The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective (original) (raw)
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Structural characterization of membrane proteins and peptides by FTIR and ATR-FTIR spectroscopy
Methods in molecular biology (Clifton, N.J.), 2013
Fourier transform infrared (FTIR) spectroscopy is widely used in structural characterization of proteins or peptides. While the method does not have the capability of providing the precise, atomic-resolution molecular structure, it is exquisitely sensitive to conformational changes occurring in proteins upon functional transitions or upon intermolecular interactions. Sensitivity of vibrational frequencies to atomic masses has led to development of "isotope-edited" FTIR spectroscopy, where structural effects in two proteins, one unlabeled and the other labeled with a heavier stable isotope, such as (13)C, are resolved simultaneously based on spectral downshift (separation) of the amide I band of the labeled protein. The same isotope effect is used to identify site-specific conformational changes in proteins by site-directed or segmental isotope labeling. Negligible light scattering in the infrared region provides an opportunity to study intermolecular interactions between l...
Biopolymers, 2004
As more and more high-resolution structures of proteins become available, the new challenge is the understanding of these small conformational changes that are responsible for protein activity. Specialized difference Fourier transform infrared (FTIR) techniques allow the recording of side-chain modifications or minute secondary structure changes. Yet, large domain movements remain usually unnoticed. FTIR spectroscopy provides a unique opportunity to record 1 H/ 2 H exchange kinetics at the level of the amide proton. This approach is extremely sensitive to tertiary structure changes and yields quantitative data on domain/domain interactions. An experimental setup designed for attenuated total reflection and a specific approach for the analysis of the results is described. The study of one membrane protein, the gastric H ϩ ,K ϩ -ATPase, demonstrates the usefulness of 1 H/ 2 H exchange kinetics for the understanding of the molecular movement related to the catalytic activity.
Biophysical Chemistry, 1995
transform infrared difference spectroscopy has been used extensively to probe structural changes in bacteriorhodopsin and other retinal proteins. However, the absence of a general method to assign bands to individual chemical groups in a protein has limited the application of this technique. While site-directed mutagenesis has been successful in special cases for such assignments, in general, this approach induces perturbations in the structure and function of the protein, thereby preventing unambiguous band assignments. A new approach has recently been reported (Sonar et al., Nature Struct. Biol., 1 (1994) 512-517) which involves cell-free expression of bacteriorhodopsin and site-directed isotope labeling (SDIL). We have now used this method to re-examine bands assigned in the bR + M difference spectrum to tyrosine residues. Our results show that out of 11 tyrosines in bR, only Tyr 185 is structurally active. This work further demonstrates the power of SDIL and FTIR to probe conformational changes at the level of individual amino acid residues in proteins. -free protein synthesis; M intermediate Elsevier Science B.V. SSDI 0301-4622(95)00016-X
Biophysical Reviews
H.G. Khorana’s seminal contributions to molecular biology are well-known. He also had a lesser known but still major influence on current application of advanced vibrational spectroscopic techniques such as FTIR difference spectroscopy to explore the mechanism of bacteriorhodopsin and other integral membrane proteins. In this review, I provide a personal perspective of my collaborative research and interactions with Gobind, from 1982 to 1995 when our groups published over 25 papers together which resulted in an early picture of key features of the bacteriorhodopsin proton pump mechanism. Much of this early work served as a blueprint for subsequent advances based on combining protein bioengineering and vibrational spectroscopic techniques to study integral membrane proteins.
Fourier Transform Infrared Difference Spectroscopy of Bacteriorhodopsin and Its Photoproducts
Proceedings of The National Academy of Sciences, 1982
Fourier transform infrared difference spectroscopy has been used to obtain the vibrational modes. in the chro-*mophore and apoprotein 'that change in intensity or position between light-adapted bacteriorhodopsin and the K and M-intermediates in its photocycle and between dark-adapted and lightadapted bacteriorhodopsin. Our infrared' measurements provide independent verification of resonance Raman results that in lightadapted bacteriorhodopsin the protein-chromophore linkage is a protonated Schiff base and in the M state the Schiff base is un-,protonated. Although we cannot unambiguously identify the Schiff base stretching frequency in the K state, the most'likely interpretation of deuterium shifts of the chromophore hydrogen out-ofplane vibrations is that the Schiff base in K is protonated. The intensity of the hydrogen out-of-plane vibrations in the K state compared with the intensities of.those in light-adapted and'darkadapted bacteriorhodopsin shows that the conformation of the chromophore in K is considerably distorted. In addition, we find evidence that the conformation of the protein changes during the photocycle.
Structural analysis of proteins by isotope-edited FTIR spectroscopy
Spectroscopy, 2010
Structure determination of multidomain proteins or protein–membrane complexes is one of the most challenging tasks in modern structural biology. High-resolution techniques, like NMR or X-ray crystallography, are limited to molecules of moderate size or those that can be crystallized easily. Both methods encounter serious technical obstacles in structural analysis of protein–membrane systems. This work describes an emerging biophysical technique that combines segmental isotope labeling of proteins with Fourier transform infrared (FTIR) spectroscopy, which provides site-specific structural information on proteins and allows structural characterization of protein–membrane complexes. Labeling of a segment of the protein with13C results in infrared spectral resolution of the labeled and unlabeled parts and thus allows identification of structural changes in specific domains/segments of the protein that accompany functional transitions. Segmental isotope labeling also allows determination...
Proceedings of the …, 1999
Active proton transfer through membrane proteins is accomplished by shifts in the acidity of internal amino acids, prosthetic groups, and water molecules. The recently introduced step-scan attenuated total ref lection Fourier-transform infrared (ATR͞FT-IR) spectroscopy was employed to determine transient pK a changes of single amino acid side chains of the proton pump bacteriorhodopsin. The high pK a of D96 (>12 in the ground state) drops to 7.1 ؎ 0.2 (in 1 M KCl) during the lifetime of the N intermediate, quantitating the role of D96 as the internal proton donor of the retinal Schiff base. We conclude from experiments on the pH dependence of the proton release reaction and on point mutants where each of the glutamates on the extracellular surface has been exchanged that besides D85 no other carboxylic group changes its protonation state during proton release. However, E194 and E204 interact with D85, the primary proton acceptor of the Schiff base proton. The CAO stretching vibration of D85 undergoes a characteristic pHdependent shift in frequency during the M state of wild-type bacteriorhodopsin with a pK a of 5.2 (؎0.3) which is abolished in the single-site mutants E194Q and E204Q and the quadruple mutant E9Q͞E74Q͞E194Q͞E204Q. The double mutation E9Q͞E74Q does not affect the lifetime of the intermediates, ruling out any participation of these residues in the proton transfer chain of bacteriorhodopsin. This study demonstrates that transient changes in acidity of single amino acid residues can be quantified in situ with infrared spectroscopy.
The Journal of Chemical Physics
Membranes are crucial for the functionality of membrane proteins in several cellular processes. Time-resolved infrared (IR) spectroscopy enables the investigation of interaction-induced dynamics of the protein and the lipid membrane. The photoreceptor and proton pump bacteriorhodopsin (BR) was reconstituted into liposomes mimicking the native purple membrane. By utilization of deuterated lipid alkyl chains, corresponding vibrational modes are frequency-shifted into a spectrally silent window what allows to monitor lipid dynamics during the photoreaction of BR. Our home-built quantum cascade laser (QCL)-based IR spectrometer covers all relevant spectral regions to detect both lipid and protein vibrational modes. QCL-probed transients at single wavenumbers are compared with previously performed step-scan Fourier-transform infrared (FTIR) measurements. The absorbance changes of the lipids could be resolved by QCL-measurements with a much better signal to noise and with nanosecond time ...
Nano Letters, 2019
Photosensitive proteins embedded in the cell membrane (about 5 nm thickness) act as photoactivated proton pumps, ion gates, enzymes, or more generally, as initiators of stimuli for the cell activity. They are composed of a protein backbone and a covalently bound cofactor (e.g. the retinal chromophore in bacteriorhodopsin (BR), channelrhodopsin, and other opsins). The light-induced conformational changes of both the cofactor and the protein are at the basis of the physiological functions of photosensitive proteins. Despite the dramatic development of microscopy techniques, investigating conformational changes of proteins at the membrane monolayer level is still a big challenge. Techniques based on atomic force microscopy (AFM) can detect electric currents through protein monolayers and even molecular binding forces in single-protein molecules but not the conformational changes. For the latter, Fourier-transform infrared spectroscopy (FTIR) using differencespectroscopy mode is typically employed, but it is performed on macroscopic liquid suspensions or thick films containing large amounts of purified photosensitive proteins. In this work, we develop AFM-assisted, tip-enhanced infrared differencenanospectroscopy to investigate light-induced conformational changes of the bacteriorhodopsin mutant D96N in single submicrometric native purple membrane patches. We obtain a significant improvement compared with the signal-to-noise ratio of standard IR nanospectroscopy techniques by exploiting the field enhancement in the plasmonic nanogap that forms between a gold-coated AFM probe tip and an ultraflat gold surface, as further supported by electromagnetic and thermal simulations. IR difference-spectra in the 1450−1800 cm −1 range are recorded from individual patches as thin as 10 nm, with a diameter of less than 500 nm, well beyond the diffraction limit for FTIR microspectroscopy. We find clear spectroscopic evidence of a branching of the photocycle for BR molecules in direct contact with the gold surfaces, with equal amounts of proteins either following the standard proton-pump photocycle or being trapped in an intermediate state not directly contributing to light-induced proton transport. Our results are particularly relevant for BR-based optoelectronic and energy-harvesting devices, where BR molecular monolayers are put in contact with metal surfaces, and, more generally, for AFM-based IR spectroscopy studies of conformational changes of proteins embedded in intrinsically heterogeneous native cell membranes.