processes we have recorded in addition the fluorescence and circular dichroism (CD) spectra and small-angle X-ray scattering (SAXS). For the fluorescence study a pseudo-wildtype mutant of the protein (ubiquitin) was utilized, carrying a point mutation (F45W) that introduces tryptophan as a natural fluorophor  to measure the folding dynamics of the protein. The experiment indicates a sudden barrier-free reformation in the surrounding solvation shell, almost a hundred times faster than the protein folding as measured by fluorescence and the secondary and tertiary structure formation as probed by CD spectroscopy. This clearly shows that the solvent-protein rearrangement and the secondary and tertiary structure formation are two consecutive processes, which are both involved in the overall dynamics of protein folding.

Further comparison of the KITA signals to the protein folding kinetics measured by SAXS and CD sheds more light on the processes occurring at short and long timescales: The SAXS measurements indicate an initial collapse of the protein’s radius of gyration on the millisecond timescale, while CD indicates excessive formation of helical secondary structure elements. The processes, taking place on a timescale of 10 milliseconds similar to the hydration dynamics, go hand in hand with the formation of dynamical hydration shells as observed in the THz response. The slow process occurring on timescales of hundreds of milliseconds to seconds, as observed in the fluorescence signal, is connected with the formation of the final tertiary structure, during which the inner core of the protein becomes even more densely packed, thus changing the local environment of the fluorescent tryptophan probe. Thus kinetic THz absorption spectroscopy has opened a new window on the mechanism of protein folding. The observation of collective water network motions during a biological function is providing complementary information compared to other techniques and will shed a new light on the interplay between water and proteins in living systems.

Thanks to HFSP support of our ideas, a risky decision since few to no preliminary data were available, we were able to adapt THz technology and computational simulations to the study of biomolecule-solvent dynamics.  We now know that Terahertz absorption is sensitive to subtle changes in the dynamical orientation of water molecules and that it gives information at distances beyond current NMR techniques. In addition, mutagenesis can be used to ask questions about the influence of protein structure on water dynamics. The use of Terahertz light has thus led to a breakthrough in our understanding of picosecond protein-water dynamics at a timescale that is found to be critical in initiating protein folding.


Meeting of the HFSP THz team at a meeting in Bochum in 2006.