Vibrational spectroscopy in life science-Friedrich Siebert and Peter Hildebrandt

Friedrich Siebert and Peter Hildebrandt Vibrational Spectroscopy in Life Science Vibrational Spectroscopy in Life Science Friedrich Siebert and Peter Hildebrandt Copyright 2008 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim ISBN: 978-3-527-40506-0 Related Titles Heimburg, T Thermal Biophysics of Membranes approx 330 pages 2007 Hardcover ISBN: 978-3-527-40471-1 Wartewig, S IR and Raman Spectroscopy Fundamental Processing 191 pages with 177 figures and tables 2003 Hardcover ISBN: 978-3-527-30245-1 Chalmers, J., Griffiths, P (Eds.) Handbook of Vibrational Spectroscopy 3880 pages in volumes 2002 Hardcover ISBN: 978-0-471-98847-2 Friedrich Siebert and Peter Hildebrandt Vibrational Spectroscopy in Life Science The Authors Friedrich Siebert Institut fuăr Molekulare Medizin und Zellforschung Albert-Ludwigs-Universitaăt Freiburg e-mail: frisi@biophysik.uni-freiburg.de Peter Hildebrandt Institut fuăr Chemie Technische Universitaăt Berlin e-mail: hildebrandt@chem.tu-berlin.de Cover Picture Vibrational spectroscopy, i.e Raman (bottom) and infrared (top) spectroscopy, has considerably contributed to the understanding of the function of proteins, here of the light-driven proton pump bacteriorhodopsin All books published by Wiley-VCH are carefully produced Nevertheless, authors, editors, and publisher not warrant the information contained in these books, including this book, to be free of errors Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Bibliographic information published by the Deutsche Nationalbibliothek Die Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at hhttp://dnb.d-nb.dei 2008 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim All rights reserved (including those of translation into other languages) No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers Registered names, trademarks, etc used in this book, even when not specifically marked as such, are not to be considered unprotected by law Printed in the Federal Republic of Germany Printed on acid-free paper Typesetting Asco Typesetter, North Point, Hong Kong Printing betz-druck GmbH, Darmstadt Binding Litges & Dopf GmbH, Heppenheim Wiley Bicentennial Logo Richard J Pacifico ISBN: 978-3-527-40506-0 V Contents Preface IX 1.1 1.2 1.3 1.4 1.5 Introduction Aims of Vibrational Spectroscopy in Life Sciences Vibrational Spectroscopy – An Atomic-scale Analytical Tool Biological Systems Scope of the Book Further Reading References 10 2.1 2.1.1 2.1.2 2.1.3 2.1.4 2.2 2.2.1 2.2.2 2.2.3 2.3 2.3.1 2.3.2 Theory of Infrared Absorption and Raman Spectroscopy Molecular Vibrations 12 Normal Modes 15 Internal Coordinates 18 The FG-Matrix 19 Quantum Chemical Calculations of the FG-Matrix 23 Intensities of Vibrational Bands 25 Infrared Absorption 25 Raman Scattering 28 Resonance Raman Effect 32 Surface Enhanced Vibrational Spectroscopy 38 Surface Enhanced Raman Effect 39 Surface Enhanced Infrared Absorption 43 References 60 3.1 3.1.1 3.1.1.1 3.1.1.2 3.1.2 3.1.3 3.1.4 Instrumentation 63 Infrared Spectroscopy 63 Fourier Transform Spectroscopy 64 Interferometer 67 Infrared Detectors 69 Advantages of Fourier Transform Infrared Spectroscopy 70 Optical Devices: Mirrors or Lenses? 71 Instrumentation for Time-resolved Infrared Studies 72 Vibrational Spectroscopy in Life Science Friedrich Siebert and Peter Hildebrandt Copyright 2008 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim ISBN: 978-3-527-40506-0 11 VI Contents 3.1.4.1 Time-resolved Rapid-scan Fourier Transform Infrared Spectroscopy 3.1.4.2 Time-resolved Studies Using Tunable Monochromatic Infrared Sources 74 3.1.4.3 Time-resolved Fourier Transform Infrared Spectroscopy Using the Step-scan Method 74 3.1.5 Time-resolved Pump-probe Studies with Sub-nanosecond Time-resolution 76 3.2 Raman Spectroscopy 79 3.2.1 Laser 80 3.2.1.1 Laser Beam Properties 81 3.2.1.2 Optical Set-up 83 3.2.2 Spectrometer and Detection Systems 84 3.2.2.1 Monochromators 84 3.2.2.2 Spectrographs 86 3.2.2.3 Confocal Spectrometers 87 3.2.2.4 Fourier Transform Raman Interferometers 89 References 97 4.1 4.1.1 4.1.2 4.1.2.1 4.1.2.2 4.2 4.2.1 4.2.1.1 4.2.1.2 4.2.1.3 4.2.2 4.2.2.1 4.2.2.2 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.3.6 4.3.7 4.4 4.4.1 4.4.1.1 Experimental Techniques 99 Inherent Problems of Infrared and Raman Spectroscopy in Life Sciences 99 The ‘‘Water’’ Problem in Infrared Spectroscopy 99 Unwanted Photophysical and Photochemical Processes in Raman Spectroscopy 101 Fluorescence and Raman Scattering 102 Photoinduced Processes 104 Sample Arrangements 105 Infrared Spectroscopy 106 Sandwich Cuvettes for Solution Studies 106 The Attenuated Total Reflection (ATR) Method 108 Electrochemical Cell for Infrared Spectroscopy 113 Raman and Resonance Raman Spectroscopy 116 Measurements in Solutions 116 Solid State and Low-temperature Measurements 117 Surface Enhanced Vibrational Spectroscopy 118 Colloidal Suspensions 119 Massive Electrodes in Electrochemical Cells 120 Metal Films Deposited on ATR Elements 122 Metal/Electrolyte Interfaces 123 Adsorption-induced Structural Changes of Biopolymers 127 Biocompatible Surface Coatings 128 Tip-enhanced Raman Scattering 130 Time-resolved Vibrational Spectroscopic Techniques 131 Pump–Probe Resonance Raman Experiments 132 Continuous-wave Excitation 133 72 Contents 4.4.1.2 4.4.1.3 4.4.2 4.4.2.1 4.4.2.2 4.4.3 4.4.4 4.5 Pulsed-laser Excitation 138 Photoinduced Processes with Caged Compounds 141 Rapid Mixing Techniques 141 Rapid Flow 144 Rapid Freeze–Quench 145 Relaxation Methods 146 Spatially Resolved Vibrational Spectroscopy 148 Analysis of Spectra 149 References 151 5.1 5.2 5.3 Structural Studies 155 Basic Considerations 155 Practical Approaches 158 Studies on the Origin of the Sensitivity of Amide I Bands to Secondary Structure 161 Direct Measurement of the Interaction of the Amide I Oscillators 167 UV-resonance Raman Studies Using the Amide III Mode 169 Protein Folding and Unfolding Studies Using Vibrational Spectroscopy 171 References 178 5.4 5.5 5.6 Retinal Proteins and Photoinduced Processes 181 Rhodopsin 183 Resonance Raman Studies of Rhodopsin 185 Resonance Raman Spectra of Bathorhodopsin 188 Fourier Transform Infrared Studies of the Activation Mechanism of Rhodopsin 195 6.1.3.1 Low-temperature Photoproducts 197 6.1.3.2 The Active State Metarhodopsin II (MII) 201 6.2 Infrared Studies of the Light-driven Proton Pump Bacteriorhodopsin 206 6.3 Study of the Anion Uptake by the Retinal Protein Halorhodopsin Using ATR Infrared Spectroscopy 214 6.4 Infrared Studies Using Caged Compounds as the Trigger Source 217 References 222 6.1 6.1.1 6.1.2 6.1.3 7.1 7.1.1 7.1.2 7.1.3 7.1.4 7.1.5 7.2 7.2.1 Heme Proteins 227 Vibrational Spectroscopy of Metalloporphyrins 228 Metalloporphyrins Under D4h Symmetry 228 Symmetry Lowering 231 Axial Ligation 232 Normal Mode Analyses 233 Empirical Structure–Spectra Relationships 234 Hemoglobin and Myoglobin 236 Vibrational Analysis of the Heme Cofactor 237 VII VIII Contents 7.2.2 7.2.3 7.3 7.3.1 7.3.2 7.3.3 7.3.4 7.3.5 7.3.6 7.4 7.4.1 7.4.2 7.4.3 7.4.4 8.1 8.2 8.3 8.4 Iron–Ligand and Internal Ligand Modes 239 Probing Quaternary Structure Changes 240 Cytochrome c – a Soluble Electron-transferring Protein 244 Vibrational Assignments 245 Redox Equilibria in Solution 246 Conformational Equilibria and Dynamics 248 Redox and Conformational Equilibria in the Immobilised State 253 Electron Transfer Dynamics and Mechanism 260 The Relevance of Surface-enhanced Vibrational Spectroscopic Studies for Elucidating Biological Functions 267 Cytochrome c Oxidase 268 Resonance Raman Spectroscopy 268 Redox Transitions 271 Catalytic Cycle 274 Oxidases from Extremophiles and Archaea 277 References 278 Non-heme Metalloproteins 283 Copper Proteins 284 Iron–Sulfur Proteins 290 Di-iron Proteins 296 Hydrogenases 300 References 302 Index 305 IX Preface Vibrational spectroscopy and life sciences, how they fit together? For more than 30 years vibrational spectroscopy was the classical tool used for the study of small molecules and an analytical tool to characterise unknown chemical compounds, and therefore, it is not obvious that these two subjects would indeed fit together Nevertheless, the fact that K P Hofmann asked us to write a book on the application of vibrational spectroscopy in life sciences, within the newly created series Tutorials in Biophysics, clearly demonstrates that this subject has reached a mature stage The success of vibrational spectroscopy in life sciences is certainly due, largely, to technical developments leading, for instance, to the commercial availability of lasers for Raman spectroscopy and rapid-scan interferometric detection systems for Fourier transform infrared (IR) spectroscopy In this way, the sensitivity of vibrational spectroscopy increased considerably, allowing experiments that were hitherto unimaginable to be carried out However, it is still not clear how these developments made it possible for the basic questions on protein function to be addressed, considering that proteins are very complex systems consisting of thousands of atoms Thus, the main goal of this tutorial is to provide arguments as to why vibrational spectroscopy is successful in biophysics research Both of us have had the privilege of taking active roles in these exciting scientific developments right from the beginning Thus, it should be understood that the material in this book has been influenced by our personal experiences When we started to devise the content of the book, we soon realised that, when considering the application of vibrational spectroscopy in life sciences, we had to focus on molecular biophysics This meant leaving out the exciting fields in which vibrational spectroscopy is used as a diagnostic tool for the identification of bacteria, cancerous cells and metabolites in living cells In addition, within the field of molecular biophysics, we had to make compromises, mainly dictated by space limitations We, therefore, decided to restrict the applications of vibrational spectroscopy to selected classes of proteins and enzymes for the benefit of an instructive illustration of the principles of the most important methodologies The selection of examples was – inevitably – subjective and governed by didactic considerations Thus, not all colleagues who have made important contributions to this field could be adequately referenced Vibrational Spectroscopy in Life Science Friedrich Siebert and Peter Hildebrandt Copyright 2008 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim ISBN: 978-3-527-40506-0 210 Retinal Proteins and Photoinduced Processes Fig 6.17 Time-slice of the time-resolved difference spectra of bacteriorhodopsin shown in Fig 6.16, times given in ms (R€ odig et al 1999) reactions and branching have been proposed Very often it is assumed that the reactions follow 1st order kinetics and that, therefore, the time course of the data (here of the spectra) can be approximated by a sum of exponentials Thus, it is assumed that everywhere in the spectrum the same time-dependence is observed In order to derive the time constants, the time-traces are described over the complete spectral range to a corresponding sum of exponentials, of which the prefactors ak ðnÞ are termed amplitude spectra This is therefore called a global fit Sðn; tÞ ẳ n X ak nị expt=tk ị ỵ a nị 6:2ị kẳ1 Sn; tị denote the time-resolved spectra There are several commercial software packages available to perform this nonlinear global fitting procedure As a result, the time constants tk and the ampli- 6.2 Infrared Studies of the Light-driven Proton Pump Bacteriorhodopsin tude spectra ak ðnÞ are obtained Even if in this way a reasonably accurate description of the spectra can be often obtained, to derive a more complicated reaction scheme containing back-reactions and/or branching, additional assumptions have to be made Here is not the place to discuss this complicated issue We will just show that a simple reaction scheme is described by such a sum of exponentials with corresponding amplitude spectra We assume the following reaction: k1 k2 k nÀ1 P1 À! P2 À! P3 Á Á Á À! Pn ð6:3Þ which is termed the unidirectional reaction scheme, i.e., no back-reactions are included and all steps are first-order reactions The last species may represent the first species, as is the situation for cyclic reactions It is clear that this simple scheme is not adequate for more complicated reactions However, any linear reaction scheme can be transformed into the scheme above As a consequence, in general, the species Pi not represent pure states with respect to their spectral properties but may be mixtures of pure states, that are in a fast equilibrium which is established as soon as Pi is formed It is straightforward to show that the spectra of species Pi can be calculated from the amplitude spectra obtained by the global t procedure (Roădig et al 1999) This simple correlation is a great advantage of this method It has been applied for the analysis of the time-resolved spectra described above (Figs 6.16 and 6.17) The fitting procedure yielded the amplitude spectra shown in Fig 6.18 Eight exponentials were necessary to describe the time-dependence In Fig 6.19 the spectra of the Pi states are shown, labelled with the corresponding decay time constants It is beyond the scope of this presentation to discuss all the different aspects of the spectra, and more details can be found in the original publication (Roădig et al 1999) The rst spectrum corresponds to the early redshifted intermediate (K or KL), the next spectrum characterises the L-state, both states exhibiting the 15-HOOP band at 983 cmÀ1 mentioned above This shows that the C14 aC15 single bond is twisted in these two states The twist in the L-state could be responsible for the reduction of the pK a of the Schiff base, which would, at least partially, initiate the proton transfer to Asp85 It is interesting that for the low-temperature L-state this HOOP band is not seen, and the consequences for the proton transfer step have bee discussed (Roădig et al 1999) This example demonstrates that in many instances it is important to obtain time-resolved IR spectra The proton transfer to Asp85 can be observed, although still with low amplitude, in the next spectrum, where a small positive band at 1761 cmÀ1 can clearly be discerned The band increases in the next two spectra, which, in addition, show decreasing contributions from the HOOP band Thus the three spectra with decay time constants of 40, 134, and 544 ms represent L–M mixtures with increasing contributions from M By subtracting the L contributions, it could be shown that the size of the bands at 1616 and 1657 cmÀ1 is very small in the Mstate with 40-ms decay time, considerably larger in the next M-state, and an additional small increase of these two bands is observed for M with a decay time of 211 212 Retinal Proteins and Photoinduced Processes Fig 6.18 Amplitude spectra of the global fit to the data set shown in Fig 6.16 using eight exponentials (R€ odig et al 1999) 544 ms These bands, which increase progressively, can be interpreted as amide I and amide II bands Otherwise, the three spectra of the M-states are very similar Thus, it appears that the M-states differ in the extent of conformational changes reflected by these two amide bands, confirming the hypothesis of M-states with differing protein conformation These protein conformations may be required to accomplish a vectorial proton transport It is appropriate to discuss under which conditions this time-resolved step– scan technique can be applied to other systems In the measurements described here, or 16 signals have been averaged at each mirror position Because of the short cycling time of bacteriorhodopsin, the repetition rate of the laser could be kept at Hz, resulting in times of 1.6 and 3.2 s, respectively, for the acquisition of the time-resolved change of the interferogram at each mirror position With a resolution of cmÀ1 and a high-frequency cut-off of 1900 cmÀ1 the number of mirror positions (see section 3.1.4.3) amounts to approximately 540 Further, if one takes into account the time needed to change the mirror position, which is approximately 0.5 s, the total measuring time amounts to approximately 19 6.2 Infrared Studies of the Light-driven Proton Pump Bacteriorhodopsin Fig 6.19 Spectra of the intermediates calculated from the amplitude spectra of Fig 6.18 (R€ odig et al 1999) for averages and 34 for 16 averages With such a run the signal to noise ratio is usually not sufficient, and the measurements have to be repeated until the number of flashes at each mirror position is around 64 Thus, the total measuring time is finally  19 ¼ 152 and  34 ¼ 136 min, respectively This estimate holds for a time-resolution of approximately 600 ns If the time-resolution is increased to 30 ns, the number of spectra to be averaged has to be increased by a factor of 600=30ị 1=2 ẳ 4:5, increasing the total measuring time correspondingly For bacteriorhodopsin, with its short cycling time, such measurements could still be performed From this discussion it is clear that the complete time of the photoreaction is a critical parameter for the application of the time-resolved step–scan technique If the reaction is not reversible, means would have to be developed to allow the quantitative exchange of the sample after each excitation As with the amide I changes in the FTIR difference spectra of rhodopsin, for the bands described here only a qualitative interpretation is possible: they indicate structural changes of the peptide backbone For the MII spectra of rhodopsin we have argued that the strong amide I bands might reflect the tilting of helix 213 214 Retinal Proteins and Photoinduced Processes identified, for example, by the spin label technique (Farrens et al 1996) Structural studies have indicated a tilt of helix of bacteriorhodopsin in the late Mstate (see reviews on bacteriorhodopsin Lanyi and Luecke 2001; Haupts et al 1999) We have some indication that the amide I changes observed in bacteriorhodopsin could be caused by the distortion of the helix in the hinge region of the helical tilt (Hauser et al 2002) Thus, it might be that the larger amide I changes observed in the two later M-states described here reflect the tilt of helix However, direct proof is not available Therefore, a better understanding of the amide I changes is highly desirable In Chapter we focussed on the molecular interpretation of the amide I band of proteins and peptides Up to now the analysis has mainly been restricted to how the secondary structural elements and solvent molecules interacting with the peptide backbone influence the amide I band However, if a more detailed description of the amide I band is available, which includes local geometries and distortions of the peptide backbone, it appears feasible that a molecular description of the amide I changes in the FTIR difference spectra becomes possible In most instances, the protein conformations of the corresponding protein states not differ in secondary structure but rather in the local rearrangements of the protein backbone 6.3 Study of the Anion Uptake by the Retinal Protein Halorhodopsin Using ATR Infrared Spectroscopy Halorhodopsin is a light-driven anion pump and shares many homologies with the light-driven proton pump bacteriorhodopsin However, remarkably, the counterion to the Schiff base and proton acceptor in bacteriorhodopsin, Asp85, is replaced by a threonine, and Asp96, the proton donor for reprotonation of the Schiff base, is replaced by an alanine Halorhodopsin is found in two types of archaebacteria, in Halobacterium salinarum (termed HsHR) and in Natronobacterium pharaonis (termed NpHR) Although there are some differences in the amino acid sequence, most of the amino acids in the inner part of the protein, and especially those around the retinal chromophore, have homologue counterparts Anions are pumped from the cell exterior to the cytosol, i.e., opposite to the direction of proton pumping in bacteriorhodopsin The anion pumping is initiated by the light-induced alltrans ! 13-cis isomerisation The vectorial anion translocation is accomplished by first ejecting the anion into the cytosol and later by the uptake of the anion from the extracellular side This latter step completes the photochemical reaction, which, as with bacteriorhodopsin, is a photocycle Thus, there is a state in which the protein is anion-free This state is called O, and it appears in about ms, and it decays via a bimolecular reaction with the uptake of the anion Apart from chloride, iodide, bromide, and nitrate are pumped by NpHR In view of the very different shape of nitrate as compared with the mono-atomic anions, it appears that the path along which the anion moves through the protein must be unspecific 6.3 Study of the Anion Uptake by the Retinal Protein Halorhodopsin The crystal structure of HsHR has shown that in the dark state the anion is bound in the neighbourhood of the protonated Schiff base (Kolbe et al 2000) Thus, the negative charge of the anion serves as the counterion for the protonated Schiff base, because Asp85, the counterion in bacteriorhodopsin, is no longer present A review on halorhodopsin based on the structure of HsHR has recently been published (Essen, 2002) An anion-free state of NpHR can also be produced by lowering the anion concentration below the binding constant This is 1, 2.5, 3, and 16 mM, for bromide, chloride, iodide, and nitrate, respectively By anion depletion, the colour of NpHR changes from purple to blue (blue NpHR), as the absorption maximum is redshifted to 600 nm This is in agreement with the anion being the counterion of the protonated Schiff base, as removal of the counterion usually causes a red-shift of the absorption maximum Of particular interest, is whether the static, anionfree state produced by low anion concentration agrees with the anion-free O-state produced during the photocycle For this, we compare the time-resolved difference spectrum of the O-intermediate with the difference spectrum obtained by following the binding of anions to blue NpHR The ATR IR method is the most suitable for this (see section 4.2.1.2) ATR samples of membrane proteins are usually prepared in the following way: the surface of the ATR crystal is overlaid with the suspension of membranes containing the membrane protein The aqueous solvent is dried-off, causing the membranes to more-or-less stick firmly to the ATR surface This process has been described well for bacteriorhodopsin and is shown in Fig 6.20 (Heberle and Zscherb 1996) Initially, the spectrum is very similar to the spectrum of water Upon drying-off the water, the bands for water at 3300 cmÀ1 (OH stretching), the broad band around 2130 cmÀ1 (combination band), and the broad fea- Fig 6.20 ATR-FTIR spectra monitoring the adsorption of bacteriorhodopsin, containing purple membranes, to the surface of the ATR element while drying off the aqueous solvent Insert shows the ATR-FTIR spectrum of the dried film (dashed line), and of the film after the addition of water (solid line) (Heberle and Zscherb 1996) 215 216 Retinal Proteins and Photoinduced Processes ture below 1000 cmÀ1 (intermolecular libration) disappear However, the bands around 3290 cmÀ1 (amide A), 1650 cmÀ1 amide I, 1550 cmÀ1 amide II, and numerous bands below 1550 cmÀ1 caused by the protein and the membrane lipids increase, because the membrane comes into close contact with the ATR surface Water also has an absorption band at 1650 cmÀ1 (OH bending mode) As the band intensity at this position increases upon drying, one can conclude that the water contributes less to the absorption as compared with the amide I band of bacteriorhodopsin If the membrane stack on the ATR surface is now overlayed with aqueous buffer, the spectrum shown in the insert is obtained Because the amide II band decreases considerably, swelling of the membrane stack takes place As has been emphasised in section 4.2.1.2, the thickness of the aqueous layer above the membrane stack is not important because of the limited penetration depth of the IR beam If the swelling has reached a stable state, binding studies can be performed by adding the respective compound to the buffer with no nonspecific distortion of the sample This sample form guarantees the native conditions at defined pH and salt concentration It has been used for time-resolved step–scan measurements of bacteriorhodopsin under well-defined pH conditions (Heberle and Zscherp 1996) Films have been prepared from NpHR, similar to those for bacteriorhodopsin, with the anion binding to the anion-depleted form of halorhodopsin (i.e., blue NpHR) being followed The corresponding spectra are shown in Fig 6.21 (Guijarro et al 2006) The uppermost spectrum shows the difference spectrum induced by chloride binding Negative bands are due to blue NpHR, whereas positive bands reflect NpHR The largest band is caused by the ethylenic mode (CbC stretching vibration) of the retinal chromophore, which has a lower position (1511 cmÀ1 ) in blue NpHR as compared with NpHR (1525 cmÀ1 ), in agreement with the different absorption maxima (600 versus 578 nm) It is remarkable that the chromophore in blue NpHR shows pronounced HOOP (hydrogen-out-ofplane) modes located around 960 cmÀ1 As has been explained for rhodopsin and bacteriorhodopsin, the HOOP modes indicate that the retinal is twisted stronger in blue NpHR as compared with NpHR Very unexpectedly, the binding of the anion is accompanied by large amide I bands, which indicate that the protein backbone experiences considerable rearrangements It will be interesting to compare these backbone changes with those triggered by the chromophore isomerisation The next three spectra compare the molecular changes induced by chloride uptake with those induced by bromide and iodide They are virtually identical As could be shown (Guijarro et al 2006), the nitrate binding spectrum is also very similar This shows that the protein does not so much react on the size and form of the anion but on the negative charge For a comparison of blue NpHR with the O-intermediate, the negative of this spectrum is shown in the lowest trace Here the negative bands are due to the O-state, whereas the positive bands are caused by NpHR This spectrum has been obtained with the time-resolved step–scan technique (see Guijarro et al 2006) It is obvious that this spectrum is very similar to the anion-uptake spectra The same amide I changes take place, and the O-intermediate is characterised by the HOOP modes Thus, one can con- 6.4 Infrared Studies Using Caged Compounds as the Trigger Source Fig 6.21 Monitoring the binding of halide anions to halorhodopsin NpHR by ATR-FTIR difference spectroscopy: (a) binding of chloride; (b) binding of chloride, bromide, and iodide; (c) inverse of the difference spectrum of the O-intermediate, i.e., bands of O point downwards and bands of the dark state of halorhodopsin point upwards (Guijarro et al 2006) clude that the last step of the halorhodopsin photocycle, i.e., the decay of the O-intermediate and the uptake of the anion, is only a passive binding process In agreement with this, the O-decay is slowed down by lowering the anion concentration, as is expected for a bimolecular reaction Further conclusions on the anion translocation process can be found in the original publication (Guijarro et al 2006) 6.4 Infrared Studies Using Caged Compounds as the Trigger Source In section 4.4.1.3 Experimental Techniques, we described the basic idea of caged compounds and how they can be used to trigger reactions in an infrared cuvette, enabling the molecular changes evolving after the photolysis of the caged com- 217 218 Retinal Proteins and Photoinduced Processes pound to be followed, i.e., after cleavage of the protecting group Here we will describe some characteristic experiments From the large number of caged compounds, those consisting of phosphorylated nucleotides in which the terminal phosphate group is protected, were the first to be used in reaction-induced IR spectroscopy Caged ATP, ADP, and GTP belong to this group of caged compounds They are particularly valuable, as many biological reactions are started by the corresponding uncaged compounds Although triggering the Ca 2ỵ ATPase from sarcoplasmatic reticulum had been among the first systems studied (Barth et al 1990), the system is fairly complex and for tutorial purposes, a somewhat simpler protein appears to be more suitable The family of Ras proteins are GTP-binding proteins representing molecular switches The GTP-bound form is the active state interacting with downstream effectors regulating various cellular responses In the GDP-bound form, the protein is switched off It exhibits an intrinsic GTPase activity, i.e., the conversion into the inactive state occurs spontaneously, although with the slow rate of 5:1  10À4 s In order to study the molecular changes triggered by the binding of GTP to the system, one has to take into account the molecular changes due to the photolysis process The reaction sequence of the photolysis of caged GTP is shown in Scheme 6.1 (Cepus et al 1998) The rate-limiting step for formation of active GTP is the decay of the intermediate 7, which is called the aci-nitro anion state of caged GTP The photolysis has been monitored with time-resolved rapid-scan FTIR spectroscopy (Cepus et al 1998), and some of the results are shown in Fig 6.22 Spectrum A covers the time range from 10 to 26 ms, and spectrum B that Scheme 6.1 Reaction sequence of caged GTP photolysis (Cepus et al 1998) 6.4 Infrared Studies Using Caged Compounds as the Trigger Source Fig 6.22 Time-resolved rapid-scan FTIR spectra of the photolysis of caged GTP: A, time range from 10 to 26 ms; and B, time range from 86 to 105 ms (Cepus et al 1998) from 86 to 105 ms It is important to mention that the measurements were performed in the presence of 250 mM DTT, that scavenges reaction product 10, which reacts with SH groups and could in biological applications distort proteins Spectrum A represents the difference spectrum between the aci-nitro anion state and the dark state of caged GTP, whereas spectrum B shows that between the final products and caged GTP The final products are GTP and the reaction product of compound 10 with DTT The three-band feature around 1124 cmÀ1 is characteristic of the three phosphate groups of GTP (and also of ATP) Specific 18 O-labelling at the a-, b-, and g-phosphates has shown that the band composition is very complex The bands at 1124 and 1093 cmÀ1 mainly represent a- and b-phosphates (PO2 À ) stretching vibrations coupled to the modes of g-phosphate (PO3 2À ) Depending on the time range, the spectra in Fig 6.22 have to be taken into account when measuring difference spectra of biological reactions triggered by GTP According to Allin and Gerwert (Allin and Gerwert 2001), the photolysis of caged GTP within the Ras protein can be described by the following, Eq (6.3): hn Ras  cgGTPðAÞ ! Ras  aci À nitro À anionBị ỵ Hỵ ! Ras  GTPCị ỵ oNAP ! Ras  GDPDị ỵ Pi 6:3ị where oNAP is compound 10 in Scheme 6.1 (o-nitrosoacetophenone), and Pi is inorganic phosphate It is important to mention that caged GTP also binds to Ras Therefore, there are no diffusion processes involved in the generation of Ras  GTP The formation of the aci-nitro anion is too fast to be resolved by the time-resolved rapid-scan technique 219 220 Retinal Proteins and Photoinduced Processes Fig 6.23 Time-resolved rapid-scan spectra of the photolysis of Ras*caged-GTP Difference spectrum between the initial state (A) and GTP bound to Ras (C) [Eq (6.3)], dotted line, difference spectrum between the 1st photoproduct (B) and GTP bound to Ras (C), solid line [Eq (6.3)] (Allin and Gerwert 2001) In Fig 6.23, the time-resolved spectrum between the initial state (A) and GTP bound to Ras (C) is shown (dotted line) In addition, the spectrum between the 1st photoproduct (B) and C is shown (solid line) The intermediate B could be captured with the rapid-scan technique, as the measurements had been performed at 260 K, the low temperature slowing-down the reaction to a rate constant of approximately sÀ1 Positive bands are caused by species C, whereas the negative bands are due to A (dotted line) and B (solid line) Therefore, the positive bands are in good agreement in the two spectra (taking into account some overlap with differing negative bands), whereas the caged GTP in particular shows some characteristic negative bands as indicated in the figure As compared with GTP complexed to Mg 2ỵ in aqueous solution (Fig 6.22), there is a pronounced upshift of the main band at 1124 cmÀ1 to 1142 cmÀ1 More insights into the assignment of these modes can be obtained by 18 O isotopic labelling, and a typical experiment is shown in Fig 6.24 Here, the spectra of the B ! C transition are compared for unlabelled and labelled caged GTP, the oxygen atoms of the g-phosphate being labelled, including the bridging oxygen between b- and g-phosphate Such experiments with additional labelling and extending the measurements to the C ! D transition has allowed the following assignment to be derived: It is clear that upon binding to the Ras protein considerable changes in the force constants take place These changes are interpreted in terms of charge displacements induced by positive charges to the protein environment It is concluded that these charge displacements result in weakening of the bond to the gphosphate, facilitating its hydrolysis Thus, these studies have provided deeper 6.4 Infrared Studies Using Caged Compounds as the Trigger Source Fig 6.24 Comparison of the spectrum of the B ! C transition (Fig 6.23) with 18 O-labelled caged GTP (Allin and Gerwert, 2001) insights into the catalysis of GTP hydrolysis by Ras, although, as has been mentioned, the reaction is still very slow The hydrolysis is accelerated considerably by the physiologically interacting Ras-GAP protein, forming a complex with Ras The mechanism of this acceleration has also been investigated by time-resolved rapid-scan FTIR spectroscopy The basic results are shown in a 3D representation in Fig 6.25 (Koătting and Gerwert 2005) The upper spectrum shows the intrinsic GTP hydrolysis reaction of Ras alone, whereas the lower spectrum shows the time-evolution starting with the photolysis of GTP within the GAP-Ras complex (note the different time scales) The much faster hydrolysis in the GAP-Ras system is evident (for these time-resolved difference spectra, the final products, i.e., Ras-GDP þ Pi and GAPRas-GDP þ Pi , respectively, have been taken as reference) In the lower representation, in addition to 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Siebert, F., 1999, ‘‘Time-resolved step-scan FTIR spectroscopy reveals differences between early and late M intermediates of bacteriorhodopsin’’, Biophys J 76, 2687– 2701 ...Friedrich Siebert and Peter Hildebrandt Vibrational Spectroscopy in Life Science Vibrational Spectroscopy in Life Science Friedrich Siebert and Peter Hildebrandt Copyright 2008 WILEY-VCH... frisi@biophysik.uni-freiburg.de Peter Hildebrandt Institut fuăr Chemie Technische Universitaăt Berlin e-mail: hildebrandt@ chem.tu-berlin.de Cover Picture Vibrational spectroscopy, i.e Raman (bottom) and infrared (top) spectroscopy, ... ISBN: 978-0-471-98847-2 Friedrich Siebert and Peter Hildebrandt Vibrational Spectroscopy in Life Science The Authors Friedrich Siebert Institut fuăr Molekulare Medizin und Zellforschung Albert-Ludwigs-Universitaăt