Time-resolved methods in biophysics. 10. Time-resolved FT ...€¦ · lecture given at the X School of Pure and Applied Biophysics “Time-resolved spectroscopic methods in biophysics”

PERSPECTIVE www.rsc.org/pps | Photochemical & Photobiological Sciences

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

Ionela Radu,a Michael Schleeger,a Carsten Bolwienb and Joachim Heberle*a,c

Received 6th July 2009, Accepted 14th September 2009First published as an Advance Article on the web 6th October 2009DOI: 10.1039/b9pp00050j

The introduction of time-resolved Fourier transform infrared (FT-IR) spectroscopy to biochemistryopened the possibility of monitoring the catalytic mechanism of proteins along their reaction pathways.The infrared approach is very fruitful, particularly in the application to membrane proteins whereNMR and X-ray crystallography are challenged by the size and protein hydrophobicity, as well as bytheir limited time-resolution. Here, we summarize the principles and experimental realizations oftime-resolved FT-IR spectroscopy developed in our group and compare with aspects emerging fromother laboratories. Examples of applications to retinal proteins and energy transduction complexes arereviewed, which emphasize the impact of time-resolved FT-IR spectroscopy on the understanding ofprotein reactions on the level of single bonds.

1. Introduction

One of the fundamental and demanding quests in bioscienceconcerns the structure of proteins at atomic level. To performtheir biological function, the proteins may adopt several relatedconformations. Such changes are often induced by binding ofsubstrates to physical regions of the protein and/or specificinteractions with cofactors and bound ligands. All intra- andintermolecular effects sensitively modulate the infrared spectrum

aBielefeld University, Department of Chemistry, Biophysical Chemistry,33615, Bielefeld, GermanybFraunhofer Institute for Physical Measurement Techniques IPM, 79110,Freiburg, GermanycFree University, Department of Physics, Exp. Molecular Biophysics, 14195,Berlin, Germany† Edited by T. Gensch and C. Viappiani. This paper is derived from thelecture given at the X School of Pure and Applied Biophysics “Time-resolved spectroscopic methods in biophysics” (organized by the ItalianSociety of Pure and Applied Biophysics), held in Venice in January 2006.

Ionela Radu

Ionela Radu received her diplomain physics from the Universityof Iassy, Romania. In 2006, sheobtained her PhD in biophysicsat the University of Freiburg(Germany) under the supervi-sion of Friedrich Siebert. Afterthree years of post-doctoral re-search in biophysical chemistryat Bielefeld University, she wentwith Joachim Heberle to the FreeUniversity of Berlin (2009). Herresearch focuses on the structuraland functional investigations of

the photosensory proteins by time-resolved FT-IR and resonanceRaman spectroscopy.

Michael Schleeger

Michael Schleeger received hisdiploma in chemistry from theUniversity of Bonn (2004). Forhis PhD thesis he joined the groupof Joachim Heberle at BielefeldUniversity. The main focus ofhis work were functional stud-ies of the cytochrome c oxi-dase using steady-state and time-resolved FT-IR-spectroscopy. Hereceived a PhD degree in 2009and is currently a postdoc in thegroup of Joachim Heberle, wherehe studies a terminal bo3 oxidaseby surface enhanced infraredspectroscopy.

of biological macromolecules because the vibrational modes aredetermined by the protein structure.

Overcoming difficulties encountered by X-ray crystallographyand NMR spectroscopy, infrared spectroscopy set the stage fora multitude of structural and functional investigations of solubleand membrane proteins. During their activity only certain partsof the proteins undergo molecular changes and their identificationfrom the infrared spectrum is impeded by the strong backgroundabsorbance of the whole protein. This obstacle is elegantlyovercome by forming the IR difference between two stable reactionstates, an ingenious procedure which renders IR spectroscopyselective. The difference spectrum exhibits bands characteristiconly for the molecular groups that are modified during proteinactivity.

Since the resulting absorbance changes are usually very small, ofthe order of 10-4 and less, their accurate detection requires accurateand sensitive instrumentation. Consequently, the applications ofvibrational spectroscopy to the elucidation of protein structure

This journal is © The Royal Society of Chemistry and Owner Societies 2009 Photochem. Photobiol. Sci., 2009, 8, 1517–1528 | 1517

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and functionality make use of the advantages of the Fouriertransform technique.

Valuable insights into the mechanism of protein function alsoimply the resolution of the evolution of intermediates along thereaction pathway in a non-invasive manner. For this purpose, time-resolved FT-IR spectroscopy has proven to be superior to manyother spectroscopies. Evidently, the major challenge which governseach dynamic experiment is the attainment of high time resolutionand sensitivity. Single-wavelength techniques using pulsed pump–probe or continuous wave (cw) lasers can interrogate dynamicprocesses with ultrafast time scales up to femtoseconds. Althoughthe sensitivity of these methods is incontestable, they do notimpart the multiple advantages of FT-IR spectroscopy. Duringthe past three decades, two effective FT-IR techniques had agrowing impact on time-dependent investigations: the rapid-scanand the step-scan techniques.1,2 Both methods evidence notableadvantages and can be exploited in conjunction. Yet, step-scanachieves higher time resolution (microseconds to nanoseconds).

In the present review we will provide a basic introductionto illustrate the principles of time-resolved FT-IR spectroscopytogether with some instrumental considerations and realizations.Finally, we exemplify how the application to several complexmembrane proteins can provide remarkable advances to theelucidation of the reaction mechanisms of this important classof proteins.

2. FT-IR techniques

2.1 Rapid-scan FT-IR Spectroscopy

In the first resort, the benefits of the FT-IR over the dispersivetechnique are reflected in the device itself. This is schematicallyshown in Fig. 1. The infrared light emitted from a glowingbroadband source (Globar) passes an aperture which controlsthe amount of energy falling on the sample. The beam entersthe interferometer comprising a fixed and a moving mirror anda beamsplitter. A HeNe laser beam controls the position of the

Fig. 1 Experimental setup for laser-induced time-resolved experimentsusing an FT-IR spectrometer.

moving mirror relative to that of the fixed mirror, in terms oflHeNe/2. The interferometer is the centre of spectral decoding.Subsequently, the light passes through the sample and, finally, isfocused on a semiconducting detector (MCT, mercury cadmiumtelluride). The intensity recorded at the detector as a function ofthe moving mirror position defines the interferogram. The outputof the detector is fed into a personal computer which computesthe infrared spectrum by means of Fourier transform (spectraldecoding).

The central challenge of the kinetic FT-IR spectroscopy is theachievement of time resolution. An idea would be to record thespectra uninterruptedly while the temporal process is executed. Inrapid-scan FT-IR the moving mirror of the interferometer rapidlyscans back and forth. Its velocity should be selected so that theduration of a scan is at least an order of magnitude shorter than thehalf-life of the perturbed system. Fig. 2 shows the data acquisitionscheme in rapid-scan FT-IR spectroscopy.

The experiment starts by collecting a reference interferogramI t0 (x). After a certain delay the reaction is initiated for instance by alaser pulse and the time-resolved interferograms I ti (x) are recordedone after another. In order to improve the signal-to-noise ratiothe experiment can be repeated many times. After completing the

Carsten Bolwien

Carsten Bolwien studiedphysics at the RWTH Aachen(Germany) and received hisdiploma (1998) in semiconductorphysics with thesis work on aspectroelectrochemical setup forvibrational spectroscopy. He thenjoined Joachim Heberle’s groupat the Research Centre Julichworking on infrared and Ramanspectroscopy of retinal proteins.After completion of his PhD(Dusseldorf University, 2002) hejoined the Fraunhofer-Institute

for Physical Measurement Techniques in Freiburg in 2003 as ascientific associate. His current research spans a variety of projectspertaining to the commercialization of spectroscopic techniqueswith major focus on Raman spectroscopy in quality control.

Joachim Heberle

Joachim Heberle studied chem-istry at the Universities ofStuttgart and Wurzburg. Afterreceiving his diploma in phys-ical chemistry from Wurzburg(1988), he did his PhD in bio-physics at the Free University ofBerlin (1991). His postdoc workat the Hahn-Meitner-Institutelead him to research biomolecu-lar spectroscopy at the ResearchCentre Julich (1993). After hisHabilitation at Dusseldorf Uni-versity (1998), he became Full

Professor at Bielefeld University (2005). Recently, he joined thePhysics Department of the Free University of Berlin as Full Professor(2009). His group develops and applies vibrational spectroscopictechniques to resolve functional mechanisms of photosensory (mem-brane) proteins and bioenergetics.

1518 | Photochem. Photobiol. Sci., 2009, 8, 1517–1528 This journal is © The Royal Society of Chemistry and Owner Societies 2009

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Fig. 2 The principle of rapid-scan FT-IR technique: After collectingreference interferograms It0

(x), the experiment is triggered by a laser pulseand transient interferograms Iti (x) are recorded continuously during thetime course of the event. With this technique, the duration of a scan (blacktraces) should be at least an order of magnitude shorter than the durationof the dynamic event (red trace).

measurement, the interferograms are phase corrected, apodized,zero-filled, and Fourier-transformed3 to yield the reference St0 (n)and the time-resolved spectra Sti (n), respectively. Finally, theabsorption changes during the reaction cycle are calculated bythe relation DAti (n) = -log(Sti (n)/St0 (n)).

Evidently, in rapid-scan experiments the time resolution isdetermined by the speed of the moving mirror. The maximum time-resolution is limited by the coplanarity of the moving mirror to thefixed mirror because the inertia of the accelerated mass may lead totilting of the mirror and the interference condition will be violated.Moreover, the time-resolution depends on the optical resolutionbecause the better the spectral resolution, i.e. the longer the scanlength, the worse the time resolution will be at a given velocity ofthe movable mirror. In the latter dependence, the definition of thescan can be exploited in order to increase the time resolution for acertain velocity of the mirror. During the movement of the mirrorrelative to the beamsplitter (forward and backward) two symmet-ric interferograms are recorded. Further, each double-sided inter-ferogram can be split in two single-sided with a small common partaround the centerburst used for phase correction. In this manner,the time resolution is increased because the time to return the mov-ing mirror is effectively used for data recording. As an example oftypical parameters used in the spectroscopy of condensed phases,the time-dependent phenomena can be resolved to only 10 ms witha scanning speed of 10.1 cm s-1 and a spectral resolution of 4 cm-1.

2.2 Step-scan FT-IR spectroscopy

In dealing with the spatio-temporal properties of proteins, one hasto cope with molecular motions on time scales from femtosecondsto milliseconds to even hours. Therefore, a new strategy wasrequested in order to decouple the time dependence of the dynamicevent from the scan duration of the conventional rapid scanninginterferometer.

2.2.1 The method. Step-scan FT-IR spectroscopy benefitsfrom the multiple advantages of the continuous-scan FT-IR spec-troscopy while imposing no mechanical restrictions to achievinghigh time resolutions. During a step-scan experiment, the movablemirror moves step-wise from one sampling position to the next(Fig. 3, vertical lines). At each position the mirror is held fixedand the kinetic process is initiated, for example by a laser pulse. Atransient recorder digitizes the intensity changes over time. Once

Fig. 3 Schematic representation of step-scan FT-IR technique: The dataare collected along the time axis and after the complete execution of thetemporal process, they are rearranged into interferograms at certain timepoints. These data are then subjected to Fourier transformation to yieldsingle-channel spectra.

the process is completed, the mirror is stepped to the next positionand after a delay which enables the monitored system to recoverand the mirror to settle, the reaction is initiated anew. Signalaveraging is usually applied to increase the signal-to-noise ratio.The kinetic data are collected, averaged and, after covering theentire set of sampling positions, they are passed to the acquisitionprocessor (AQP). Here, the kinetics are rearranged into interfero-grams; before sample excitation I t0 (x), the reference interferogram,and at different time points after sample excitation I ti (x), the time-resolved interferograms. Further, the single-channel spectra, St0 (n)and Sti (n) are computed with the mathematical operations used forFT-IR data processing (vide supra). Finally, absorption differencespectra DAti (n) are calculated at times after triggering the reactionwith the single-channel spectra before the reaction was initiated(pre-trigger region).

In order to reduce the acquisition time one can take advantage ofundersampling.3 The total number of step points in the interfero-gram depends on the spectral resolution and bandwidth, as well ason the acquisition mode. Since for protein conformational changesthere is little spectral information above 1800 cm-1, an optical low-pass filter can be used that limits the free spectral change from1950 to 950 cm-1. Recording the spectra with 4.5 cm-1 resolution,the number of sampling points for a one-sided interferogram isreduced to 844. One hundred additional sampling points on theopposite side of the centerburst are collected that are used forphase correction.

Before starting the step-scan measurement, the centerburst andstart position of the mirror, as well as the total length of thestep-scan movement, are calculated in a short continuous-scanexperiment. The upper frequency limit determines the samplingpoint spacing as an integer of lHeNe/2. Then, the interferometercontrol electronics are set into step-scan mode and the measure-ment begins.

2.2.2 Step-scan device. Fig. 1 shows the experimental step-scan device employed by our group. It comprises a Bruker IFS 66vspectrometer with an MCT detector connected to a DC-coupledpreamplifier, a 200 KHz, 16 bit on-board transient recorder, andan external programmable digital pulse generator (Model 39,Wavetek, Ismaning, Germany). The optical bench is evacuateddown to 3 mbar and is mounted on a vibrationally isolated table.These are the prerequisites for an improved stability of the movingmirror in the step-scan mode.


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For pulsed sample excitation, a Q-switched Nd:YAG laser(GCR 12S, Spectra Physics, Darmstadt, Germany; frequency-double output at 532 nm, pulse duration of 8 ns, maximumexcitation energy of 100 mJ cm-2) is used. Quartz fibre bundlesare used to direct the laser emission to the sample. The repetitionrate of the excitation is adjusted depending on the decay time of thetransient species. The pulse-to-pulse intensity varies by as muchas 10% causing variations of the number of excited molecules withevery repeated event. To compensate for these variations, whichultimately lead to additional noise in the spectra, several laser shotsare co-added at each sampling position to ensure a reproducibleaverage value.

2.2.3 Step-scan timing. In a step-scan experiment, the mirrormovements, laser trigger, and data acquisition must be synchro-nized. A suitable method is to use an external pulse generator tomaster the exact sequence of the timing. The interferometer con-troller steps and stabilizes the mirror at a certain position and sendsa signal to the on-board analogue-to-digital converter (ADC) tostart data collection with the next signal from the external exper-iment trigger source. After an appropriate delay, this generates aTTL-pulse which is sent concomitantly to the laser flashlamps andthe digitizer. The flashlamps pump the laser medium (Nd3+) andafter 140 ms the Q-switch is activated and laser emission starts.Data recorded during the pre-triggering time are used as referencefor difference spectroscopy. After sample excitation, the digitizeris triggered by the logarithmic clock of the external programmabledigital pulse generator. In a typical experimental run, 1000 timepoints cover the time range from 7 ms to 200 ms.

2.2.4 Step-scan parameters. With the step-scan technique,the time resolution is not restricted by the velocity of theinterferometer mirror, but is limited by the response time of thedetection system as well as by the digitization rate of the transientrecorder. Thus, it is important that the electronic components ofthe step-scan device have comparable bandwidths and speeds toperform the experiment. It is desirable to fit the time resolutionto the dynamics of the studied event, because the noise willincrease with higher time resolution. Taking these observationsinto account, Chen and Palmer4 reported practical realizations ofstep-scan FT-IR measurements with 10 ns time resolution. Theycharacterized the decay kinetics of a transition metal complex afterphotodissociation of ligated CO. It is pointed out, however, thatthe difference absorbance observed in experiments with proteinsis at least one order of magnitude less intense.

When it comes to the digitization of the signal at the detector, thebit resolution of the transient recorder is an important parameter.It should be remembered that the interferogram is characterized bythe large intensity of the centerburst that exponentially decays. Atlarge pathlength difference, the signal is very small. Yet, these datapoints determine the spectral resolution of the experiment. If thesedata are digitized by an analogue-to-digital (ADC) converter withless bit resolution, the resulting spectrum after Fourier-transformwill be of low spectral resolution. In general, a low resolution ADCwill lead to additional noise (so called bit-noise) in the resultingspectrum. However, the higher the bit resolution, the lower thesampling frequency of the ADC, which is a critical parameterfor the time-resolution (vide supra). State-of-the-art digitizerscompromise between bit and time resolution. For example, theinternal 16 bit ADC (216 voltage steps) of the Bruker IFS 66

(which has since been replaced by a 24 bit ADC in the currentVertex spectrometers) limits the digitization frequency to 200 kHz.If faster spectral variations are to be recorded, an external digitizer(8 bit to 10 bit) with higher sampling frequency (100 MHz to200 MHz) is interfaced to the spectrometer.

The signal recorded at the detector comprises both the staticinterferogram (DC signal) and the time-dependent interferogram(AC signal). The latter is usually 104 times smaller than the formerand contains the desired spectral changes. Therefore, to realize thehigh sensitivity of the internal 16 bit ADC of the FT-IR bench,the static signal should be separated from the dynamic changes.This is done either by compensating the offset of the DC-coupledpreamplifier, or by switching to AC coupling. The latter choice isusually preferred by the manufacturers because it fully exploits theamplitude resolution of the ADC and, thus, reduces spectral noise.On the other hand, AC coupling, acting as a high-pass amplifier,eliminates the low frequencies and typically truncates the kineticsin the millisecond time domain.

A disadvantage associated with AC-coupled measurements isthe fact that the transient interferogram comprises both positiveand negative components, which prohibits the application ofclassical phase correction methods (Mertz) exploited in thefast Fourier-transform (FFT). However, a DC signal collectedsimultaneously at each mirror position is added to the AC signaland the resulting interferogram is conventionally phase corrected.

In the Siebert laboratory, the first group who applied step-scanFT-IR to biological systems,2 the detector signal is amplified by aDC-coupled preamplifier. They implemented a self-compensatingamplifier with an AC/DC switch that is controlled by thesoftware. By these means, the static interferogram (DC) andthe time-resolved interferogram (AC) are recorded in the sameexperiment, eliminating phase mismatch which might occur whentwo experiments are successively collected. The transient signal isfurther amplified to fit the dynamic range of the 8 bit, 200 MHzand 8 bit, 50 MHz boards connected to the host computer. Duringthe step-scan experiment the transient recorders concomitantlyregister the kinetic data at each mirror position.

Rammelsberg et al.5 carried out time-resolved experimentson bacteriorhodopsin using a commercial FT-IR spectrometerequipped with a home-built preamplifier with a DC output(200 kHz) and an AC output (10 MHz). The outputs are separatelydigitized by a 12 bit, 200 kHz transient recorder and by an 8 bit,200 MHz board, respectively.

3. Sampling techniques

3.1 Transmission cells

The most common and widespread infrared cell is the transmissioncuvette. Typically, a suspension containing approximately 100 mgprotein is dried onto an infrared transparent window with adiameter of 20 mm to obtain a homogeneous film. The film isrehydrated via the vapour phase and sealed by a counter windowof the same material. Finally, the cell is screwed in a brass holderplaced in the sample chamber.

Several FT-IR measurements on biological molecules per-formed with film samples anticipated the necessity of higher watercontent. This requirement is ensured by the so-called sandwichsample. The protein solution is dried on a bottom window, inside


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of a circular groove and approximately 30 ml buffer are overlaid. Acounter window squeezes the construct, forming a reservoir, withthe thickness between 3 and 7 mm.

3.2 ATR cells

The overlapping of the amide I band and the scissoring bandof water often results in insufficient infrared transmission of thesandwich cells at around 1650 cm-1. Therefore, a trade-off ismade between high protein content and sufficient hydration of thesample. A significant advance in the understanding of membraneprotein structure and functionality demands investigations of thesample in aqueous environments. With attenuated total reflection(ATR) FT-IR spectroscopy, the protein reconstituted in lipids isdeposited on the surface of a highly refractive crystal material(diamond, Ge, Si, ZnSe or ZnS). The infrared beam is focused intothe crystal at an angle above the critical angle for total internalreflection and after several total reflections the beam reaches theend. In this manner a standing wave emerges within the crystal,whereas at the interface with the rarer medium a non-radiativeevanescent field evolves which decays exponentially outside theinternal reflection element (IRE). The decay of the intensity ofthe wave is exponential. The depth to which the evanescent wavespenetrate the sample is characterized by the distance at which theintensity has decayed to 1/e:

dn

n np [ ]=

−l

q/

sin ( / ) /1

22 1

2 1 22p

At a wavelength of 6.06 mm (=1650 cm-1), using an angle ofincidence q of 45◦, ZnSe (n1) as IRE and water (n2) as sample,a penetration depth of 1.1 mm is calculated from the aboveequation. If the physical dimensions of the IRE are chosen suchthat 6 internal reflections are used, an effective pathlength of6.6 mm results. This is comparable to the pathlength of commonlyused transmission cells with the prominent advantage that thepathlength is always constant in ATR experiments.

A gentle flow of the aqueous buffer over the sample facil-itates the study of infrared spectra under a wide variety ofsolution conditions.6 The micro-ATR unit used in our laboratory(RESULTEC analytic equipment, Illekirchberg, Germany) em-ploys ZnSe as focusing element and is capped with a thin diamonddisc (Fig. 4). ZnS or diamond are preferred IRE materials whenlight-induced processes are studied by ATR spectroscopy because

Fig. 4 Schematic of a micro-ATR cell. The internal reflection elementis made of ZnSe and is capped with a diamond disc. The diameter ofactive area is 4.3 mm and 5 reflections are used for probing the sample.Advantages of the ATR cell are the exchangeability of the crystal materialand low consumption of the sample.

these materials are transparent to visible light whereas Si or Ge aresemiconductor materials that exhibit transmission changes uponlight excitation.7 A disadvantage of the ATR technique is the lowersignal-to-noise ratio of the FT-IR difference spectra comparedto those recorded with the transmission technique. This is thechief consequence of the multiple reflections in the ATR crystal.However, the open access to the sample and the precise controlover the sample conditions exceeds this optical disadvantage.

3.3 Surface-enhanced infrared absorption (SEIRA)

Most time-resolved spectroscopic techniques rely on the factthat the molecules can be synchronized. Light is an exquisitetrigger to synchronize many protein molecules due to the wave-like character. However, few proteins respond to light but manyare stimulated by electrons, membrane potential or binding ofother proteins or external ligands. Surface-enhanced spectroscopy,the logical advance in ATR methodology, can be used to probeproteins on the level of a single monolayer.8,9 In surface-enhancedinfrared absorption (SEIRA) spectroscopy, the infrared activevibrations of a sample deposited on a thin gold surface areenhanced by the strong electromagnetic field induced on the metalparticles by the incident light (Fig. 5). The enhancement decaysrapidly with the distance from the surface (decay length in theorder of 10 nm). As a great advantage, the near-field effect ofthe surface enhancement eliminates contribution from the bulkaqueous phase to the IR spectrum and selectively detects signalsfrom the adsorbed monolayer of a membrane or a protein evenwhen immersed in water. An important role for the SEIRA effect isplayed by the roughness of the gold layer because the enhancementfactor depends on the size, shape and density of the gold particles.Typically, the enhancement factor in SEIRA spectroscopy rangesbetween 10 and 100, yet several orders of magnitude smaller thanthat observed with surface-enhanced Raman scattering.10

Fig. 5 Schematic representation of a monolayer of membrane proteinadsorbed to a chemically modified gold film and probed by the infraredbeam. The SEIRA effect decays rapidly with the distance from thesubstrate.

In order to preserve the structure and functionality ofthe adsorbed biomolecules, the gold film must be chemicallymodified.11 Molecules that contain thiol groups (as dithio-bis(succinimidylpropionate)) spontaneously form a stable self-assembly monolayer (SAM) with the bare gold surface throughcovalent linkage to the sulfur group. The detergent-solubilizedmembrane protein (2 to 5 mM) is adhered to the chemicallymodified metal film via an organic group connected to a His


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tag. In the last step, the oriented attached protein is reconstitutedin artificial lipids. Proper orientation of the protein relative tothe reactant is achieved by this approach which is crucial for theanalysis of the function of the protein. This is superior to the ATRtechnique where an arbitrarily oriented multilayer of proteins isformed on the surface of the internal reflection element.

All surface modification steps to tether proteins to the solidsurface are monitored in situ by SEIRA spectroscopy. Based onthe controlled assembly of such a biomimetic system, a novelconcept is introduced which combines functional IR spectroscopyand electrochemistry. As redox-active proteins when tethered tothe solid surface are readily activated by electron injection fromthe electrode surface, the difference spectroscopic approach canbe applied which has been proven to be extremely useful instudying the functionality of light-driven systems (vide infra).We succeeded in monitoring redox-induced infrared changes ofa cytochrome c monolayer.11,12 Infrared bands of the order of 10-4

were detected and assigned to structural changes in the wholeprotein that take place after electron transfer. Implementation ofthe SEIRA technique is advantageous not only with respect tosensitivity but also for providing fast electrochemical response.Pulsed electron injection from the electrode enables kinetic IRstudies of monolayers of redox-active proteins.

4. Applications

Membrane proteins reside in cell membranes and participate intransport, recognition and transduction processes that are vitalto living systems. Understanding of the physiological mechanismof a membrane protein emerges from structural details about keyintermediate steps of enzymatic action. Progress has been impededby difficulties in nearly every step, from heterologous expressionand isolation to crystallization difficulties that arise from the con-straints posed by their amphiphilic character.13 The advent of novelcrystallization methods targeted specifically to membrane proteinshas spurred success in their crystallography.14–16 Yet, the crystal-lographic models cannot trace many of the structural changeswhich occur during the protein function, such as changes in theprotonation state of specific residues or the formation of hydrogenbonds. Here, FT-IR difference spectroscopy can add very usefulinformation about their functionality, in particular because it isable to resolve the kinetics between the structural intermediates.

4.1 Retinal proteins

Numerous time-resolved infrared studies were focused on thesmall integral membrane protein bacteriorhodopsin (BR) which isthe best-understood member of the seven-helical transmembraneprotein family (Fig. 6a). When illuminated, this small (26 kDa) butvery robust protein pumps protons out of the cell to establish theproton gradient across the membrane that drives ATP synthesis.Besides bacteriorhodopsin, the family of archaeal rhodopsinsincludes halorhodopsin (HR), which harvests light to pumpchloride ions from the extracellular medium into cytoplasm, andtwo sensory rhodopsins SRI and SRII that are the primaryreceptors of phototaxis.

Upon light excitation, the archaeal rhodopsins undergo a seriesof light-induced cyclic reactions, the primary event being theisomerization of the all-trans retinal chromophore to the 13-cis

Fig. 6 (a) Structural view of retinal proteins. The all-trans retinal(pink) is harboured by seven-transmembrane helices (ribbons). Upon lightexcitation the retinal isomerizes to 13-cis which initiates a set of proteinconformation changes. (b) The photocycle of bacteriorhodopsin.

configuration. Photo-excitation of BR initiates a series of statetransitions with intermediates referred as J, K, L, M, N and O,which were well characterized by visible spectroscopy (Fig. 6b).17,18

The photocycle of BR is very intricate. There is generalconsensus on the main photocycle intermediates but the transitionfrom one state to the other is still controversial. This may be dueto the fact that the intermediate states are not well defined becausethe commonly applied strategy to trap intermediate states may failas in the late states of the photocycle or may lead to mixtures ofintermediate states.

The first applications of step-scan FT-IR spectroscopy investi-gated the KL-to-L transition at room temperature, demonstratingthat the retinal is twisted in the Schiff base region as indicatedby an intense HOOP (hydrogen-out-of-plane) band at 983 cm-1,present in both KL and L spectra.19,20 This band is lacking in the Lspectrum recorded at cryogenic temperatures indicating differentconfigurations of the retinal Schiff base at room and cryogenictemperatures. Hage et al.21 extended the interpretation in terms ofa process during the KL to L transition where structural changesof the b-ionone ring end of the chromophore and of D115 occurfaster than changes at the Schiff base region of the chromophore.The presence of two K species in the photocycle of BR at roomtemperature was corroborated by two other independent time-resolved FT-IR studies.22,23

A clear-cut separation of the subsequent L, M, N and O in-termediates was achieved by time-resolved step-scan ATR/FT-IRspectroscopy by varying temperature and pH values (Fig. 7).24

On the basis of the IR difference spectra in the carbonyl region(Fig. 8a), the assignment of the bands in the various intermediatestates leads to a thorough understanding of proton translocationacross BR. The pioneering work of Engelhard et al.25 revealedthat besides the retinal Schiff base, aspartic acids play a dominantrole in proton transfer within BR. IR spectroscopy is particularlyuseful when studying the role of acidic amino acids because theC=O stretching frequency is well-isolated from other vibrationsof the protein.

As the present review puts the focus on the application of time-resolved FT-IR spectroscopy, we refrain from explicitly discussingthe extensive work on the tedious assignment of the differencebands but refer to reviews on the topic.26–29 The negative bandfeature in the L–BR difference spectrum arises from the C=Ostretching vibration of two aspartic acids. D96 and D115 are both


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Fig. 7 Time-resolved infrared difference spectra of the photointermedi-ates of bacteriorhodopsin. The experiments were performed with an ATRcell under the following conditions: L–BR (10 ms, pH 6.6, 20 ◦C), M–BR(300 to 400 ms, pH 8.4, 20 ◦C), N–BR (80 to 100 ms, pH 8.4, 20 ◦C), andO–BR (5 to 10 ms, pH 4.0, 40 ◦C).

Fig. 8 (a) Expansion of the carbonyl region of the time-resolvedATR/FT-IR difference spectra of bacteriorhodopsin (see Fig. 7 for ex-perimental details). The band assignment (dashed vertical lines) illustratesthe transient acid/base reactions of particular amino acids along with theenvironmental changes in the vicinity of the respective carboxylic acid.Negative bands are due to the deprotonation of an aspartic acid whereasthe protonation is indicated by a positive band. (b) Sequence of protontransfer events in bacteriorhodopsin (steps 1 to 6). The dark green arrowsdenote proton transfer reactions derived from FTIR spectroscopic data inpanel (A).

protonated in ground-state BR and their terminal carboxylic acidsundergo a change in hydrogen bonding but in opposite direction.The M state which forms in about 50 ms, is characterized by protontransfer from the retinal Schiff base to D85 (step 1 in Fig. 8b).A strong band at 1761 cm-1 characterizes this proton transferevent which is due to the C=O stretch of the sidechain of D85(yellow spectrum in Fig. 8A). The proton release reaction of anexcess proton from the cytoplasm to the extracellular side of

the membrane was surveyed by nanosecond time-resolved FT-IRspectroscopy.5,30–32 The authors proposed a proton release pathwayin the shape of a transient hydrogen-bonded network comprisingseveral polar amino acids and water molecules (step 2 in Fig. 8b).Evidence is growing that such a dynamic water-assisted local areanetwork (WLAN)33,34 may operate also in other ion pumps.35

In line with these observations, Maeda’s group demonstrated bymeans of step-scan FT-IR spectroscopy that, during the L-to-Mtransition, internal water molecules accommodated in a cavitybetween D96 and the Schiff base are perturbed by the structuralchanges of the protein.36,37 Focusing on the structural changesdue to the deprotonation of the proton release group upon theformation of the M intermediate, the authors suggest that theinteraction between the protonated D85 and D212 is stabilizedby the disconnection between helix G, R82 and the retinal Schiffbase. In a very recent work, Lorenz-Fonfrıa and Kandori provideevidence that the proton release group deprotonates upon thetransition between two M substrates and, as a result, the protonaffinity of D85 increases.38

It is evident from Fig. 8a that the C=O stretching vibration ofD85 shifts down by about 7 cm-1 when the later intermediates Nand O are formed. In the N state, whose maximum concentrationis reached at about 3 ms after photoexcitation (step 3), a negativeband is discernible at 1742 cm-1 (green spectrum in Fig. 8a).This band was assigned to the C=O stretch of D96, the internalproton donor to the Schiff base. Reprotonation of D96 fromthe cytoplasmic surface takes place in the millisecond timescale(step 4) and is strongly pH-dependent.39 Protons from the cy-toplasm are attracted by negatively charged residues along thesurface and funneled to the entrance of the proton uptake pathwaywhere D38 is located.40 In the final stages of the photoreaction,D85 deprotonates and the released proton is transferred to thenearby residue D212 (step 5 in Fig. 8b) for which the C=Ostretch is assigned to the positive band at 1713 cm-1 (blue tracein Fig. 8a). Under physiological conditions the residence time forthe proton at D212 is short and the proton is readily transferredto the initial proton release complex (step 6). This reactioncompletes the proton transfer reaction across BR. In conclusion,the electro-mechanical driving force for proton translocation isprovided by photon absorption of the retinal and the consequentstructural changes of the cofactor and the apo-protein. All of thesereactions are monitored by time-resolved FT-IR spectroscopy byinterpreting the difference spectra shown in Fig. 7.

The interpretation of the results of IR spectroscopy with respectto details of the reaction mechanism relies also on structuraldata. The elucidation of the molecular structure of BR atnearly atomic resolution,41 however, raises the pertinent questionof whether the catalytic activity of the protein(s) is preservedin the microcrystals. Initially, the crystals were investigated bysteady-state IR spectroscopy and the difference spectra indicatedunperturbed intermediate states.42 In a further step, we succeededin studying single BR crystals with a diameter of 50 mm by time-resolved step-scan FT-IR microspectroscopy.43 It was found thatsimilar structural changes occur both in the crystal and in thepurple membrane though with slightly different kinetics.

Besides the extensive work on bacteriorhodopsin, time-resolvedFT-IR spectroscopy contributed to the understanding of othermicrobial rhodopsins. In the following, we will present a shortintroduction into these studies.


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The molecular mechanism and the bioenergetic function of theother light–energy converter halorhodopsin (HR) are, in spite ofdifferent investigations, less well understood than those of BR.44–47

During the catalytic cycle of HR, anions like chloride, bromideor nitrate are imported into the cell, preserving in this way theosmotic balance which is required for cell growth. The crystalstructure of HR revealed the binding site of chloride at the positionof Asp85 in BR and provided several clues about the mechanismof chloride pumping.48 Time-resolved step-scan FT-IR differencespectroscopy on HR was initiated by the Braiman group.49–51 Atransition between two L species was identified which only involvesdistortions of the apo-protein whereas the chromophore bindingpocket does not change.52 The authors proposed that the L1-to-L2

conversion represents the switch of the anion accessibility fromextracellular to the cytoplasmic site. However, the resolution ofthe halide transport into single sequential steps of ion transferanalogous to the emerging mechanism of proton pumping in BR,has not been achieved yet.

Whereas BR and HR function as ion pumps, two photosensors(sensory rhodopsin I and II) serve the bacteria in searchingfor favourable light conditions at low oxygen concentration.SRII recognizes the repellent blue light and enables the host-cell to prevent the photo-oxidative stress in case of high oxygenconcentrations in the bright sunlight. The structure of SRII fromNatronobacterium pharaonis is very similar to that of BR.53,54

Differences are found in the retinal binding pocket and in thecytoplasmic channel where potentially charged amino acids ofBR are replaced by neutral amino acids. Time-resolved step scanFT-IR experiments on SRII highlighted the formation of M-like state when the Schiff base deprotonates and the counterionAsp75 becomes protonated.55 The spectra proved the existenceof two M intermediates and during the M1-to-M2 transition onlythe apo-protein underwent structural changes. This conversionwas proposed to be the signal to activate the cognate transducerprotein.

Originally thought to be confined to Archaea, microbialrhodopsins have now been detected in all three kingdoms of life.56,57

Among the microbial rhodopsins, step-scan spectroscopy has beenapplied to study the proton pumping steps in proteorhodopsin(PR) from marine bacteria.58 It was demonstrated by photocurrentmeasurements that PR can operate as an outwardly and aninwardly directed light-driven proton pump. Yet, the most distinctdifference between these two operational modes is related to theprotonation of D97 (corresponding to D85 in BR). The otherconformational changes appear to be largely pH-independent. Theinternal proton donor D96 of BR is replaced by E108 in PR. Thelonger amino acid side chain does not impair the functional roleof the latter but the lower frequency of the C=O stretch of E108(1728 cm-1) as compared to D96 of BR (1742 cm-1) is indicativefor a stronger hydrogen-bonded environment.

4.2 Energy transduction complexes

Step-scan spectroscopy requires the molecular reaction underinvestigation to be repeated about 100 000 times. Proteins witha reaction cycle are, therefore, ideal for the application of thestep-scan technique, like the microbial rhodopsins. However, thereaction cycle need not to be longer then a few seconds. Otherwise,the recording time will be very long and the limited stability of

the setup will finally prevent a successful experiment. Yet, theenergy transduction complexes of photosynthetic and respiratorychains are challenging from an IR spectroscopic view because oftheir sheer size which is at least 10 times larger then the retinalproteins. Despite this obstacle, spectrometric fine-tuning hand-in-hand with progress in sample preparation provided the cluefor step-scan experiments on the very relevant class of energytransduction membrane proteins.

4.2.1 Photosynthetic reaction center (RC). In photosynthe-sis, light excitation of the special pair of bacteriochlorophyllsinitiates charge separation over the membrane in less than 200 ps.Electron transfer occurs mainly from the primary electron donorP through a bacteriochlorophyll monomer and a bacteriopheo-phytin to two ubiquinone molecules QA and QB which are coupledby hydrogen bonds to histidine residues ligated to a non-heme Fe2+.The crystallographic structure of the reaction center pinpointedthat the secondary quinone QB is localized in a cluster of polarand acidic residues which acts as a proton transfer center.59 QB isloosely bound to the active site and after transfer of two electronsand two protons the reduced quinol diffuses from the RC into thecell membrane.

In a pioneering work, Hienerwadel et al. applied time-resolvedIR spectroscopy to investigate the photooxidation of the primaryelectron donor as well as the kinetics of the QA

-QB → QAQB-

electron transfer with a time resolution of 500 ns.60 Three yearslater, the same group analyzed the spectral domain between 1780and 1695 cm-1 for the native RC and D212N mutant and proposedthat D212 in the binding pocket of QB undergoes proton uptakeduring the QA

-QB → QAQB- transition.61

The first step-scan FT-IR experiments of the 100 kDa RCfrom the Rhodobacter sphaeroides were performed by the Bretongroup.62 They monitored the photooxidation process of theprimary electron donor P and its photoconversion to the tripletstate with a time resolution of 10 ms. Later, they applied thistechnique to investigate the primary electron donor of the reactioncenter from the green sulfur bacterium Chlorobium tepidum.63 Thetransient spectra showed the formation and decay of the tripletstate and a band could be unambiguously attributed to 9-ketoC=O stretching vibration of P. The position of the band shiftsupon the formation of the triplet state or photooxidation reflectingdifferent interactions with the surrounding environment.

The Gerwert group used the step-scan technique with a timeresolution of 30 ns to explore the mechanism of electron transferduring the QA

-QB → QAQB- transition.64 Surprisingly, the data in-

dicate that the QB- formation precedes QA

- oxidation. The authorssuggest that the ultrafast reduction of QA induces protonation ofindividual His and Asp residues located along the proton-uptakepathway within 12 ms. In contrast to the crystallographic results,the infrared data indicate that the reduction of QB is caused bythe enhanced positive charge in its proximity rather than by theconformational movement from the distal to proximal position.53

QB- appears with a time constant of 150 ms and the authors

propose that the active electron originates from the non-hemeFe2+ via histidine ligands. Finally, the ferric ion is reduced by theprimary quinone at 1.1 ms. A subsequent time-resolved infraredstudy on the mutant D210N showed that the proton movement inthe proton uptake pathway influences the reoxidation of QA in theQA

-QB → QAQB- transition but not the reduction of QB.65


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4.2.2 Cytochrome c oxidase (CcO). Cytochrome c oxidaseis the terminal electron acceptor of the respiratory chain andoperates as redox-linked proton pump across the inner mitochon-drial membrane of eukaryotes or the cytoplasmic membrane ofArchaea and Eubacteria, respectively. The large protein complex(204 kDa of CcO from bovine heart) catalyzes the overall reductionof dioxygen to two molecules of water in an intricately coupledreaction mechanism. Time-resolved UV/Vis measurements usingthe flow-flash photolysis technique66 showed that the initialelectrons are furnished by cytochrome c on the periplasmic side.Their entry site to the enzyme is a dinuclear copper center CuA

located in subunit II. Electrons continue to flow via a low-spinheme a into the heme a3/CuB binuclear center, where O2 is reducedto water in a chemical process accompanied by proton uptake fromthe cytoplasm (Fig. 9a).

Fig. 9 (a) The active site of cytochrome c oxidase from R. sphaeroides withthe cofactors involved in the electron transfer and the putative pathways ofthe proton transport (based on the crystal structure at 2.8 A resolution ofcytochrome c oxidase71). (b) R2CO–E difference spectrum recorded 30 msafter photolysis in rapid-scan mode with an optical resolution of 4.5 cm-1.

Whereas the identification of a pathway for proton release is stillobscure, mutagenesis experiments in combination with a varietyof biophysical techniques suggested two proton uptake pathwayswithin the membrane-ingrown portion of the protein.67,68 Thesepathways were also identified in the X-ray structures of bovineCcO69 and of several bacterial oxidases.70,71 The K pathway ofthese organisms links the suface of subunits II and I through astring of hydrophilic amino acids and the hydroxyethylfarnesylmoiety of heme a3 ending up with Y288 of subunit I (Rhodobactersphaeroides numbering). This tyrosine residue seems to play acritical role in catalyzing the splitting of the O=O bond afterit binds to reduced heme a3 at the active site.72 The D pathwayextends along a row of polar residues and water molecules tothe highly conserved glutamate E286 which functions as a protonshuttle during the catalytic cycle.

The concerted proton and electron traffic results in alterationsof amino acid side chain orientations, hydrogen bond lengths,electron density, and protonation states. In the process of reductionof molecular oxygen to water the physiological intermediate states

move through fully oxidized O, the partially reduced termed E, R2

and the oxoferryl intermediates Pm and F.Transmission rapid-scan spectroscopy in combination with

ATR techniques on stable reaction intermedeates of CcO ex-amined the protonation states of the central residues E286 andY288.73 As a consequence of pKa shifts and location in ahydrophobic milieu, the glutamic acid E286 is deprotonated inthe E and Pm intermediate and protonated in R2, F and O state.The E–R2 difference FTIR spectrum recorded with the rapid-scan technique in the presence of carbon monoxide (CO) exhibitslarge band distortions in amide I domain (Fig. 9b). These spectralaspects give evidence that proton translocation accompanying thetransition is induced by conformational changes of the proteinbackbone. Additionally, the steady-state spectra acquired withATR technique clearly show that Y288 is protonated in the fullyoxidized enzyme, is a neutral radical in state Pm, and forms areduced anion in state F. Finally, the authors proposed a plausiblereaction scheme of catalysis by CcO, thus demonstrating theunique capability of IR difference spectroscopy to gain informa-tion about the well-orchestrated interplay of particular residuesand the chronological events governing the protein machinery.

Subtle structural changes assisting the catalytic turnover can beprovided by a deeper and specific intrusion into the active site.This task can be elegantly accomplished by FT-IR spectroscopyof photolysed CO-bound states R2CO and R4CO, respectively.Here, R4 represents the fully reduced protein which is of non-physiological character. In the absence of O2, carbon monoxidebinds to CcO at reduced heme a3 and Fe-CO bound can betransiently photo-dissociated by an intense laser pulse. When CO isphotolysed from R4CO, the released CO binds to the nearby CuB

+,equilibrates with CO in solution, and finally rebinds to the fullyreduced heme a3. Interestingly, at cryogenic temperatures, after COphotodissociation only the transfer of CO from heme a3 to CuB isobserved.74,75 Dyer et al. took advantage of single-wavelength time-resolved infrared spectroscopy to present first evidence on CObinding to CuB

+ at room temperature.76 They observed the decayof CuB

+–CO species with a half-life of 1.5 ms and the subsequentrebinding of CO to the heme a3. This result suggests that CuB

might have an additional function in the catalytic reaction ofthe enzyme as ligand shuttle to heme a3. Several years later, thesame group proposed that the picosecond binding of CO to CuB

induces the transfer of an endogenous ligand L from CuB+ to heme

a3.77 As a result, a transient five-coordinate high-spin heme a3 isformed with the ligand at the position of the proximal histidine.The breakage of the Fe–L bond (0.7 ms) is the rate-determiningstep for the return of CO to the heme a3. The authors suggest thatthe property of CuB as a ligand shuttle may also manifest duringthe binding of other small molecules (as O2) at the active siteand that the ligand-exchange processes could control and couplethe electro-transfer and proton-translocation steps of the enzyme.Since the overall process takes about 20 ms, its investigationcould be addressed by step-scan IR spectroscopy.78 The differencespectra of the photodissociation of the CO-complex of bovine CcOshow a negative band at 1962 cm-1 characteristic for CO depletedfrom heme a3 and a positive band at 2062 cm-1 which proves thatthe ligand binds to CuB (Fig. 10). It is noteworthy that the datastress that the decay of the latter band is temporally connectedto absorption changes attributed to E286 in the D pathway. Thisapproach is of particular relevance because it demonstrates the


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Fig. 10 Step-scan FT-IR difference spectra of CO bound to fully-reducedbovine cytochrome c oxidase (300 mm CcO, 40 mM ascorbate, 100 mMphosphate buffer, pH 8.6, -20 ◦C).

applicability of microsecond FT-IR spectroscopy to larger andmuch more demanding protein complexes such as cytochromec oxidase. Moreover, the study represented the first step-scanFT-IR difference spectroscopy in the congested region below1800 cm-1 where the mechanistically relevant stretching modesof the peptide bond and amino acid side chains absorb. Step-scan FT-IR measurements on the ba3-CcO from the eubacteriumThermus thermophilus reveal that the ring A propionate of hemea3 undergoes significant changes upon transient formation of theCuB

+–CO complex.79,80 Furthermore, the transient binding of COto CuB seems also to be linked to the deprotonation of D372(residue numbering from Thermus thermophilus) which belongs tothe Q-proton pathway.80,81 The infrared data suggest a hydrogen-bonding connectivity among the ring A propionate of heme a3,D372 and a water molecule that is a part of the Q-proton pathway.The authors postulate that this proton connected group, which isstructurally conserved in the heme-copper oxidase family, forms anoutput proton channel. A subsequent step-scan FT-IR approachprovides evidence that 15–20% of photodissociated CO does notbind to CuB but is funneled in a docking site near ring A of hemea3 propionate where it remains for 35 ms. The trapped CO causeslocal protein fluctuations, which in response seem to hamper therecombination of CO to the heme a3 for few miliseconds.82 As anexception from the observed behaviour of CO photodissociationfrom R4CO, step-scan FT-IR data on cytochrome cbb3 oxidasefrom the proteobacterium Pseudomonas stutzeri indicated thatin this enzyme the decay of the transient CuB

+–CO species isconcurrent with the CO rebinding to the heme b3.83

The photodissociation of CO from the half-reduced state R2COis more complex and involves electron backward transfer from thereduced heme a3 to the oxidized heme a via two separate reactionswith different rate constants.73,84–86 The slower reaction takes placein approximately 150 ms and is coupled with proton-transfer steps.The rebinding of CO to CcO from Paracoccus denitrificans wasprobed by time-resolved rapid-scan FT-IR spectroscopy.87 Bothfor fully-reduced and half-reduced enzyme this process is biphasicat 268 K but for the latter is much slower. Furthermore, the infrareddata bring evidence that E278 (E286 in Rhodobacter sphaeroides)undergoes changes which are coupled to the electron transferbetween heme a3 and heme a.

Recently, the protonation changes of E278 upon F-to-O tran-sition were directly detected by rapid-scan technique with a timeresolution of 46 ms.88 Here, the authors use a slow mutant enzymefrom Paracoccus denitrificans and investigate the reaction of R4COwith oxygen initiated by a laser flash.

5. Perspectives

Proteins are dynamic entities and their mechanistic descriptionalso requires abilities to monitor how amino acids fold into three-dimensional structures. During the folding process the proteinsundergo changes in the polypeptide backbone conformation andhydrogen-bonding, two parameters which can be addressed byinfrared spectroscopy. Indeed, laser-induced temperature-jumpexperiments in combination with time-resolved infrared spec-troscopy have characterized dynamics and specificity of helix–coiltransitions in small peptide fragments in nanosecond regime.89,90

Emerging from multiple quantum NMR, the benefits of phase-controlled vibrational photon echoes techniques providing two-dimensional IR spectra can be elegantly used to observe the timeevolution of structural changes and the coupling between differentparts of macromolecules.91

The recent development of femtosecond-stimulated Ramanspectroscopy, another alternative to the pump–probe technique,appears to offer a particular promise for the study of primaryevents in the visual pigment rhodopsin. This approach investigatedtransient vibrational frequencies of structures along the pathwayfrom photorhodopsin to bathorhodopsin revealing spatial rear-rangements in the retinal backbone that activate the receptor.92

Notes and references

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Time-resolved methods in biophysics. 10. Time-resolved FT ...€¦ · lecture given at the X School of Pure and Applied Biophysics “Time-resolved spectroscopic methods in biophysics”