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The allosteric protein MCL-1 and its natural inhibitors, the BH3-only proteins PUMA, BIM, and NOXA regulate apoptosis by interacting promiscuously within an entangled binding network. Little is known about the transient processes and dynamic conformational fluctuations that are the basis for the formation and stability of the MCL-1/BH3-only complex. In this study, we designed photoswitchable versions of MCL-1/PUMA and MCL-1/NOXA, and investigated the protein response after an ultrafast photo-perturbation with transient infrared spectroscopy. We observed partial α-helical unfolding in all cases, albeit on strongly varying timescales (1.6 ns for PUMA, 9.7 ns for the previously studied BIM, and 85 ns for NOXA). These differences are interpreted as a BH3-only-specific "structural resilience" to defy the perturbation while remaining in MCL-1's binding pocket. Thus, the presented insights could help to better understand the differences between PUMA, BIM, and NOXA, the promiscuity of MCL-1, in general, and the role of the proteins in the apoptotic network. [ABSTRACT FROM AUTHOR]

Protein dynamics form a critical bridge between protein structure and function, yet the impact of evolution on ultrafast processes inside proteins remains enigmatic. This study delves deep into nanosecond-scale protein dynamics of a structurally and functionally conserved protein across species separated by almost a billion years, investigating ten homologs in complex with their ligand. By inducing a photo-triggered destabilization of the ligand inside the binding pocket, we resolved distinct kinetic footprints for each homolog via transient infrared spectroscopy. Strikingly, we found a cascade of rearrangements within the protein complex which manifest in time points of increased dynamic activity conserved over hundreds of millions of years within a narrow window. Among these processes, one displays a subtle temporal shift correlating with evolutionary divergence, suggesting reduced selective pressure in the past. Our study not only uncovers the impact of evolution on molecular processes in a specific case, but has also the potential to initiate a field of scientific inquiry within molecular paleontology, where species are compared and classified based on the rapid pace of protein dynamic processes; a field which connects the shortest conceivable time scale in living matter (10-9 s) with the largest ones (1016 s). [ABSTRACT FROM AUTHOR]

We present experimental and simulated 2D IR spectra of some high-pressure forms of isotope-pure D2O ice and compare the results to those of ice Ih published previously [F. Perakis and P. Hamm, Phys. Chem. Chem. Phys. 14, 6250 (2012); L. Shi et al., ibid. 18, 3772 (2016)]. Ice II, ice V, and ice XIII have been chosen for this study, since this selection covers many aspects of the polymorphism of ice. That is, ice II is a hydrogen-ordered phase of ice, in contrast to ice Ih, while ice V and ice XIII are a hydrogen-disordered/ordered couple that shares essentially the same oxygen structure and hydrogenbonded network. For the transmission 2D IR spectroscopy, a novel method had to be developed for the preparation of ultrathin films (1-2 μm) of high-pressure ices with good optical quality. We also simulated 2D IR spectra based on molecular dynamics simulations connected to a vibrational exciton picture. These simulations agree with the experimental results in a semi-quantitative manner for ice II, while the same approach failed for ice V and ice XIII. From the perspective of 2D IR spectroscopy, ice II appears to be more inhomogeneously broadened than ice Ih, despite its hydrogen-order, which we attribute to the fact that ice II is structurally more complex with four distinguishable hydrogen bonds that mix due to exciton coupling. Ice V and ice XIII, on the other hand, behave as expected with the hydrogen-disordered case (ice V) being more inhomogenously broadened. Furthermore, in all hydrogen-ordered forms (ice II and ice XIII), cross peaks could be identified in the anisotropic 2D IR spectrum, whose signs reveal the relative direction of the corresponding excitonic states. [ABSTRACT FROM AUTHOR]

The solvent dynamics around fluorinated acetonitrile is characterized by 2-dimensional infrared spectroscopy and atomistic simulations. The lineshape of the linear infrared spectrum is better captured by semiempirical (density functional tight binding) mixed quantum mechanical/molecular mechanics simulations, whereas force field simulations with multipolar interactions yield lineshapes that are significantly too narrow. For the solvent dynamics, a relatively slow time scale of 2 ps is found from the experiments and supported by the mixed quantum mechanical/molecular mechanics simulations. With multipolar force fields fitted to the available thermodynamical data, the time scale is considerably faster--on the 0.5 ps time scale. The simulations provide evidence for a well established CF-HOH hydrogen bond (population of 25%) which is found from the radial distribution function g(r) from both, force field and quantum mechanics/molecular mechanics simulations. [ABSTRACT FROM AUTHOR]

We present two-dimensional infrared (2D IR) spectra of organic monolayers immobilized on thin metallic films at the solid liquid interface. The experiments are acquired under Attenuated Total Reflectance (ATR) conditions which allow a surface-sensitive measurement of spectral diffusion, sample inhomogeneity, and vibrational relaxation of the monolayers. Terminal azide functional groups are used as local probes of the environment and structural dynamics of the samples. Specifically, we investigate the influence of different alkyl chain-lengths on the ultrafast dynamics of the monolayer, revealing a smaller initial inhomogeneity and faster spectral diffusion with increasing chain-length. Furthermore, by varying the environment (i.e., in different solvents or as bare sample), we conclude that the most significant contribution to spectral diffusion stems from intra- and intermolecular dynamics within the monolayer. The obtained results demonstrate that 2D ATR IR spectroscopy is a versatile tool for measuring interfacial dynamics of adsorbed molecules. [ABSTRACT FROM AUTHOR]

We apply two-dimensional infrared spectroscopy to differentiate between the two polyamorphous forms of glassy water, low-density (LDA) and high-density (HDA) amorphous ices, that were obtained by slow vapor deposition at 80 and 11 K, respectively. Both the vibrational lifetime and the bandwidth of the 1-2 transition of the isolated OD stretch vibration of HDO in H2O exhibit characteristic differences when comparing hexagonal (Ih), LDA, and HDA ices, which we attribute to the different local structures - in particular the presence of interstitial waters in HDA ice - that cause different delocalization lengths of intermolecular phonon degrees of freedom. Moreover, temperature dependent measurements show that the vibrational lifetime closely follows the structural transition between HDA and LDA phases. [ABSTRACT FROM AUTHOR]

While allostery is of paramount importance for protein regulation, the underlying dynamical process of ligand (un)binding at one site, resulting time evolution of the protein structure, and change of the binding affinity at a remote site are not well understood. Here the ligand-induced conformational transition in a widely studied model system of allostery, the PDZ2 domain, is investigated by transient infrared spectroscopy accompanied by molecular dynamics simulations. To this end, an azobenzene derived photoswitch is linked to a peptide ligand in a way that its binding affinity to the PDZ2 domain changes upon switching, thus initiating an allosteric transition in the PDZ2 domain protein. The subsequent response of the protein, covering four decades of time, ranging from ∼1 ns to ∼10 µs, can be rationalized by a remodeling of its rugged free-energy landscape, with very subtle shifts in the populations of a small number of structurally well-defined states. It is proposed that structurally and dynamically driven allostery, often discussed as limiting scenarios of allosteric communication, actually go hand-in-hand, allowing the protein to adapt its free-energy landscape to incoming signals. [ABSTRACT FROM AUTHOR]

Using classical molecular dynamics simulations, the 2D infrared (IR) spectroscopy of CN- solvated in D2O is investigated. Depending on the force field parametrizations, most of which are based on multipolar interactions for the CN- molecule, the frequency-frequency correlation function and observables computed from it differ. Most notably, models based on multipoles for CN- and TIP3P for water yield quantitatively correct results when compared with experiments. Furthermore, the recent finding that T1 times are sensitive to the van der Waals ranges on the CN- is confirmed in the present study. For the linear IR spectrum, the best model reproduces the full widths at half maximum almost quantitatively (13.0 cm-1 vs. 14.9 cm-1) if the rotational contribution to the linewidth is included. Without the rotational contribution, the lines are too narrow by about a factor of two, which agrees with Raman and IR experiments. The computed and experimental tilt angles (or nodal slopes) α as a function of the 2D IR waiting time compare favorably with the measured ones and the frequency fluctuation correlation function is invariably found to contain three time scales: a sub-ps, 1 ps, and one on the 10-ps time scale. These time scales are discussed in terms of the structural dynamics of the surrounding solvent and it is found that the longest time scale (≈10 ps) most likely corresponds to solvent exchange between the first and second solvation shell, in agreement with interpretations from nuclear magnetic resonance measurements. [ABSTRACT FROM AUTHOR]

We utilize two-color two-dimensional infrared spectroscopy to measure the intermolecular coupling between azide ions and their surrounding water molecules in order to gain information about the nature of hydrogen bonding of water to ions. Our findings indicate that the main spectral contribution to the intermolecular cross-peak comes from population transfer between the asymmetric stretch vibration of azide and the OD-stretch vibration of D2O. The azide-bound D2O bleach/stimulated emission signal, which is spectrally much narrower than its linear absorption spectrum, shows that the experiment is selective to solvation shell water molecules for population times up to ∼500 fs. The waters around the ion are present in an electrostatically better defined environment. Afterwards, ∼1 ps, the sample thermalizes and selectivity is lost. On the other hand, the excited state absorption signal of the azide-bound D2O is much broader. The asymmetry in spectral width between bleach/stimulated emission versus excited absorption has been observed in very much the same way for isotope-diluted ice Ih, where it has been attributed to the anharmonicity of the OD potential. [ABSTRACT FROM AUTHOR]

We present experimental 2D IR spectra of isotope diluted ice Ih (i.e., the OH stretch mode of HOD in D2O and the OD stretch mode of HOD in H2O) at T = 80 K. The main spectral features are the extremely broad 1-2 excited state transition, much broader than the corresponding 0-1 groundstate transition, as well as the presence of quantum beats. We do not observe any inhomogeneous broadening that might be expected due to proton disorder in ice Ih. Complementary, we perform simulations in the framework of the Lippincott-Schroeder model, which qualitatively reproduce the experimental observations. We conclude that the origin of the observed line shape features is the coupling of the OH-vibrational coordinate with crystal phonons and explain the beatings as a coherent oscillation of the O···O hydrogen bond degree of freedom. [ABSTRACT FROM AUTHOR]

We demonstrate a method to collect purely absorptive three-dimensional (3D) fifth-order vibrational spectra on the model system CO2 in H2O. The six beam interferometer is described, as well as a method to experimentally determine the phase of the 3D spectrum. The measured spectra agree very well with simulations of the data based on the cumulant expansion. There are five peaks corresponding to different paths up and down the vibrational ladder. The positions, signs, and amplitudes of the peaks agree with theoretical predictions, and the intensities of the peaks scale linearly with concentration. Based on the concentration dependence and agreement between the simulations and measurements, we conclude that cascaded lower order signals contribute negligibly to the observed signal. [ABSTRACT FROM AUTHOR]

With the help of ultrafast time-resolved infrared spectroscopy, we investigate the temperature dependence of the IR driven cis→trans isomerization of nitrous acid (HONO) in solid Kr. We find that the lifetime of the OH-stretch vibration, as well as the final cooling into the matrix, is affected only minimally (if at all) by temperature. Nevertheless, the quantum yield of the cis→trans isomerization reaction increases by ≈30% to a total of 50%–70% when lowering the temperature from 30 to 15 K, whereas the trans→cis back yield is reduced by ≈40%. The results are discussed in analogy to Marcus theory of nonadiabatic electron transfer for the essentially barrierless case. We present a unified view of this important prototype proton transfer reaction that can explain the high cis→trans quantum yield of close to 1. [ABSTRACT FROM AUTHOR]

With the help of ultrafast time-resolved infrared spectroscopy, we investigate rotational diffusion of cis- and trans-nitrous acid (HONO) in solid Kr at 30 K, as well as its reorientation upon the IR-driven cis→trans isomerization. We find different mobilities for the two isomers: cis-HONO is pinned to the matrix with no decay of the anisotropy on the 100 ns time scale, whereas trans-HONO rotates around its long axis, reducing its anisotropy partially on that time scale. The long axis itself, defined by the terminal oxygen and hydrogen atoms of HONO, stays fixed on even a minute time scale. Accompanying molecular dynamics simulations reproduce the anisotropic rotational diffusion of trans-HONO correctly, although on a completely wrong time scale, whereas they would predict complete reorientation of cis-HONO within ≈10 ps, in harsh disagreement with the experiment. We attribute the mismatch of orientational time scales to either too soft interaction potentials or to the fact that HONO occupies an interstitial rather than a monosubstitutional matrix site. The experiments furthermore show that the direction of the OH bond hardly changes during the IR-driven cis→trans isomerization, in contrast to the intuitive picture that it is mostly the light hydrogen which moves. Rather, it is the two central nitrogen and oxygen atoms that are removed during isomerization in a hula hoop fashion, whereas the terminal atoms are still pinned to the matrix cage. [ABSTRACT FROM AUTHOR]

Three-dimensional-IR spectroscopy is proposed as a new spectroscopic technique that is sensitive to three-point frequency fluctuation correlation functions. This will be important when the statistics of the underlying stochastic process is non-Gaussian, and hence when the system does not follow the linear response hypothesis. Furthermore, a very general classification of nonlinear spectroscopy in terms of higher order frequency fluctuation correlation functions is introduced, according to which certain moments of a multidimensional spectrum are related to certain frequency fluctuation correlation functions. The classification is rigorous in the so-called inhomogenous limit, but remains valid approximately also when motional narrowing becomes important. The work also puts a recent paper [J. Bredenbeck et al., Phys. Rev. Lett. 95, 083201 (2005)] onto solid theoretical grounds, where we have shown for the first time that fifth-order spectroscopy—in this case transient two-dimensional spectroscopy—is indeed sensitive to the three-point frequency fluctuation correlation function. [ABSTRACT FROM AUTHOR]

In this study we focus on the differences and analogies of two experimental implementations of two-dimensional infrared (2D-IR) spectroscopy: double-resonance or dynamic hole burning 2D-IR spectroscopy and pulsed Fourier transform or heterodyne detected photon echo spectroscopy. A comparison is done theoretically as well as experimentally by contrasting data obtained from both methods. As an example we have studied the strongly coupled asymmetric and symmetric carbonyl stretching vibrations of dicarbonylacetylacetonato rhodium dissolved in hexane. Both methods yield the same peaks in a 2D-IR spectrum. Within certain approximations we derive an analytic expression which shows that the 2D-IR spectra are broadened in one frequency dimension in the double-resonance experiment by convolution with the pump pulse spectral width, while the spectral resolution in the other frequency direction is the same in both cases. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]

We present a general expression for the polarization dependence of transient two-dimensional IR spectroscopy (T2D-IR), a technique designed to measure 2D-IR spectra of transient species. T2D-IR is a UV pump narrowband-IR-pump broadband-IR-probe experiment of fifth order in the laser field which involves up to three different transition dipole moments. The UV pulse adds an additional degree of freedom in polarization as compared to 2D-IR spectroscopy and increases the versatility of signal manipulation and the potential structural information content of the signals. The polarization conditions leading to a maximum of structural information are discussed. Important special cases of polarization conditions are formulated. The application of polarization selectivity is demonstrated for different types of T2D-IR experiments on photo triggered metal-to-ligand charge transfer in the model system [Re(CO)3(dmbpy)Cl]. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]

The potential information content of two-dimensional infrared (2D-IR) spectroscopy of the amide-I band as a structure analysis method of small peptides is explored in a computational study, applying it to a cyclic penta peptide as an example. In the presence of realistic homogeneous and inhomogeneous broadening, the structure resolution power in the case of a nonisotope labeled molecule would be vanishingly small. However, 2D-IR spectroscopy can reveal the structure of the peptide uniquely if using a sufficiently large set of isotope labeled compounds. Design strategies for isotope labeling are developed. In the case of the cyclic penta peptide studied here, at least three single [SUP13]C labeled compounds would be needed to determine the structure. While double [SUP13]C labeling does not offer any advantage compared to single [SUP13]C labeling, mixed [SUP13]C [SUP16]O- [SUP12]C [SUP18]O or [SUP13]C [SUP16]O- [SUP13]C [SUP18]O double labeling does. It is furthermore explored to what extent a structure can still be determined even under nonideal conditions, i.e., if systematic errors in the molecular models are allowed or if the molecule is allowed to coexists in different conformations simultaneously. [ABSTRACT FROM AUTHOR]

Ultrafast two-dimensional infrared spectroscopy (2D IR) has been advanced in recent years toward measuring signals from only a monolayer of sample molecules at solid-liquid and solid-gas interfaces. A series of experimental methods has been introduced, which in the chronological order of development are 2D sumfrequency-generation (2D SFG), transmission 2D IR, and reflection 2D IR, the latter in either internal, attenuated total reflection (ATR), or external reflection configuration. The different variants of 2D vibrational spectroscopy are based on either the even-order or the odd-order nonlinear susceptibility, and all allow resolving similar molecular temporal and spectral information. In this review, we introduce the basic principles of the different methods of 2D vibrational spectroscopy at surfaces along with a balanced overview on the technological aspects as well as benefits and shortcomings. We furthermore discuss the current scope of applications for 2D vibrational surface spectroscopy, which spans an impressively broad range of samples from biological molecules to heterogeneous catalysts. The emphasis is on the ultrafast structural dynamics of molecules at interfaces, environmental interactions, and intermolecular interactions. We furthermore consider important recent technological developments of 2D vibrational surface spectroscopy, which employ (i) surface enhancement, (ii) methods for studying electrochemical interfaces, and (iii) extensions for resolving nonequilibrium processes (transient 2D IR). A detailed outlook is finally given regarding important future applications and technological developments of 2D vibrational surface spectroscopy. [ABSTRACT FROM AUTHOR]

We present two-dimensional infrared (2D IR) spectra of the OD stretch vibration of isotope diluted water (HOD/H2O) from ambient conditions (293 K) down to the metastable supercooled regime (260 K). We observe that spectral diffusion slows down from 700 fs to 2.6 ps as we lower the temperature. A comparison between measurements performed at the magic angle with those at parallel polarization shows that the 2D IR line shape is affected by the frequency-dependent anisotropy decay in the case of parallel polarization, altering the extracted correlation decay. A fit within the framework of an Arrhenius law reveals an activation energy of Ea = 6.2 ± 0.2 kcal/mol and a pre-exponential factor of 1/A = 0.02 ± 0.01 fs. Alternatively, a power law fit results in an exponent γ = 2.2 and a singularity temperature Ts = 221 K. We tentatively conclude that the power law provides the better physical picture to describe the dynamics of liquid water around the freezing point. [ABSTRACT FROM AUTHOR]

This paper reviews our experimental and theoretical efforts toward understanding vibrational self-trapping of the amide I and N–H mode of crystalline acetanilide (ACN), other similar hydrogen-bonded crystals, as well as of model peptides. In contrast to previous works, we used nonlinear IR spectroscopy as the experimental tool, which is specifically sensitive to the anharmonic contributions of the intramolecular interactions (as the nonlinear IR response of set of harmonic oscillators vanishes exactly). Our work reconfirms the previous assignment of the two bands of the amide I mode of ACN as being a self-trapped and a free exciton state, but in addition also establishes the lifetimes of these states and identifies the relevant phonons. Furthermore, we provide evidence for vibrationally self-trapped states also in model α-helices. However, given the short lifetime, any biological relevance in the sense of Davydov’s initial proposal can probably be ruled out. [ABSTRACT FROM AUTHOR]

Using time-resolved IR spectroscopy, we monitored the kinetics of folding and unfolding processes of a photoswitchable 16-residue alanine-based α-helical peptide on a timescale from few picoseconds to almost 40 μs and over a large temperature range (279–318 K). The folding and unfolding processes were triggered by an ultrafast laser pulse that isomerized the cross linker within a few picoseconds. The main folding and unfolding times (700 ns and 150 ns, respectively, at room temperature) are in line with previous T-jump experiments obtained from similar peptides. However, both processes show complex, strongly temperature-dependent spectral kinetics that deviate clearly from a single-exponential behavior. Whereas in the unfolding experiment the ensemble starts from a well defined folded state, the starting ensemble in the folding experiment is more heterogeneous, which leads to distinctly different kinetics of the experiments, because they are sensitive to different regions of the energy surface. A qualitative agreement with the experimental data-set can be obtained by a model where the unfolded states act as a hub connected to several separated ‘misfolded’ states with a distribution of rates. We conclude that a rather large spread of rates (k1 : kn ≈9) is needed to explain the experimentally observed stretched exponential response with stretching factor β = 0.8 at 279 K. [ABSTRACT FROM AUTHOR]

X-ray crystallography and nuclear magnetic resonance measurements provide us with atomically resolved structures of an ever-growing number of biomolecules. These static structural snapshots are important to our understanding of biomolecular function, but real biomolecules are dynamic entities that often exploit conformational changes and transient molecular interactions to perform their tasks. Nuclear magnetic resonance methods can follow such structural changes, but only on millisecond timescales under non-equilibrium conditions. Time-resolved X-ray crystallography has recently been used to monitor the photodissociation of CO from myoglobin on a subnanosecond timescale, yet remains challenging to apply more widely. In contrast, two-dimensional infrared spectroscopy, which maps vibrational coupling between molecular groups and hence their relative positions and orientations, is now routinely used to study equilibrium processes on picosecond timescales. Here we show that the extension of this method into the non-equilibrium regime allows us to observe in real time in a short peptide the weakening of an intramolecular hydrogen bond and concomitant opening of a β-turn. We find that the rate of this process is two orders of magnitude faster than the ‘folding speed limit’ established for contact formation between protein side chains. [ABSTRACT FROM AUTHOR]

Using three-dimensional infrared (3D-IR) spectroscopy, we investigate the vibrational dynamics of isotope-diluted ice Ih. By probing the OD stretch mode of HOD in H2O, we observe an extremely rapid decay (≈200 fs) of the population from the second vibrational excited state. Quantum simulations based on a two-dimensional Lippincott-Schroeder potential agree nearly quantitatively with the experimental 3D-IR lineshapes and dynamics. The model suggests that energy dissipation is enhanced due to nonadiabatic effects between vibrational states, which arise from strong mode-mixing between the OD stretch mode with lattice degrees of freedom. Furthermore, we compare the simulation results to ab initio based potentials, in which the hydrogen bond anharmonicity is too small to reproduce the experimental 3D-IR spectra. We thus conclude that the Lippincott-Schroeder potential effectively coalesces many degrees of freedom of the crystal into one intermolecular coordinate. [ABSTRACT FROM AUTHOR]

1D and 2D-IR spectroscopy was used to resolve the structural transition that is responsible for the gating mechanism of the influenza A M2 channel (). This report constitutes a milestone in the development of both ATR-FTIR and 2D-IR spectroscopies as precise tools for structural biology. [Copyright &y& Elsevier]

We explore the capability of the non-natural amino acid azidohomoalanine (AHA) as an IR label to sense relatively small structural changes in proteins with the help of 2D IR difference spectroscopy. To that end, we AHA-labeled an allosteric protein (the PDZ2 domain from human tyrosine-phosphatase 1E) and furthermore covalently linked it to an azobenzene-derived photoswitch as to mimic its conformational transition upon ligand binding. To determine the strengths and limitations of the AHA label, in total six mutants have been investigated with the label at sites with varying properties. Only one mutant revealed a measurable 2D IR difference signal. In contrast to the commonly observed frequency shifts that report on the degree of solvation, in this case we observe an intensity change. To understand this spectral response, we performed classical MD simulations, evaluating local contacts of the AHA labels to water molecules and protein side chains and calculating the vibrational frequency on the basis of an electrostatic model. Although these simulations revealed in part significant and complex changes of the number of intraprotein and water contacts upon trans-cis photoisomerization, they could not provide a clear explanation of why this one label would stick out. Subsequent quantum-chemistry calculations suggest that the response is the result of an electronic interaction involving charge transfer of the azido group with sulfonate groups from the photoswitch. To the best of our knowledge, such an effect has not been described before. [ABSTRACT FROM AUTHOR]

Ultrafast two-dimensional infrared spectroscopy (2D-IR) is a promising tool to investigate molecular structure and its equilibrium fluctuations with subpicosecond time resolution. Recently, the method has been extended to the non-equilibrium regime to investigate the conformational transition of a photoswitchable cyclic peptide by transient two-dimensional infrared spectroscopy (2D-IR). This article reports on a nove laser pulse sequence that allows "labeling" certain vibrational modes in the reactant of a photoreaction by an infrared pulse and tracks their evolution on the way to the product.

2D-IR spectroscopy has matured to a powerful technique to study the structure and dynamics of peptides, but its extension to larger proteins is still in its infancy, the major limitations being sensitivity and selectivity. Site-selective information requires measuring single vibrational probes at sub-millimolar concentrations where most proteins are still stable, which is a severe challenge for conventional (FT)IR spectroscopy. Besides its ultrafast time-resolution, a so far largely underappreciated potential of 2D-IR spectroscopy lies in its sensitivity gain. The present paper sets the goals and outlines strategies how to use that sensitivity gain together with properly designed vibrational labels to make IR spectroscopy a versatile tool to study a wide class of proteins. [ABSTRACT FROM AUTHOR]

We explore the capability of the azidohomoalanine (Aha)as a vibrationallabel for 2D IR spectroscopy to study the binding of the target peptideto the PDZ2 domain. The Aha label responds sensitively to its localenvironment and its peak extinction coefficient of 350–400M–1cm–1is high enough to routinelymeasure it in the low millimolar concentration regime. The centralfrequency, inhomogeneous width and spectral diffusion times deducedfrom the 2D IR line shapes of the Aha label at various positions inthe peptide sequence is discussed in relationship to the known X-raystructure of the peptide bound to the PDZ2 domain. The results suggestthat the Aha label introduces only a small perturbation to the overallstructure of the peptide in the binding pocket. Finally, Aha is amethionine analog that can be incorporated also into larger proteinsat essentially any position using protein expression. Altogether,Aha thus fulfills the requirements a versatile label should have forstudies of protein structure and dynamics by 2D IR spectroscopy. [ABSTRACT FROM AUTHOR]

Increased long-term performance was found for photocatalytic H2 production in a homogeneous combination of [Re(NCS)(CO)3bipy] (1; bipy = 2,2'-bipyridine), [Co(dmgH)2] (dmgH2 = dimethylglyoxime), tdethanolamine (TEOA), and [HTEOA][BF4I in N,N-dimethylforniamide, achieving TONRe up to 6000 (H/Re). The system proceeded by reductive quenching of *1 by TEOA, followed by fast (k1 = 1.3 x 108 M-1 s-1) electron transfer to (CoII (dmgH)2] and subsequent protonation (K2) and elimination (k3, second-order process in cobalt) of H2. Observed quantum yields were up to ∼90% (H produced per absorbed photon). The type of acid had a substantial effect on the long-term stability. A decomposition pathway involving cobalt is limiting the long-term performance. Time-resolved infrared (IR) spectroscopy confirmed that photooxidized TEOA generates a second reducing equivalent, which can be transferred to 1(70%, k2e = 3.3 x 108 M-1 s-1) if no [CoII(dmgH)2] is present. [ABSTRACT FROM AUTHOR]

A series of photoswitchable, α-helical peptides were studied using two-dimensional infrared spectroscopy (2D-IR). Single-isotope labeling with 13C18O at various positions in the sequence was employed to spectrally isolate particular backbone positions. We show that a single 13C18O 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. [ABSTRACT FROM AUTHOR]