Time-resolved methods in biophysics. 10. Time-resolved FT-IR difference spectroscopy and the application to membrane proteins

Radu, Ionela; Schleeger, Michael; Bolwien, Carsten; Heberle, Joachim

doi:10.1039/b9pp00050j

Cited by 58 publications

(70 citation statements)

References 90 publications

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“…The former spectroscopy is the standard method to characterize intermediate states of retinal proteins from the electronic structure of the chromophore. The latter spectroscopy is particularly informative about the dynamics of conformational and protonation changes in proteins when probing vibrational modes of the protein backbone and of the side chain of amino acids (30,31). Two bands are resolved at 375 and 520 nm in the photocycle of CrChR2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

Lórenz-Fonfrı́a

Bamann

Resler

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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106

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The discovery of channelrhodopsins introduced a new class of lightgated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time-resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(−) and 1,648(+) cm. These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance.channelrhodopsin | optogenetics | channel gating | infrared spectroscopy | time-resolved spectroscopy I on channels are membrane proteins that mediate the passive movement of cations and anions across biological membranes, a process not only central for most living organisms but key to electrical excitability of cells. Upon activation, ion channels constitute a transient pathway to facilitate the permeation of ions across the cellular membrane. Ion channels can be switched, or gated, from a closed (nonconductive) to an open (conductive) state by external stimuli, such as ligands, voltage, or mechanical stress (1-3). Available structural information indicates that the ion-conducting pathway generally comprises a pore with wide regions solvated by water and constrictions sites formed by specific polar groups that confer ion selectivity (4). In the nonconductive state, the permeation of ions is prevented by energy barriers along the pore, either from physical occlusion or from the hydrophobic nature of residues lining the pore: the pore does not need to be physically occluded to be functionally closed (5). Habitual suspects for the gating mechanism, i.e., how the protein transits from the nonconductive to the conductive state, are diverse types of structural changes, such as orientation/rotation changes of transmembrane helices or of bulky (aromatic) side chains (6). Several computational studies have suggested a more subtle gating mechanism, where the thermodynamics and kinetics of the hydration of the ion-conducting pore might play a vital role (5,7,8). However, experimental support for this last proposal relies largely on studies on synthetic nanometric pores (9).Channelrhodopsins (ChRs) belong to the new class of lightgated ion channels, i.e., ion channels activated by light...

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Section: Resultsmentioning

confidence: 99%

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

Lórenz-Fonfrı́a

Bamann

Resler

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

106

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“…Independent of that, the azide group is attractive also because of the simple routes it enables to chemically modify the resulting MLs by using straightforward "click"-chemistry. 37,41,49 This will allow for structural and electrostatic investigations of samples with higher complexity such as immobilized proteins 38,66 or polymers, 67,68 and will open up a broad range of future applications of the 2D ATR IR technique such as ultrafast spectro-electrochemistry or multi-dimensional spectroscopy at biological interfaces.…”

Section: Resultsmentioning

confidence: 99%

2D attenuated total reflectance infrared spectroscopy reveals ultrafast vibrational dynamics of organic monolayers at metal-liquid interfaces

Kraack

Lotti

Hamm

2015

The Journal of Chemical Physics

101

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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. 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. C 2015 AIP Publishing LLC. [http://dx

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“…In this context, ultrafast vibrational dynamics in membrane proteins, polymer films or at electrochemical interfaces can be envisioned which have up to now only been studied routinely with comparatively low temporal resolution (approximately nanoseconds). 21,33 In the context of low absorbing samples, possible signal enhancement effects gain significant attraction. In particular Au nanoparticles and roughened surfaces have been widely applied to obtain increased sensitivity in stationary as well as ultrafast spectroscopy.…”

Section: Discussionmentioning

confidence: 99%

Surface-enhanced, multi-dimensional attenuated total reflectance spectroscopy

2015

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Ultrafast two-dimensional infrared spectroscopy (2D IR) spectroscopy is performed in attenuated total reflectance (ATR) geometry with the Kretschmann configuration in order to measure femtosecond to picosecond dynamics of selfassembled monolayers on gold-coated solid-liquid interfaces. In the monolayers low-absorbing (<200 M-1 cm-1) nitrile functional groups are used as local vibrational probes to monitor vibrational relaxation and spectral diffusion in dependence of different environments of the nitrile group. By comparing spectral diffusion dynamics of the vibrational probe in bulk solution and in the monolayer we find that the dynamics are slowed down by more than a factor of 20 upon immobilization of the sample. Moreover, spectral diffusion dynamics are affected by the local environment within the monolayers as evidenced by 2D ATR IR experiments on mixed monolayers with different aliphatic and aromatic coadsorbates. The results are interpreted in terms of absent excitation energy-transfer as well as solvation dynamics around the nitrile vibrational probe. Our results demonstrate that 2D ATR IR spectroscopy offers the possibility to obtain ultrafast dynamics from sub-monolayer coverages of even low-absorbing vibrational probes such as nitrile functional groups. ABSTRACTUltrafast two-dimensional infrared spectroscopy (2D IR) spectroscopy is performed in attenuated total reflectance (ATR) geometry with the Kretschmann configuration in order to measure femtosecond to picosecond dynamics of selfassembled monolayers on gold-coated solid-liquid interfaces. In the monolayers low-absorbing (<200 M -1 cm -1 ) nitrile functional groups are used as local vibrational probes to monitor vibrational relaxation and spectral diffusion in dependence of different environments of the nitrile group. By comparing spectral diffusion dynamics of the vibrational probe in bulk solution and in the monolayer we find that the dynamics are slowed down by more than a factor of 20 upon immobilization of the sample. Moreover, spectral diffusion dynamics are affected by the local environment within the monolayers as evidenced by 2D ATR IR experiments on mixed monolayers with different aliphatic and aromatic coadsorbates. The results are interpreted in terms of absent excitation energy-transfer as well as solvation dynamics around the nitrile vibrational probe. Our results demonstrate that 2D ATR IR spectroscopy offers the possibility to obtain ultrafast dynamics from sub-monolayer coverages of even low-absorbing vibrational probes such as nitrile functional groups.

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Time-resolved methods in biophysics. 10. Time-resolved FT-IR difference spectroscopy and the application to membrane proteins

Cited by 58 publications

References 90 publications

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

2D attenuated total reflectance infrared spectroscopy reveals ultrafast vibrational dynamics of organic monolayers at metal-liquid interfaces

Surface-enhanced, multi-dimensional attenuated total reflectance spectroscopy

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