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Master's Thesis
Study of hydrogen-bonding energetics and dynamics of biological water using ultrafast electronic
spectroscopy
Young Jae Kim
Department of Chemistry
Graduate School of UNIST
2017
Study of hydrogen-bonding energetics and dynamics of biological water using ultrafast
electronic spectroscopy
Young Jae Kim
Department of Chemistry
Graduate School of UNIST
Study of hydrogen-bonding energetics and dynamics of biological water using ultrafast
electronic spectroscopy
A thesis/dissertation
submitted to the Graduate School of UNIST in partial fulfillment of the
requirements for the degree of Master of Science
Young Jae Kim
6. 9. 2017 Approved by
_________________________
Advisor
Oh-Hoon Kwon
Study of hydrogen-bonding energetics and dynamics of biological water using ultrafast
electronic spectroscopy
Gil-Dong Hong
This certifies that the thesis/dissertation of Young jae Kim is approved.
6/9/2017
signature
___________________________
Advisor: Oh-Hoon Kwon signature
___________________________
typed name: Yung-Sam Kim signature
___________________________
typed name: Bum Suk Zhao signature
___________________________
Abstract
There are growing lines of evidence indicating the importance of water dynamics in the function of biological architectures. Yet, molecular mechanisms describing how water exerts these biological activities are still missing. In this paper, we seek to address this issue by characterizing the transient properties of biomolecule-associated water, which is well known as biological water, on engineered model proteins with two analogous fluorescent probes, tryptophan (W) and 7- azatryptophan (AW), in real time with a femtosecond time resolution. In particular, solvent catalyzed excited-state proton tranfer (ESPT) of AW have been studied a lot, while W lacks the photochemical property of ESPT. By comparing two system, with only one system interacts vivaciously with water, the hydrogen-bonding characteristics of biological water can be analyzed. Especially, we repots a new methodology that monitors the energetics of hydrogen bonds among biological water molecules.
Contents
I. Introduction --- 4
II. Method and Materials --- 5
1.1 Sample preparation--- 5
1.2 Steady-state/time-resolved fluorescence measurements --- 5
III. Results and Discussion --- 7
3.1 Model system --- 7
3.2 Structural integrity of the designed systems --- 7
3.3 Ultrafast hydration dynamics --- 8
3.4 Energetics of hydrogen-bonding of biological water --- 9
IV. Conclusion --- 11
V. Figures, Schemes and Tables --- 12
VI. References --- 17
VII. Acknowledgements --- 20
List of Figures & Tables
Figure 1. Steady-state circular dichroism (CD) (a), UV-visible absorption (b), and fluorescence emission (c) spectrum. All the samples were prepared at 0.2 mM concentration and excitation wavelength was 285 nm.
Figure 2. The time-resolved anisotropy decay (r(t)) and emission-maximum functions of W (a and e), A1m (b and f), AW (c and g), and A1m-AW (d and h). Especially for protein samples, triple exponential function was used as fitting function for both r(t) and emission-maximum functions.
Figure 3. Representative normalized femtosecond-resolved upconversion transients of W, A1m, AW, and A1m-AW in left and center columns. All the samples were excited at 285 nm. These upconversion transients were transposed to wavenumber vs intensity in order to show the femtosecond-resolved fluorescence emission maximum shifts. Normalized femtosecond-resolved fluorescence spectrum with selected time delays of W, A1m, AW, and A1m-AW in right column. These spectrum were fitted to log-normal function.
Figure 4. The mechanism of the solvent-catalyzed tautomerization of AW in bulk water (a) and biological water (b). The water molecule need to lose one hydrogen bond with either bulk water (a) or biological water (b) so as to form cyclic configurations, which can undergo solvent mediated tautomerization. Illustration of the relationship between bulk water and biological water. A water molecule keeps exchanging from bound to free or free to bound with having energy difference of ΔΔG.
Table 1. Photophysical parameters of AW and A1m-AW
Introduction
Water molecules, which are very tiny, have crucial role in biological systems in microscopic view. Almost all of biological macromolecules can have their activity/inactivity controls and sustain their structural stability only with the presence of water molecules in their vicinities [1-6]. For examples, water molecules mediate the maintenance and participate in all over the central dogma, from replication to translation of DNA [7-9]. Furthermore, water molecules participate in the protein- substrate or protein-protein interactions [10-12]. Therefore, It is important to characterize the dynamics and energetics of water molecules in molecular level.
The behavior of water molecules becomes unique when they exist in the environs of a biological macromolecule compared to that of water molecules in bulk [6,13]. Those water molecules are called biological water. At the biological surface, the hydrogen bond of a water molecule among them can be either replaced by that to a hydrophilic protein surface or limited depending on the topology, hydrophobicity, and/or the charged state of the protein surface. This intimates that the biological water molecules are energetically different from water molecules in bulk water, which is well known through many previous researches. Lots of scientific works have been done on the hydration dynamics of biological water in biological systems for both experimental and theoretical ways [14-19]. Through these works, many scientists revealed the substructures of proteins and their dynamics, which will give clues for protein-substrate or protein-protein interactions. On the other hand, the energetics of hydrogen bonds of biological water has been overlooked, which is essential for understanding the hydration dynamics on biological surface.
In this paper, hydrogen bonds of biological water through solvation dynamics on protein surface are analyzed. The fluorescent, non-canonical amino acid, 7-azatryptophan (AW) was used in this study as a hydrogen (H)-bonding probe. The chromophore of AW, 7-azaindole (AI), is a well- known molecule which undergoes excited-state proton transfer (ESPT) catalyzed by a water molecule, for which AI competes with other water molecules to form hydrogen bonds [20-23]. This fluorescent probe is inserted to chosen coiled-coil protein which have highly exposed to water environment site.
For the control experiment, tryptophan (W) was adopted because it has similar morphology with substitution of one carbon to nitrogen in chromophoric ring but does not show the photochemical property of ESPT. It is also inserted to identical site in identical coiled-coil protein with AW case.
From the measurements of the femtosecond (fs)-resolved fluorescence transients of the two proteins with the fluorescent amino acids installed, The difference in ultrafast hydration dynamics of the two analogous systems, but with only one undergoing ESPT with forming double hydrogen bonds with a biological water molecule, was compared. The difference of the hydration dynamics and the change of the ESPT rate on the protein surface are analyzed to give the dynamics and energetics information on biological water.
Methods and Materials
1. Sample preparation
DL-Tryptophan was purchased and used as received (≥99%, Sigma-Aldrich, T3300). DL-7- Azatryptophan was also purchased and used as received (98%, TCI, A0557). Phosphate buffer saline (PBS) solution was prepared by adding 10 mM KH2PO4 and 150 mM NaCl into pure water. This solution then titrated to pH 7.4 at room temperature. All the fluorescence probes and proteins are dissolved in this PBS solution. Both DL-tryptophan and DL-7-azatryptophan was dissolved in PBS solution with the concentration of 0.2 mM.
In order to get the modified coiled-coil protein, a fragment of EcoRI/HindIII from pQEA1, which includes the coding sequence of A1. Then, it was ligated into EcoRI/ HindIII-digested pQE- 80L (Qiagen) to produce pQE-80L/A1. The 34th position, aspartic acid (D) residue, was substituted with W or AW by site-directed mutagenesis to synthesize A1m-W or A1m-AW. Finally, the outcome plasmids were appointed as pQE-80L/A1m-W or pQE-80L/A1m-AW, respectively.
2. Steady-state/time-resolved fluorescence measurements
For the UV absorption spectra, UV/vis spectrophotometer (V-730, JASCO) was used.
Fluorescence spectra were obtained with a fluorescence spectrophotometer (QM-400, Photon Technology International). Circular Dichroism (CD) spectrum for protein samples were taken from spectropolarimeter (J-1100, JASCO). Femtosecond-resolved fluorescence transients were gained by applying the fluorescence-upconversion (FU) technique described below. An amplified Ytterbium- based laser system (Pharos, Light Conversion), which produces light of 1030 nm center wavelength with pulse width of ~170 fs at the repetition rate of 200 kHz with an output power of 6000 mW.
Through the pulse picker divider, the repetition rate was reduced down to 50 kHz without pulse energy change. Then the output beam was divided into two parts in order to generate pump beam and gate beam, respectively. For the pump-beam, the fundamental beam is directed into an optical parametric amplifier (OPA) (Orpheus, Light Conversion). After passing through the OPA, resulting idler beam was sum-frequency generated with the residual fundamental beam at β-barium borate (BBO) crystal (type II) in order to make the intermediate beam of 570 nm. This intermediate beam passed through the other BBO crystal (type II) to make pump beam at 285 nm. The 285 nm pump beam got into a fluorescence spectrometer (Chimera, Light Conversion) which is equipped with a monochromator (MSA-130, Sloar Laser System) and a photomultiplier tube (PMC-100, Becker &
Hickl). All the sample were contained in the 2 mm pass-length UV quartz cuvettes and were being stirred while the pump-beam were excitation beam illuminating. The resulting fluorescence was separated from pump beam with using long-pass filter, which only absorbs the pump beam. After the filtering, fluorescence lights were focused on a BBO crystal (type II). The gate beam went through a computer-controlled optical delay line was overlapped with the fluorescence lights on the BBO crystal
so as to make upconverted signal. The UG5 filter was equipped after the BBO crystal for separation of upconverted signal from pump beam and fluorescence. Further mechanical separation of upconverted signal was achieved by the array of some iris apertures and a prism. In order to eliminate the influence of anisotropy, the pump beam polarization was set at the magic angle (54.7°) with regard to the upconversion crystal axis. Picosecond (ps)-resolved fluorescence lifetime measurements, time- correlated single-photon counting (TCSPC) in the Chimera was perfomed through a TCSPC module (SPC-130, Becker & Hickl). For time-dependent fluorescence anisotropy, 𝐼(𝑡) = (𝐼∥− 𝐼⊥)/(𝐼∥+ 2𝐼⊥), the pump beam was changed to parallel or perpendicular polarization with respect to the angle of upconversing BBO crystal. Then the fluorescence spectrum were gained as the same way with femtosecond transients with excitation wavelength of 285 nm.
Results and Discussion
1. Model System
Commercially available 7AW has similar molecular structure and size as W does, but exhibits strong H-bonding chemical interactions with water. The chromophoric moiety of AW has been reported to undergo a reversible photochemical reaction with water molecules and the rates for the reaction depend on a hydration environment [20-23]. Thus AW is adapted for probing the energetic and the dynamics of water after installation on the surface of engineered proteins.
The choice of the engineered proteins is a coiled-coil protein, which is named as A1, based on its structural integrity in terms of exposure of the hydration probes [16,24-25]. The secondary structure of A1 protein is α-helix with six copies of seven amino acid repeating, which can be described as (abcdefg)n. In here, each alphabet represent different amino acids. Especially, A1 has two hydrophobic amino acid residues, a and d positions, and together, they makes hydrophobic site in α- helix. In solution, the hydrophobic site of two A1 molecules are attracted and forms A1m dimers, with having hydrophobic core. On the other hand, A1 does not have W nor AW. Thus, single highly water- exposed site, which is the opposite site of a and d, of A1 is modified by installing either W or AW.
Particularly, the substituted residues were at the center of the amino acid sequences. One system has substitution with W, which is called A1m-W, and the other system has substitution with AW, which is called A1m-AW.
2. Structural integrity of the designed systems
Fig. 2(a) shows the circular dichroism (CD) spectrum of the two engineered coiled-coil proteins with the wavelength range covers from 200 nm to 250 nm. These spectrum were analyzed with K2D3 software, which showed the α-helicity of A1m and A1m-AW as 52% and 57%, respectively [26]. Compared to the previous α-helicity of A1 and their modifications [16,27], these values suggest that the structural change after substitution was minimized. Indeed, observed high α- helicities indicate both A1m and A1m-AW have more than half of their amino acids in their α-helix secondary structure regions. The probes are located in the center of protein sequences, which means they are located in the center of rigid α-helix directly toward the exposure to water. Therefore it can be said that the probes are successfully inserted in the rigid and hydrophilic site of target systems.
In Fig. 2(b), absorption spectra of W and A1m show their absorption maxima around 280 nm while absorption maxima of AW and A1m-AW locate around 290 nm. For both the fluorescent probes, A and AW, there is negligible difference between the spectra of each probe and the corresponding coiled-coil protein system. This shows that the environments of probe in protein systems are similar with that of probes in bulk water. Therefore, this information from UV spectrum further demonstrates that the fluorescent probes are located in the hydrophilic region of target system and at the same time, they are highly exposed to water environments.
The time-resolved anisotropic decay profiles were measured and the results showed three components with hundreds of fs, tens of ps, and several nanoseconds (ns) decays. From these profiles, wobbling semiangles (θ) were obtained with using the equation previously reported [15,28]. The wobbling semiangles of A1m and A1m-AW were 22% ± 2% and 22% ± 3%, each. Small differences between the wobbling semiangles leads to the conclusion that the fluorescent probes are in the similar structural environment. Finally, information from UV, CD, and anisotropic decay spectrum proved that there was negligible difference in their environmental factor, which can differentiate unique ESPT from observed hydration and photophysical dynamics. This will lead us to the calculation of H- bonding energetics on protein surfaces. The detailed anisotropic dynamics will be covered in the later part of this article.
3. Ultrafast hydration dynamics
In Figure 3, emission maximum of both W and A1m locate near 350 nm and their shapes are similar. On the other side, the emission maximum of AW and A1m-AW show significant difference, 400 nm for AW and 380 nm for A1m-AW. For the case of AW, AW interacts with water molecules more intensely than W because of two H-bonding sites in AW, resulting in ESPT. Unlike probe only system in bulk water, water molecules near A1m-AW interact with a protein surface, forming H-bonds.
Consequently, AW, which was inserted in protein, interacts with water molecule which have H-bond with protein and this makes the differences in the emission profiles. Especially, the major decay component, ESPT, which is the result of interaction with AW and water, usually occurs in time range of ~800 ps while the major decay component of W is about 3 ns or longer [23,29]. Therefore, the solvation dynamic dramatically affects on the A1m-AW, resulting in the noticeable change in emission spectra.
Fig.3 shows several representative fs-resolved fluorescence upconversion transients. These upconversion transients were transposed to wavenumber versus intensity plot with regards to the time elapses. This spectrum was fitted by log-normal function, f(X) = 𝐼 × exp(−ln2 × (ln(1 + 2𝑏 × (X − νmax)/𝑊)/𝑏)^2 ). Then the solvation correlation functions (c(t)) were constructed, which is defined as 𝑐(𝑡) = [𝜈max(𝑡) −νmax(∞)]/[𝜈max(0) − 𝜈max(∞)] [30]. Based on the solvation correlation functions, which estimates the Stoke shifts of probe with time, triple exponential function for both amino acids and protein systems are constructed from these transposed data. For protein systems, which have probes exposed to water, three time scales of τ1, τ2, and τ3 were obtained to be ~500 fs, 5 ps, and ~160 ps, respectively. The ultrafast solvation component is the result of interaction with inner- layer water molecules in hydration shell. The second components with several ps range comes from the interaction with outer-layer water molecules in hydration shell. The slowest components with tens to a few hundred of ps are the outcomes of water-protein rearrangements. These characterizations of
The time-resolved anisotropy with triple exponential decay function was measured so as to detect the protein side-chain relaxation. The initial ultrafast component (faster than 1 ps) is internal conversion of two excited electronic state. The second decay component with ~50 ps was come from the local wobbling relaxations. The slow component with several ns was originated from protein tumbling motions [3,37-38]. If these time components are compared with components come from solvation, which describes the motion of water molecules, local wobbling relaxations follows the fast two solvation components. Therefore, once the inner-layer and outer-layer water molecules are rearranged, then the protein side-chain starts rearranging in order to stabilize system [15,39-41].
4. Energetics of hydrogen-bonding of biological water
By comparing the fluorescence lifetime of AW and A1m-AW, the H-bonding energy of biological water can be evaluated. AW undergoes ESPT via forming a cyclically H-bonded complex with water from non-cyclically H-bonded configuration [22-23]. In order to form a cyclically H- bonded AW-H2O complex in bulk water, the water molecule should lose one hydrogen bond with other water molecule and need to have hydrogen bond with AW. The rate of ESPT then can be written as follow equation :
kobs,bulk = kPT exp[-ΔGbulk‡
/kBT]
…Equation 1 In Eq.[1], kobs,bulk is an observed reaction rate in bulk water; kPT is an intrinsic rate for proton movements; -ΔGbulk‡ is the free energy change for the probe molecule to take a water molecule from bulk; kB is the Boltzmann constant; T is the temperature in Kelvin scale.
In the case of AW-H2O complex in biological water, the water molecule should lose one hydrogen bond with biological water and gain hydrogen bond with AW. As the same way the rate of ESPT can be described as below :
kobs,bio = kPT exp[-ΔGbio‡
/kBT]
…Equation 2 where kobs,bio is an observed reaction rate on a protein surface and -ΔGbio‡
is the free energy change for the probe molecule to take a water molecule form biological water. Together, the observed photochemical kinetic parameters on the surface of protein will follow the equation:
kobs,bio/kobs,bulk = exp[-ΔΔG/kBT]
...Equation 3 In here, -ΔΔG is the difference of the H-bonding energy of bulk water and biological water. Therefore, by comparing the reaction rate of ESPT of AW and A1m-AW, the H-bonding energy of biological water can be calculated.
In order to get the ESPT reaction rate of AW and A1m-AW, ns-resolved transient through TCSPC technique. We could not observe the fast rise/decay components which depend on the
detection wavelength. This is because of the time-resolution limit of our detector. The spectrum were therefore fitted by single exponential function for AW and double exponential function for A1m-AW.
Table 1 shows the abridgment of these results. Then kobs,bio and kobs,bulk were compared based on the Eq.3 to get the energy difference, -ΔΔG. The calculated -ΔΔG was 0.1 kcal/mol. Because the H- bonding energy of water in bulk is 4.9 kcal/mol [42], the H-bonding energy of biological water is 4.8 kcal/mol.
The weaker H-bonding strength among biological water makes easier to exchange their hydrogen bonds with one another. This increases the mobility of biological water. Hence the AW confined in A1m-AW can receive hydrogen bond from biological water to form cyclic configuration easily than the case of AW in bulk water, resulting in faster ESPT rate. Also, we suggest that the small energy difference of biological water is due to the extremely hydrophilic environment, which is accomplished by both inserting the probes into residue highly exposed to water and fairly hydrophilic neighboring residues of the probe like charged (Arg and Glu) or polar (Ser). This is the case for A1 protein, which have probe located in greatly hydrophilic surface domain. If this series of experiments were done at the different type of domain or protein, the energetics and dynamics of biological water is not alike with our case.
Conclusion
In summary, we report the dynamics and energetics on H-bonding of biological water through ultrafast spectroscopy. In addition, we suggest new methodology for calculating H-bonding energetics of biological water using AW and its analogous probe, W. These two probes are inserted into coiled-coil protein, A1, so as to study the dynamics and energetics of biological water. The selected residue is hydrophilic site of the α-helix and this position is highly exposed to water when it forms dimeric coiled-coil protein in solution. For protein samples, constructed solvation correlation functions were fitted by triple exponential function. Each components represents inner-layer water relaxation, outer-layer water motion, and coupled water-protein recombination, respectively. Also, we received the reaction rate of ESPT from ns-resolved transient of AW and A1m-AW. Through comparison of the reaction rates, as described in equation 3, the difference of energy between H- bonding of water in bulk and H-bonding of biological water on the surface of A1m-AW. Finally, the calculated energy difference was 0.1 kcal/mol. This much difference is only for A1m-AW protein, especially restricted in hydrophilic and rigid site which is highly exposed to water. Of course the energy difference will be different when the observing site or protein is changed. Therefore, in this way, we expect to calculate H-bonding energies other types of biological water that locate in different environment such as hydrophobic residue or water channel, which will give valuable knowledge to understand the operation of water molecule to structures and activities of biomacromolecules.
Figures and Tables
Figure 1. Steady-state circular dichroism (CD) (a), UV-visible absorption (b), and fluorescence emission (c) spectrum. All the samples were prepared at 0.2 mM concentration and excitation wavelength was 285 nm.
Figure 2. The time-resolved anisotropy decay (r(t)) and emission-maximum functions of W (a and e), A1m (b and f), AW (c and g), and A1m-AW (d and h). Especially for protein samples, triple exponential function was used as fitting function for both r(t) and emission-maximum functions.
Figure 3. Representative normalized femtosecond-resolved upconversion transients of W, A1m, AW, and A1m-AW in left and center columns. All the samples were excited at 285 nm. These upconversion transients were transposed to wavenumber vs intensity in order to show the femtosecond-resolved fluorescence emission maximum shifts. Normalized femtosecond-resolved fluorescence spectrum with selected time delays of W, A1m, AW, and A1m-AW in right column. These spectrum were fitted to log-normal function.
Figure 4. The mechanism of the solvent-catalyzed tautomerization of AW in bulk water (a) and biological water (b). The water molecule need to lose one hydrogen bond with either bulk water (a) or biological water (b) so as to form cyclic configurations, which can undergo solvent mediated tautomerization. Illustration of the relationship between bulk water and biological water. A water molecule keeps exchanging from bound to free or free to bound with having energy difference of ΔΔG.
Table 1. Photophysical parameters of AW and A1m-AW Sample λem τ1 (ns) τ2 (ns)
AW 330 0.71±0.03 (100%) N.A.
340 350 360 370 380 400 420 440 460 480
Sample λem τ1 (ns) τ2 (ns) A1m-AW 330 0.63±0.02(60%) 4.45±0.79(40%)
340 0.63±0.02(61%) 4.45±0.79(39%) 350 0.63±0.02(66%) 4.45±0.79(34%) 360 0.63±0.02(60%) 4.45±0.79(40%) 370 0.63±0.02(76%) 4.45±0.79(34%) 380 0.63±0.02(87%) 4.45±0.79(13%) 400 0.63±0.02(92%) 4.45±0.79(8%) 420 0.63±0.02(96%) 4.45±0.79(4%) 440 0.63±0.02(97%) 4.45±0.79(3%) 460 0.63±0.02(98%) 4.45±0.79(2%) 480 0.63±0.02(97%) 4.45±0.79(3%)
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Acknowledgements
I thank to Bung-Sung Lee and prof. Tae-Hyun Yoo at Ajou university for synthesizing the protein samples. Also, I appreciates Kyung Min Lee, Su Hwan Kim, and prof. Sang Kyu Kwak for their help in receiving and analyzing computational data. This research was funded by 2013 Research Fund of UNIST, NRF Korea funded by the Ministry of Science, ICT and Future Planning (MSIP), and the Institute for Basic Science (IBS-R020-D1) Korea. I specially appreciate prof. Oh-Hoon Kwon for his kind advising during my M.S. degree. I am also grateful to my colleagues, the members of ULSaN laboratory.
