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The vibrational coupling between protein backbone modes and the role of water interactions are important topics in biomolecular spectroscopy. Our work reports the first study of the coupling between amide I and amide A modes within peptides and proteins with secondary structure and water contacts. We use two-color two-dimensional infrared (2D IR) spectroscopy and observe cross peaks between amide I and amide A modes. In experiments with peptides with different secondary structures and side chains, we observe that the spectra are sensitive to secondary structure. Water interactions affect the cross peaks, which may be useful as probes for the accessibility of protein sites to hydration water. Moving to two-color 2D IR spectra of proteins, the data demonstrate that the cross peaks integrate the sensitivities of both amide I and amide A spectra and that a two-color detection scheme may be a promising tool for probing secondary structures in proteins.
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Amidas , Proteínas , Espectrofotometria Infravermelho , Água , Espectrofotometria Infravermelho/métodos , Água/química , Proteínas/química , Amidas/química , Estrutura Secundária de Proteína , Peptídeos/químicaRESUMO
Superfluid helium nanodroplets are an ideal environment for the formation of metastable, self-organized dopant nanostructures. However, the presence of vortices often hinders their formation. Here, we demonstrate the generation of vortex-free helium nanodroplets and explore the size range in which they can be produced. From x-ray diffraction images of xenon-doped droplets, we identify that single compact structures, assigned to vortex-free aggregation, prevail up to 10^{8} atoms per droplet. This finding builds the basis for exploring the assembly of far-from-equilibrium nanostructures at low temperatures.
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We demonstrate that a sodium dimer, Na_{2}(1^{3}Σ_{u}^{+}), residing on the surface of a helium nanodroplet, can be set into rotation by a nonresonant 1.0 ps infrared laser pulse. The time-dependent degree of alignment measured, exhibits a periodic, gradually decreasing structure that deviates qualitatively from that expected for gas-phase dimers. Comparison to alignment dynamics calculated from the time-dependent rotational Schrödinger equation shows that the deviation is due to the alignment dependent interaction between the dimer and the droplet surface. This interaction confines the dimer to the tangential plane of the droplet surface at the point where it resides and is the reason that the observed alignment dynamics is also well described by a 2D quantum rotor model.
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The susceptibility of aqueous dipeptides to photodissociation by deep ultraviolet irradiation is studied by femtosecond spectroscopy supported by density functional theory calculations. The primary photodynamics of the aqueous dipeptides of glycyl-glycine (gly-gly), alalyl-alanine (ala-ala), and glycyl-alanine (gly-ala) show that upon photoexcitation at a wavelength of 200 nm, about 10% of the excited dipeptides dissociate by decarboxylation within 100 ps, while the rest of the dipeptides return to their native ground state. Accordingly, the vast majority of the excited dipeptides withstand the deep ultraviolet excitation. In those relatively few cases, where excitation leads to dissociation, the measurements show that deep ultraviolet irradiation breaks the Cα-C bond rather than the peptide bond. The peptide bond is thereby left intact, and the decarboxylated dipeptide moiety is open to subsequent reactions. The experiments indicate that the low photodissociation yield and in particular the resilience of the peptide bond to dissociation are due to rapid internal conversion from the excited state to the ground state, followed by efficient vibrational relaxation facilitated by intramolecular coupling among the carbonate and amide modes. Thus, the entire process of internal conversion and vibrational relaxation to thermal equilibrium on the dipeptide ground state occurs on a time scale of less than 2 ps.
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Dipeptídeos , Raios Ultravioleta , Dipeptídeos/química , Análise Espectral , Íons , AlaninaRESUMO
The mechanical properties of biomaterials are dictated by the interactions and conformations of their building blocks, typically proteins. Although the macroscopic behavior of biomaterials is widely studied, our understanding of the underlying molecular properties is generally limited. Among the noninvasive and label-free methods to investigate molecular structures, infrared spectroscopy is one of the most commonly used tools because the absorption bands of amide groups strongly depend on protein secondary structure. However, spectral congestion usually complicates the analysis of the amide spectrum. Here, we apply polarized two-dimensional (2D) infrared spectroscopy (IR) to directly identify the protein secondary structures in native silk films cast from Bombyx mori silk feedstock. Without any additional peak fitting, we find that the initial effect of hydration is an increase of the random coil content at the expense of the helical content, while the ß-sheet content is unchanged and only increases at a later stage. This paper demonstrates that 2D-IR can be a valuable tool for characterizing biomaterials.
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Bombyx , Fibroínas , Animais , Seda/química , Bombyx/química , Fibroínas/química , Espectrofotometria Infravermelho , Materiais Biocompatíveis , Amidas , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The adsorption of protein to nanoparticles plays an important role in toxicity, food science, pharmaceutics, and biomaterial science. Understanding how proteins bind to nanophase surfaces is instrumental for understanding and, ultimately, controlling nanoparticle (NP) biochemistry. Techniques probing the adsorption of proteins at NP interfaces exist; however, these methods have been unable to determine the orientation and folding of proteins at these interfaces. For the first time, we probe in situ with sum frequency scattering vibrational spectroscopy the orientation of model leucine-lysine (LK) peptides adsorbed to NPs. The results show that both α-helical and ß-strand LK peptides bind the particles in an upright orientation, in contrast to the flat orientation of LKs binding to planar surfaces. The different binding geometry is explained by Coulombic forces between peptides across the particle volume.
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Peptídeos , Proteínas , Emulsões , Peptídeos/química , Adsorção , Análise Espectral/métodosRESUMO
We used two-dimensional infrared spectroscopy to disentangle the broad infrared band in the amide II vibrational regions of Bombyx mori native silk films, identifying the single amide II modes and correlating them to specific secondary structure. Amide I and amide II modes have a strong vibrational coupling, which manifests as cross-peaks in 2D infrared spectra with frequencies determined by both the amide I and amide II frequencies of the same secondary structure. By cross referencing with well-known amide I assignments, we determined that the amide II (N-H) absorbs at around 1552 and at 1530 cm-1 for helical and ß-sheet structures, respectively. We also observed a peak at 1517 cm-1 that could not be easily assigned to an amide II mode, and instead we tentatively assigned it to a Tyrosine sidechain. These results stand in contrast with previous findings from linear infrared spectroscopy, highlighting the ability of multidimensional spectroscopy for untangling convoluted spectra, and suggesting the need for caution when assigning silk amide II spectra.
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Bombyx , Amidas/química , Animais , Seda , Espectrofotometria Infravermelho/métodos , Tirosina , VibraçãoRESUMO
Strong-field ionization of nanoscale clusters provides excellent opportunities to study the complex correlated electronic and nuclear dynamics of near-solid density plasmas. Yet, monitoring ultrafast, nanoscopic dynamics in real-time is challenging, which often complicates a direct comparison between theory and experiment. Here, near-infrared laser-induced plasma dynamics in â¼600 nm diameter helium droplets are studied by femtosecond time-resolved x-ray coherent diffractive imaging. An anisotropic, â¼20 nm wide surface region, defined as the range where the density lies between 10% and 90% of the core value, is established within â¼100 fs, in qualitative agreement with theoretical predictions. At longer timescales, however, the width of this region remains largely constant while the radius of the dense plasma core shrinks at average rates of ≈71 nm/ps along and ≈33 nm/ps perpendicular to the laser polarization. These dynamics are not captured by previous plasma expansion models. The observations are phenomenologically described within a numerical simulation; details of the underlying physics, however, remain to be explored.
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Determining the secondary and tertiary structures of proteins at aqueous interfaces is crucial for understanding their function, but measuring these structures selectively at the interface is challenging. Here we demonstrate that two-dimensional infrared (2D-IR) spectroscopy of protein stabilized emulsions offers a new route to measuring interfacial protein structure with high levels of detail. We prepared hexadecane/water oil-in-water emulsions stabilized by model LK peptides that are known to fold into either α-helix or ß-sheet conformations at hydrophobic interfaces and measured 2D-IR spectra in a transmission geometry. We saw clear spectral signatures of the peptides folding at the interface, with no detectable residue from remaining bulk peptides. Using 2D spectroscopy gives us access to correlation and dynamics data, which enables structural assignment in cases where linear spectroscopy fails. Using the emulsions allows one to study interfacial spectra with standard transmission geometry spectrometers, bringing the richness of 2D-IR to the interface with no additional optical complexity.
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Proteínas , Água , Emulsões/química , Peptídeos , Conformação Proteica , Proteínas/química , Espectrofotometria Infravermelho , Água/químicaRESUMO
The SARS coronavirus 2 (SARS-CoV-2) spike protein is located at the outermost perimeter of the viral envelope and is the first component of the virus to make contact with surrounding interfaces. The stability of the spike protein when in contact with surfaces plays a deciding role for infection pathways and for the viability of the virus after surface contact. While cryo-EM structures of the spike protein have been solved with high resolution and structural studies in solution have provided information about the secondary and tertiary structures, only little is known about the folding when adsorbed to surfaces. We here report on the secondary structure and orientation of the S1 segment of the spike protein, which is often used as a model protein for in vitro studies of SARS-CoV-2, at the air-water interface using surface-sensitive vibrational sum-frequency generation (SFG) spectroscopy. The air-water interface plays an important role for SARS-CoV-2 when suspended in aerosol droplets, and it serves as a model system for hydrophobic surfaces in general. The SFG experiments show that the S1 segment of the spike protein remains folded at the air-water interface and predominantly binds in its monomeric state, while the combination of small-angle X-ray scattering and two-dimensional infrared spectroscopy measurements indicate that it forms hexamers with the same secondary structure in aqueous solution.
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COVID-19 , SARS-CoV-2 , Humanos , Glicoproteína da Espícula de Coronavírus/química , Água/químicaRESUMO
Interfaces between bulk media are often where critical molecular processes occur that can dictate the chemistry of an entire macroscopic system. Optical spectroscopy such as IR or Raman spectroscopy is often challenging to apply to interfaces due to contributions from bulk phases that dominate the spectra, masking any detail about the interfacial layer. Vibrational sum frequency generation (VSFG) spectroscopy is a nonlinear spectroscopy that allows vibrational spectra of molecules at interfaces to be directly measured. This Tutorial series is aimed at people entering the VSFG world without a rigorous formal background in optical physics or nonlinear spectroscopy. In this article, we present the fundamental theory of VSFG spectroscopy, with a focus on qualitative, intuitive explanation of the relevant physical phenomena, with minimal mathematics, to enable a newcomer to VSFG spectroscopy to quickly become conversant in the language and fundamental physics of the technique.
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Vibração , Água , Humanos , Análise Espectral Raman , Propriedades de Superfície , Água/químicaRESUMO
In this Tutorial series, we aim to provide an accessible introduction to vibrational sum frequency generation (VSFG) spectroscopy, targeted toward people entering the VSFG world without a rigorous formal background in optical physics or nonlinear spectroscopy. In this article, we describe in depth how a broadband VSFG spectrometer is designed and constructed, using the instrument in SurfLab, Aarhus University, as an illustrative case. Detailed information about specific instrumentation (together with reasons why things are the way they are) is given throughout. This information is often omitted in other descriptions of such instrumentation and so will be invaluable to people new to the field.
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Vibração , Humanos , Análise Espectral/métodos , Propriedades de SuperfícieRESUMO
We discuss how Coulomb explosion imaging (CEI), triggered by intense femtosecond laser pulses and combined with laser-induced alignment and covariance analysis of the angular distributions of the recoiling fragment ions, provides new opportunities for imaging the structures of molecules and molecular complexes. First, focusing on gas phase molecules, we show how the periodic torsional motion of halogenated biphenyl molecules can be measured in real time by timed CEI, and how CEI of one-dimensionally aligned difluoroiodobenzene molecules can uniquely identify four structural isomers. Next, focusing on molecular complexes formed inside He nano-droplets, we show that the conformations of noncovalently bound dimers or trimers, aligned in one or three dimensions, can be determined by CEI. Results presented for homodimers of CS2, OCS, and bromobenzene pave the way for femtosecond time-resolved structure imaging of molecules undergoing bimolecular interactions and ultimately chemical reactions.
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We present a simple and inexpensive liquid surface height controller that can monitor and maintain the height of a liquid surface in a surface-sensitive experiment. The system is based on a commercial laser pointer, universal serial bus webcam, syringe pump, and homemade control software. The system can sense changes in the height of the surface of ±1 µm, and the maximum range of the device without readjustment is around 2.5 mm. The intended use of the device is to maintain the height of a sample at the air-water interface in a sum-frequency generation spectroscopy measurement, which constantly changes due to water evaporation. A demonstration of the system maintaining the height of a water surface to a tolerance of ±5 µm over a period of 8 h is shown to illustrate the stability of a system controlled by this device.
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Alignment of OCS, CS_{2}, and I_{2} molecules embedded in helium nanodroplets is measured as a function of time following rotational excitation by a nonresonant, comparatively weak ps laser pulse. The distinct peaks in the power spectra, obtained by Fourier analysis, are used to determine the rotational, B, and centrifugal distortion, D, constants. For OCS, B and D match the values known from IR spectroscopy. For CS_{2} and I_{2}, they are the first experimental results reported. The alignment dynamics calculated from the gas-phase rotational Schrödinger equation, using the experimental in-droplet B and D values, agree in detail with the measurement for all three molecules. The rotational spectroscopy technique for molecules in helium droplets introduced here should apply to a range of molecules and complexes.
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Rotational dynamics of gas phase carbon disulfide (CS2) dimers were induced by a moderately intense, circularly polarized alignment laser pulse and measured as a function of time by Coulomb explosion imaging with an intense fs probe pulse. For the alignment pulse, two different temporal intensity profiles were used: a truncated pulse with a 150 ps turn-on and a 8 ps turn-off, or a 'kick' pulse with a duration of 1.3 ps. For both types of pulse, rich rotational dynamics with characteristic full and fractional revivals were recorded, showing that the intermolecular carbon-carbon axis periodically aligns along the propagation direction of the laser pulses. The truncated pulse gave the strongest alignment, which we rationalize as being due to a flat relative phase between the components in the rotational wave packet generated. Fourier analysis of the alignment dynamics gave well-spaced peaks which were fit to determine the rotational constant, B, and the centrifugal constant, DJ, for the ground state of the dimer. Our results agree with values from high-resolution IR spectroscopy. Numerical simulations of the alignment accurately reproduced the experimental dynamics when the truncated pulse or a low intensity kick pulse was used, but failed to reproduce the dynamics induced by a high intensity kick pulse. We posit that the discrepancy is due to excitation of the intermolecular torsional motion by the kick pulse.
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UV pump-extreme UV (XUV) probe femtosecond transient absorption spectroscopy is used to study the 268 nm induced photodissociation dynamics of bromoform (CHBr3). Core-to-valence transitions at the Br(3d) absorption edge (â¼70 eV) provide an atomic scale perspective of the reaction, sensitive to changes in the local valence electronic structure, with ultrafast time resolution. The XUV spectra track how the singly occupied molecular orbitals of transient electronic states develop throughout the C-Br bond fission, eventually forming radical Br and CHBr2 products. Complementary ab initio calculations of XUV spectral fingerprints are performed for transient atomic arrangements obtained from sampling excited-state molecular dynamics simulations. C-Br fission along an approximately C S symmetrical reaction pathway leads to a continuous change of electronic orbital characters and atomic arrangements. Two timescales dominate changes in the transient absorption spectra, reflecting the different characteristic motions of the light C and H atoms and the heavy Br atoms. Within the first 40 fs, distortion from C 3 v symmetry to form a quasiplanar CHBr2 by the displacement of the (light) CH moiety causes significant changes to the valence electronic structure. Displacement of the (heavy) Br atoms is delayed and requires up to â¼300 fs to form separate Br + CHBr2 products. We demonstrate that transitions between the valence-excited (initial) and valence + core-excited (final) state electronic configurations produced by XUV absorption are sensitive to the localization of valence orbitals during bond fission. The change in valence electron-core hole interaction provides a physical explanation for spectral shifts during the process of bond cleavage.
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Dimers of tetracene molecules are formed inside helium nanodroplets and identified through covariance analysis of the emission directions of kinetic tetracene cations stemming from femtosecond laser-induced Coulomb explosion. Next, the dimers are aligned in either one or three dimensions under field-free conditions by a nonresonant, moderately intense laser pulse. The experimental angular covariance maps of the tetracene ions are compared to calculated covariance maps for seven different dimer conformations and found to be consistent with four of these. Additional measurements of the alignment-dependent strong-field ionization yield of the dimer narrow the possible conformations down to either a slipped-parallel or parallel-slightly rotated structure. According to our quantum chemistry calculations, these are the two most stable gas-phase conformations of the dimer and one of them is favorable for singlet fission.
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Disulfide bonds are pivotal for the structure, function, and stability of proteins, and understanding ultraviolet (UV)-induced S-S bond cleavage is highly relevant for elucidating the fundamental mechanisms underlying protein photochemistry. Here, the near-UV photodecomposition mechanisms in gas-phase dimethyl disulfide, a prototype system with a S-S bond, are probed by ultrafast transient X-ray absorption spectroscopy. The evolving electronic structure during and after the dissociation is simultaneously monitored at the sulfur L1,2,3-edges and the carbon K-edge with 100 fs (FWHM) temporal resolution using the broadband soft X-ray spectrum from a femtosecond high-order harmonics light source. Dissociation products are identified with the help of ADC and RASPT2 electronic-structure calculations. Rapid dissociation into two CH3S radicals within 120 ± 30 fs is identified as the major relaxation pathway after excitation with 267 nm radiation. Additionally, a 30 ± 10% contribution from asymmetric CH3S2 + CH3 dissociation is indicated by the appearance of CH3 radicals, which is, however, at least partly the result of multiphoton excitation.