Supplementary document for Dual Phase-Detected Infrared Photothermal Microscopy - 6790933.pdf

Supplementary notes and figure

Terahertz Chiroptical Spectroscopy of an α‑Helical Polypeptide: A Molecular Dynamics Simulation Study

Vibrational spectroscopy has provided incisive information on the structure of biological molecules. Here, using a molecular dynamics simulation method, infrared vibrational circular dichroism and vibrational optical rotatory dispersion spectra of a right-handed α-helix in the terahertz (THz) frequency range are calculated. Both the autocorrelation function of an electric dipole moment and the cross-correlation function of electric and magnetic dipole moments of the α-helix are calculated and Fourier-transformed to obtain THz absorption and optical activity spectra, which reveal characteristic features of the helical polypeptide structure. The anharmonicity and delocalized nature of the low-frequency modes in the THz frequency domain are taken into account to obtain statistically convergent results on the THz optical activity spectra. In addition, the magnitude of the THz vibrational optical activity signal of the α-helix is directly compared with those of typical, previously studied mid- and near-infrared chiral molecules. We anticipate that THz chiroptical spectroscopy that has not yet been demonstrated experimentally would provide highly important and complementary information on protein structure and dynamics

Role of Solvent Water in the Temperature-Induced Self-Assembly of a Triblock Copolymer

Water-soluble triblock copolymers have received much attention in industrial applications and scientific fields. We here show that femtosecond mid-IR pump–probe spectroscopy is useful to study the role of water in the temperature-induced self-assembly of triblock copolymers. Our experimental results suggest two distinct subpopulations of water molecules: those that interact with other water molecules and those involved in the hydration of a triblock copolymer surface. We find that the vibrational dynamics of bulk-like water is not affected by either micellation or gelation of triblock copolymers. The increased population of water interacting with ether oxygen atoms of the copolymer during the unimer to micelle phase transition is important evidence for the entropic role of water in temperature-induced micelle formation at a low copolymer concentration. In contrast, at the critical gelation temperature and beyond, the population of surface-associated water molecules interacting with ether oxygen atoms decreases, which indicates important enthalpic control by water. The present study on the roles of water in the two different phase transitions of triblock copolymers sheds new light on the underlying mechanisms of temperature-induced self-aggregation behaviors of amphiphiles that are ubiquitous in nature

Effect of Osmolytes on the Conformational Behavior of a Macromolecule in a Cytoplasm-like Crowded Environment: A Femtosecond Mid-IR Pump–Probe Spectroscopy Study

Osmolytes found endogenously in almost all living beings play an important role in regulating cell volume under harsh environment. Here, to address the longstanding questions about the underlying mechanism of osmolyte effects, we use femtosecond mid-IR pump–probe spectroscopy with two different IR probes that are the OD stretching mode of HDO and the azido stretching mode of azido-derivatized poly­(ethylene glycol) dimethyl ether (PEGDME). Our experimental results show that protecting osmolytes bind strongly with water molecules and dehydrate polymer surface, which results in promoting intramolecular interactions of the polymer. By contrast, urea behaves like water molecules without significantly disrupting water H-bonding network and favors extended and random-coil segments of the polymer chain by directly participating in solvation of the polymer. Our findings highlight the importance of direct interaction between urea and macromolecule, while protecting osmolytes indirectly affect the macromolecule through enhancing the water–osmolyte interaction in a crowded environment, which is the case that is often encountered in real biological systems

Computational Infrared and Two-Dimensional Infrared Photon Echo Spectroscopy of Both Wild-Type and Double Mutant Myoglobin-CO Proteins

The CO stretching mode of both wild-type and double mutant (T67R/S92D) MbCO (carbonmonoxymyoglobin) proteins is an ideal infrared (IR) probe for studying the local electrostatic environment inside the myoglobin heme pocket. Recently, to elucidate the conformational switching dynamics between two distinguishable states, extensive IR absorption, IR pump–probe, and two-dimensional (2D) IR spectroscopic studies for various mutant MbCO’s have been performed by the Fayer group. They showed that the 2D IR spectroscopy of the double mutant, which has a peroxidase enzyme activity, reveals a rapid chemical exchange between two distinct states, whereas that of the wild-type does not. Despite the fact that a few simulation studies on these systems were already performed and reported, such complicated experimental results have not been fully reproduced nor described in terms of conformational state-to-state transition processes. Here, we first develop a distributed vibrational solvatochromic charge model for describing the CO stretch frequency shift reflecting local electric potential changes. Then, by carrying out molecular dynamic simulations of the two MbCO’s and examining their CO frequency trajectories, it becomes possible to identify a proper reaction coordinate consisting of His64 imidazole ring rotation and its distance to the CO ligand. From the 2D surfaces of the resulting potential of mean forces, the spectroscopically distinguished A₁ and A₃ states of the wild-type as well as two more substates of the double mutant are identified and their vibrational frequencies and distributions are separately examined. Our simulated IR absorption and 2D IR spectra of the two MbCO’s are directly compared with the previous experimental results reported by the Fayer group. The chemical exchange rate constants extracted from the two-state kinetic analyses of the simulated 2D IR spectra are in excellent agreement with the experimental values. On the basis of the quantitative agreement between the simulated spectra and experimental ones, we further examine the conformational differences in the heme pockets of the two proteins and show that the double mutation, T67R/S92D, suppresses the A₁ population, restricts the imidazole ring rotation, and increases hydrogen-bond strength between the imidazole N_ε–H and the oxygen atom of the CO ligand. It is believed that such delicate change of distal His64 imidazole ring dynamics induced by the double mutation may be responsible for its enhanced peroxidase catalytic activity as compared to the wild-type myoglobin

Azido Homoalanine is a Useful Infrared Probe for Monitoring Local Electrostatistics and Side-Chain 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 to be 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

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

Water Dynamics in Cytoplasm-Like Crowded Environment Correlates with the Conformational Transition of the Macromolecular Crowder

Polyethylene glycol (PEG) is a unique polymer material with enormous applicability in many industrial and scientific fields. Here, its use as macromolecular crowder to mimic the cellular environment <i>in vitro</i> is the focus of the present study. We show that femtosecond mid-IR pump–probe spectroscopy using three different IR probes, HDO, HN₃, and azido-derivatized crowder, provides complete and stereoscopic information on water structure and dynamics in the cytoplasm-like macromolecular crowding environment. Our experimental results suggest two distinct subpopulations of water molecules: those that interact with other water molecules and those that are part of a hydration shell of crowder on its surface. Interestingly, water dynamics even in highly crowded environment remains bulk-like in spite of significant perturbation to the tetrahedral H-bonding network of water molecules. That is possible because of the formation of water aggregates (pools) even in water-deficient PEGDME-water solutions. In such a crowded environment, the conformationally accessible phase space of the macromolecular crowder is reduced, similar to biopolymers in highly crowded cytoplasm. Nonetheless, the hydration water on the surface of crowders slows down considerably with increased crowding. Most importantly, we do not observe any coalescing of surface hydration water (of the crowder) with bulk-like water to generate collective hydration dynamics at any crowder concentration, contrary to recent reports. We anticipate that the present triple-IR-probe approach is of exceptional use in studying how conformational states of crowders correlate with structural and dynamical changes of water, which is critical in understanding their key roles in biological and industrial applications

Studying Water Hydrogen-Bonding Network near the Lipid Multibilayer with Multiple IR Probes

A critical difference between living and nonliving is the existence of cell membranes, and hydration of membrane surface is a prerequisite for structural stability and various functions such as absorption/desorption of drugs, proteins, and ions. Therefore, a molecular level understanding of water structure and dynamics near the membrane is important to perceive the role of water in such a biologically relevant environment. In our recent paper [J. Phys. Chem. Lett. 2016, 7, 741] on the IR pump–probe study of the OD stretch mode of HDO near lipid multibilayers, we have observed two different vibrational lifetime components of OD stretch mode in the phospholipid multibilayer systems. The faster component (0.6 ps) is associated with OD groups interacting with the phosphate moiety of the lipid, while the slower component (1.9 ps) is due to choline-associated water molecules that are close to bulklike water. Here, we additionally use hydrazoic acid (HN₃) as another IR probe of which frequency is highly sensitive to its local H-bonding water density. Interestingly, we found that the vibrational lifetime of the asymmetric azido stretch mode of HN₃ in the lipid multibilayer system is similar to that in neat water, whereas its orientational relaxation is a bit slower than that in bulk water. This indicates that due to the tight packing of lipid molecules, particularly the head parts, in the gel phase, HN₃ molecules mostly stay near the choline group of lipid and interact with water molecules in the vicinity of choline groups. This suggests that membrane surface-adsorbed molecules such as hydrophilic drug molecules may interact with choline-associated water molecules, when the membrane is in the gel phase, instead of phosphate-associated water molecules