Digital Resilience Biomarkers for Personalized Health Maintenance and Disease Prevention.

Health maintenance and disease prevention strategies become increasingly prioritized with increasing health and economic burden of chronic, lifestyle-related diseases. A key element in these strategies is the empowerment of individuals to control their health. Self-measurement plays an essential role in achieving such empowerment. Digital measurements have the advantage of being measured non-invasively, passively, continuously, and in a real-world context. An important question is whether such measurement can sensitively measure subtle disbalances in the progression toward disease, as well as the subtle effects of, for example, nutritional improvement. The concept of resilience biomarkers, defined as the dynamic evaluation of the biological response to an external challenge, has been identified as a viable strategy to measure these subtle effects. In this review, we explore the potential of integrating this concept with digital physiological measurements to come to digital resilience biomarkers. Additionally, we discuss the potential of wearable, non-invasive, and continuous measurement of molecular biomarkers. These types of innovative measurements may, in the future, also serve as a digital resilience biomarker to provide even more insight into the personal biological dynamics of an individual. Altogether, digital resilience biomarkers are envisioned to allow for the measurement of subtle effects of health maintenance and disease prevention strategies in a real-world context and thereby give personalized feedback to improve health.

Hydrophobic Collapse in N-Methylacetamide-Water Mixtures.

Aqueous N-methylacetamide solutions were investigated by polarization-resolved pump-probe and 2D infrared spectroscopy (2D IR), using the amide I mode as a reporter. The 2D IR results are compared with molecular dynamics simulations and spectral calculations to gain insight into the molecular structures in the mixture. N-Methylacetamide and water molecules tend to form clusters with "frozen" amide I dynamics. This is driven by a hydrophobic collapse as the methyl groups of the N-methylacetamide molecules cluster in the presence of water. Since the studied system can be considered as a simplified model for the backbone of proteins, the present study forms a convenient basis for understanding the structural and vibrational dynamics in proteins. It is particularly interesting to find out that a hydrophobic collapse as the one driving protein folding is observed in such a simple system.

Interplay between Hydrogen Bonding and Vibrational Coupling in Liquid N-Methylacetamide.

Intrinsically disordered proteins play an important role in biology, and unraveling their labile structure presents a vital challenge. However, the dynamical structure of such proteins thwarts their study by standard techniques such as X-ray diffraction and NMR spectroscopy. Here, we use a neat liquid composed of N-methylacetamide molecules as a model system to elucidate dynamical and structural properties similar to those one can expect to see in intrinsically disordered proteins. To examine the structural dynamics in the neat liquid, we combine molecular dynamics, response-function-based spectral simulations, and two-dimensional polarization-resolved infrared spectroscopy in the amide I (CO stretch) region. The two-dimensional spectra reveal a delicate interplay between hydrogen bonding and intermolecular vibrational coupling effects, observed through a fast anisotropy decay. The present study constitutes a general platform for understanding the structure and dynamics of highly disordered proteins.

Ligand binding studied by 2D IR spectroscopy using the azidohomoalanine label.

We explore the capability of the azidohomoalanine (Aha) as a vibrational label for 2D IR spectroscopy to study the binding of the target peptide to the PDZ2 domain. The Aha label responds sensitively to its local environment and its peak extinction coefficient of 350-400 M(-1) cm(-1) is high enough to routinely measure it in the low millimolar concentration regime. The central frequency, inhomogeneous width and spectral diffusion times deduced from the 2D IR line shapes of the Aha label at various positions in the peptide sequence is discussed in relationship to the known X-ray structure of the peptide bound to the PDZ2 domain. The results suggest that the Aha label introduces only a small perturbation to the overall structure of the peptide in the binding pocket. Finally, Aha is a methionine analog that can be incorporated also into larger proteins at essentially any position using protein expression. Altogether, Aha thus fulfills the requirements a versatile label should have for studies of protein structure and dynamics by 2D IR spectroscopy.

2D-IR study of a photoswitchable isotope-labeled alpha-helix.

A series of photoswitchable, alpha-helical peptides were studied using two-dimensional infrared spectroscopy (2D-IR). Single-isotope labeling with (13)C(18)O at various positions in the sequence was employed to spectrally isolate particular backbone positions. We show that a single (13)C(18)O label can give rise to two bands along the diagonal of the 2D-IR spectrum, one of which is from an amide group that is hydrogen-bonded internally, or to a solvent molecule, and the other from a non-hydrogen-bonded amide group. The photoswitch enabled examination of both the folded and unfolded state of the helix. For most sites, unfolding of the peptide caused a shift of intensity from the hydrogen-bonded peak to the non-hydrogen-bonded peak. The relative intensity of the two diagonal peaks gives an indication of the fraction of molecules hydrogen-bonded at a certain location along the sequence. As this fraction varies quite substantially along the helix, we conclude that the helix is not uniformly folded. Furthermore, the shift in hydrogen bonding is much smaller than the change of helicity measured by CD spectroscopy, indicating that non-native hydrogen-bonded or mis-folded loops are formed in the unfolded ensemble.

Enhancing signal detection and completely eliminating scattering using quasi-phase-cycling in 2D IR experiments.

We demonstrate how quasi-phase-cycling achieved by sub-cycle delay modulation can be used to replace optical chopping in a box-CARS 2D IR experiment in order to enhance the signal size, and, at the same time, completely eliminate any scattering contamination. Two optical devices are described that can be used for this purpose, a wobbling Brewster window and a photoelastic modulator. They are simple to construct, easy to incorporate into any existing 2D IR setup, and have attractive features such as a high optical throughput and a fast modulation frequency needed to phase cycle on a shot-to-shot basis.

Dynamical transition in a small helical peptide and its implication for vibrational energy transport.

The two-dimensional infrared spectrum of an octameric helical peptide in chloroform was measured as a function of temperature. Isotope labeling of the carbonyl group of one of the amino acids was used to obtain information for an isolated vibration. The antidiagonal width of the 2D-IR signal, which is a measure of the homogeneous dephasing time T(2), is constant from 220 to 260 K (within experimental error), and increases steeply above. The homogeneous dephasing time of the carbonyl vibration is attributed to the flexibility of the system and/or its immediate surrounding. The system undergoes a dynamical transition at about 270 K, with similarities to the protein dynamical transition. Furthermore, the temperature dependence of the antidiagonal width strongly resembles that of the efficiency of vibrational energy transport along the helix, which has been studied in a recent paper (J. Phys. Chem. B 2008, 112, 15487). The connection between the two processes, structural flexibility and energy transport mechanism, is discussed.

Polarization portraits of single multichromophoric systems: visualizing conformation and energy transfer.

A novel technique, two-dimensional (2D) polarization single-molecule imaging, is presented. It is based on measurements and analysis of fluorescence intensity as a function of excitation and emission polarization angles. The technique allows recording of full information on the steady-state polarization properties of fluorescent objects. It is particularly suitable for application to single multichromophoric systems (molecules or nanoparticles) with energy transfer (ET) between different chromophores (e.g., single fluorescent pi-conjugated polymer chains). The 2D polarization data simultaneously provide information on the conformation of the system and the efficiency of its internal excitation ET. The technique is used to characterize single chains and different kinds of chain aggregates of different conjugated polymers at different temperatures. The 2D polarization measurements reveal a dramatic difference in ET taking place in these systems. Clear temperature dependence of ET is observed for individual aggregates as well as for their statistical ensembles. Also, a dependence on solvent and aggregate size is shown. Additionally, extensive "traditional one-dimensional" polarization results on the polarization anisotropy of fluorescence excitation and emission are presented. These results and findings are discussed in relation to internal organization of the nano-objects under study.

Simulation of vibrational energy transfer in two-dimensional infrared spectroscopy of amide I and amide II modes in solution.

Population transfer between vibrational eigenstates is important for many phenomena in chemistry. In solution, this transfer is induced by fluctuations in molecular conformation as well as in the surrounding solvent. We develop a joint electrostatic density functional theory map that allows us to connect the mixing of and thereby the relaxation between the amide I and amide II modes of the peptide building block N-methyl acetamide. This map enables us to extract a fluctuating vibrational Hamiltonian from molecular dynamics trajectories. The linear absorption spectrum, population transfer, and two-dimensional infrared spectra are then obtained from this Hamiltonian by numerical integration of the Schrodinger equation. We show that the amide I/amide II cross peaks in two-dimensional infrared spectra in principle allow one to follow the vibrational population transfer between these two modes. Our simulations of N-methyl acetamide in heavy water predict an efficient relaxation between the two modes with a time scale of 790 fs. This accounts for most of the relaxation of the amide I band in peptides, which has been observed to take place on a time scale of 450 fs in N-methyl acetamide. We therefore conclude that in polypeptides, energy transfer to the amide II mode offers the main relaxation channel for the amide I vibration.

Vibrational relaxation in simulated two-dimensional infrared spectra of two amide modes in solution.

Two-dimensional infrared spectroscopy is capable of following the transfer of vibrational energy between modes in real time. We develop a method to include vibrational relaxation in simulations of two-dimensional infrared spectra at finite temperature. The method takes into account the correlated fluctuations that occur in the frequencies of the vibrational states and in the coupling between them as a result of interaction with the environment. The fluctuations influence the two-dimensional infrared line shape and cause vibrational relaxation during the waiting time, which is included using second-order perturbation theory. The method is demonstrated by applying it to the amide-I and amide-II modes in N-methylacetamide in heavy water. Stochastic information on the fluctuations is obtained from a molecular dynamics trajectory, which is converted to time dependent frequencies and couplings with a map from a density functional calculation. Solvent dynamics with the same frequency as the energy gap between the two amide modes lead to efficient relaxation between amide-I and amide-II on a 560 fs time scale. We show that the cross peak intensity in the two-dimensional infrared spectrum provides a good measure for the vibrational relaxation.