Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

Lórenz-Fonfrı́a, Vı́ctor A.

doi:10.1021/acs.chemrev.9b00449

Cited by 179 publications

(179 citation statements)

References 840 publications

(3,057 reference statements)

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“…1b), with expected bands at ~1700-1680 cm −1 and at ~1530-1500 cm −1 in aprotic organic solvents of low/medium polarity. 44 Upon considering the expected wavenumber and absorption coefficient of these vibrations relative to those of the amide backbone group, 42 we concluded that both peptides are roughly 85% α-helical.…”

Section: Resultsmentioning

confidence: 95%

A photoswitchable helical peptide with light-controllable interface / transmembrane topology in lipidic membranes

Gutiérrez-Salazar

Santamaría-Aranda

Schaar

et al. 2021

Preprint

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According to the three-step model, the spontaneous insertion and folding of helical transmembrane (TM) polypeptides into lipid bilayers is driven by three sequential equilibria: solution-to-membrane interface (MI) partition, unstructured-to-helical folding, and MI-to-TM helix insertion. However, understanding these three steps with molecular detail has been challenged by the lack of suitable experimental approaches to rapidly and reversibly perturb membrane-bound hydrophobic polypeptides out of equilibrium. Here, we report on a 24-residues-long hydrophobic α-helical polypeptide, covalently coupled to an azobenzene photoswitch (KCALP-azo), which displays a light-controllable TM/MI equilibrium in hydrated lipid bilayers. FTIR spectroscopy shows that dark-adapted KCALP-azo (trans azobenzene) folds as a TM α-helix, with its central TM region displaying an average tilt of 36 ± 4° with the membrane normal (TM topology). After trans-to-cis photoisomerization of the azobenzene moiety with UV light (reversed with blue light), spectral changes by FTIR spectroscopy indicate that the helical structure of KCALP-azo is maintained but the peptide experiences a more polar environment. Interestingly, pH changes induced similar spectral alterations in the helical peptide LAH4, with a well-characterized pH-dependent TM/MI equilibrium. Polarized experiments confirmed that the membrane topology of KCALP-azo is altered by light, with its helix tilt changing reversibly from 32 ± 5° (TM topology, blue light) to 79 ± 8° (MI topology, UV light). Further analysis indicates that, while the trans isomer of KCALP-azo is ≈100% TM, the cis isomer exists in a ≈90% TM and ≈10% MI mixture. Strategies to further increase the perturbation of the TM/MI equilibrium with the light are briefly discussed.

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Section: Resultsmentioning

confidence: 95%

A photoswitchable helical peptide with light-controllable interface / transmembrane topology in lipidic membranes

Gutiérrez-Salazar

Santamaría-Aranda

Schaar

et al. 2021

Preprint

Self Cite

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“…However, analysing bands of low absorbance or overlapping signals, efficient trigger concept must be devised to compute accurate FTIR difference spectra. 49 Besides illumination, solvent exchange, and potential jump experiments [59][60][61] , I introduced quantitative gas titrations as a novel trigger strategy.…”

Section: Discussionmentioning

confidence: 99%

“…Deuteration and hydration are mutually reversible, as demonstrated in Figure 7D. Understanding H/D isotope effects is key to understanding hydrogen bonding interations 49 and hydride chemistry. 65 The relation between sample concentration and sample film quality is not straight-forward and should be evaluated for each sample individually.…”

Section: Preparation Of Protein Filmsmentioning

confidence: 99%

“…FTIR difference spectroscopy allows extracting cofactor signals across a wide energy regime when changes are triggered by visible light, e.g., for the analysis of chromoproteins. 49 Despite singular exceptions 50 , GPMs do not show catalytic photochemistry. Irradiation experiments are important nevertheless: photochemical 'side reactions' and redox dyes allow addressing well-defined state transitions of GPMs.…”

Section: Infrared Spectroscopymentioning

confidence: 99%

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Operando Infrared Spectroscopy for the Analysis of Gas-processing Metalloenzymes

Stripp¹

2020

Preprint

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Earth-abundant transition metals like iron, nickel, copper, molybdenum, and vanadium have been identified as essential constituents of the cellular gas metabolism in all kingdoms of life. Associated with biological macromolecules, gas-processing metalloenzymes (GPMs) are formed that catalyse a variety of redox reactions. This includes the reduction of O2 to water by cytochrome c oxidase (‘complex IV’), the reduction of N2 to NH4 by nitrogenase, as well as the reduction of protons to H2 (and oxidation of the later) by hydrogenase. GPMs perform at ambient temperature and pressure, in the presence of water, and often extremely low educt concentrations, thus serving as natural examples for efficient catalysis. Facilitating the design of biomimetic catalysts, biophysicist thrive to understand the reaction principles of GPMs making use of various techniques. In this perspective, I will introduce Fourier-transform infrared spectroscopy in attenuated total reflection configuration (ATR FTIR) for the analysis of GPMs like cytochrome c oxidase, nitrogenase, and hydrogenase. Infrared spectroscopy provides information about the geometry and redox state of the catalytic cofactors, the protonation state of amino acid residues, the hydrogen-bonding network, and protein structural changes. I developed an approach to probe and trigger the reaction of GPMs by gas exchange experiments, exploring the reactivity of these enzymes with their natural reactants. This allows recording sensitive ATR FTIR difference spectra with seconds time resolution. Finally yet importantly, infrared spectroscopy is an electronically non-invasive technique that allows investigating protein samples under biologically relevant conditions, i.e., at ambient temperature and pressure, and in the presence of water.

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“…protein fold) in a label-free manner. 1,2 Applications range from assignment of bacterial species, 3 detection of light-induced changes in protein conformation, cofactors and protonation state of individual amino acid residues, 4,5 aggregation states of proteins related to neurodegenerative diseases like Alzheimer's or Parkinson's disease [6][7][8] , and label-free imaging of tissue in cancer diagnosis. [9][10][11] On top of structural information, the temporal resolution of IR spectroscopy matches the decay times of vibrational modes in the femtosecond range.…”

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confidence: 99%

Infrared Scattering-Type Scanning Near-Field Optical Microscopy in Water

Pfitzner¹,

Heberle²

2020

Preprint

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<div><div><div><p>Infrared (IR) absorption spectroscopy detects state and chemical composition of biomolecules solely by their inherent vibrational fingerprints. Major disadvantages like the lack of spatial resolution and sensitivity were compensated lately by the use of pointed probes as local sensors enabling the detection of quantities as few as hundreds of proteins with nanometer precision. This makes infrared scattering-type scanning near-field optical microscopy a very powerful tool in life science. The strong absorption of infrared radiation of liquid water, however, still prevents to simply access the measured quantity – light scattered at the probing atomic force microscope tip. Here we report on the local IR response of biological membranes immersed in aqueous bulk solution. We make use of a silicon solid immersion lens as substrate and focusing optics to achieve detection efficiencies sufficient to yield IR near-field maps of purple membranes. We scrutinized our experimental findings by applying theoretical models. Finally, we suggest a means to improve the imaging quality by laser scanning assisted scattering-type scanning near-field optical microscopy. We believe that IR scattering-type scanning near-field optical microscopy will resolve biological structures in their native environments at nm resolution without the need for labeling.</p></div></div></div>

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Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

Cited by 179 publications

References 840 publications

A photoswitchable helical peptide with light-controllable interface / transmembrane topology in lipidic membranes

A photoswitchable helical peptide with light-controllable interface / transmembrane topology in lipidic membranes

Operando Infrared Spectroscopy for the Analysis of Gas-processing Metalloenzymes

Infrared Scattering-Type Scanning Near-Field Optical Microscopy in Water

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