Structural and Functional Implications of the Instability of the ADP/ATP Transporter Purified from Mitochondria as Revealed by FTIR Spectroscopy

Lórenz-Fonfrı́a, Vı́ctor A.; Villaverde, Joaquim; Trezeguet, Véronique; Lauquin, Guy J.‐M.; Brandolin, Gérard; Padrós, Esteve

doi:10.1016/s0006-3495(03)74471-3

Cited by 9 publications

(6 citation statements)

References 43 publications

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“…Because of this experimental difficulty, it has been much less studied than the amide I and amide II vibrations. For membrane proteins adopting a helical structure the amide A is centered at around 3300 cm –1 ,, and for β-sheets at ∼3250 cm –1 . , Because of the cancellation of the bulk water absorption, amide A vibrations are often resolved in IR difference spectra ,, but rarely used to derive structural information because of the lack of detailed theoretical and experimental studies to interpret the observed changes.…”

Section: Integration and Interpretationmentioning

confidence: 99%

“…For instance, it is particularly difficult to control the final ionic strength in the sample, which can be an important parameter (a) Absorption spectrum of the detergent solubilized mitochondrial ATP/ADP carrier at 8 mg/mL in a H 2 O buffer, overlaid with the scaled spectrum of the buffer. 117 The subtraction of the sample and the scaled buffer spectra reveals the absorption from the protein (and detergent). The amide I−II region of the protein is expanded for a better visualization, showing a maximum absorbance of 0.02.…”

Section: 2)mentioning

confidence: 99%

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Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

2020

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Infrared difference spectroscopy probes vibrational changes of proteins upon their perturbation. Compared with other spectroscopic methods, it stands out by its sensitivity to the protonation state, H-bonding, and the conformation of different groups in proteins, including the peptide backbone, amino acid side chains, internal water molecules, or cofactors. In particular, the detection of protonation and H-bonding changes in a timeresolved manner, not easily obtained by other techniques, is one of the most successful applications of IR difference spectroscopy. The present review deals with the use of perturbations designed to specifically change the protein between two (or more) functionally relevant states, a strategy often referred to as reaction-induced IR difference spectroscopy. In the first half of this contribution, I review the technique of reaction-induced IR difference spectroscopy of proteins, with special emphasis given to the preparation of suitable samples and their characterization, strategies for the perturbation of proteins, and methodologies for time-resolved measurements (from nanoseconds to minutes). The second half of this contribution focuses on the spectral interpretation. It starts by reviewing how changes in H-bonding, medium polarity, and vibrational coupling affect vibrational frequencies, intensities, and bandwidths. It is followed by band assignments, a crucial aspect mostly performed with the help of isotopic labeling and site-directed mutagenesis, and complemented by integration and interpretation of the results in the context of the studied protein, an aspect increasingly supported by spectral calculations. Selected examples from the literature, predominately but not exclusively from retinal proteins, are used to illustrate the topics covered in this review.

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Section: Integration and Interpretationmentioning

confidence: 99%

Section: 2)mentioning

confidence: 99%

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

2020

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“…8C). Based on their frequencies, they can be tentatively assigned to the submicrosecond elongation of β-hairpins [1,640-1,625 cm −1 (35)] from extramembrane loops [∼1,662 cm −1 (38)]. However, the negative absorption at 1,663 cm −1 may also comprise changes in the νC=N of the protonated SB, the frequency of which has been assigned to 1,657 cm −1 by resonance Raman spectroscopy (18).…”

Section: Structural Changes Of the Protein Backbonementioning

confidence: 99%

Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating

Lórenz-Fonfrı́a

Resler

Krause

et al. 2013

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

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162

414

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The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements. We have resolved the vibrational changes associated with the open states of the channel (P 2 390 and P 3 520 ) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane α-helices with a t 1/2 = 60 μs, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t 1/2 = 10 μs), with the latter being reprotonated by aspartic acid 156 (t 1/2 = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. Previous conclusions on the involvement of glutamic acid 90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the nonconductive P 4 480 state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions and the protein conformational changes to the gating of the cation channel.O ptogenetics provides new tools to neurophysiologists to steer cellular responses with unprecedented temporal and spatial resolution. The former takes advantage of light as an ultrashort trigger, whereas the latter is achieved by genetically encoding and directing photosensitive proteins to specific cell types. The most prominent among the optogenetics tools is channelrhodopsin (ChR), which was found to be the first light-gated ion channel of its kind (1, 2). This discovery paved the way for an exponentially growing number of neurophysiological applications, ranging from single cells to living animals (3). Light-gated ion permeation by ChR expands the various modes of action of the large family of microbial rhodopsins already comprising light-driven ion pumps and sensors (4). Among the various ChRs, which differ mostly in cation selectivity (3), ChR2 is used in the majority of optogenetic applications because of the higher expression yield in mammalian cells.A projection structure of the heptahelical ChR2 showed a dimer with the contact interface between helices C and D suggested to form the cation channel (5). More recently, a chimeric ChR (C1C2) was constructed by linking the last two helices (F and G) of ChR2 to the first five (A to E) of ChR1 and resolved by X-ray crystallography to 2.3 Å (6). The high-resolution structure confirmed the dimeric arrangement and identified an electronegative extracellular pore in each monomer framed by helices A, B, C, and G. Accompanying electrophysiological experiments on point mutants indicated r...

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“…Other times, positive and negatives values should be expected in a deconvoluted spectra for physical reasons, as for infrared lineal dichroism spectra [51][52][53] or for difference spectra. For instance, several papers have reported infrared difference spectra corresponding mainly to protein conformational changes upon substrate binding.…”

Section: Introductionmentioning

confidence: 99%

Maximum Entropy Deconvolution of Infrared Spectra: Use of a Novel Entropy Expression without Sign Restriction

Lórenz-Fonfrı́a

Padrós

2005

Appl Spectrosc

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Absorbance and difference infrared spectra are often acquired aiming to characterize protein structure and structural changes of proteins upon ligand binding, as well as for many other chemical and biochemical studies. Their analysis requires as a first step the identification of the component bands (number, position, and area) and as a second step their assignment. The first step of the analysis is challenged by the habitually strong band overlap in infrared spectra. Therefore, it is useful to make use of a mathematical method able to narrow the component bands to the extent to eliminate, or at least reduce, the band overlap. Additionally, to be of general applicability this method should permit negative values for the solution. We present a maximum entropy deconvolution approach for the band-narrowing of absorbance and difference spectra showing the required characteristics, which uses the generalized negative Burg-entropy (Itakura-Saito discrepancy) generalized for difference spectra. We present results on synthetic noisy absorbance and difference spectra, as well as on experimental infrared spectra from the membrane protein bacteriorhodopsin.

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Structural and Functional Implications of the Instability of the ADP/ATP Transporter Purified from Mitochondria as Revealed by FTIR Spectroscopy

Cited by 9 publications

References 43 publications

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating

Maximum Entropy Deconvolution of Infrared Spectra: Use of a Novel Entropy Expression without Sign Restriction

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